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Damyanova KB, Nixon B, Johnston SD, Gambini A, Benitez PP, Lord T. Spermatogonial stem cell technologies: applications from human medicine to wildlife conservation†. Biol Reprod 2024; 111:757-779. [PMID: 38993049 PMCID: PMC11473898 DOI: 10.1093/biolre/ioae109] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2024] [Revised: 07/05/2024] [Accepted: 07/10/2024] [Indexed: 07/13/2024] Open
Abstract
Spermatogonial stem cell (SSC) technologies that are currently under clinical development to reverse human infertility hold the potential to be adapted and applied for the conservation of endangered and vulnerable wildlife species. The biobanking of testis tissue containing SSCs from wildlife species, aligned with that occurring in pediatric human patients, could facilitate strategies to improve the genetic diversity and fitness of endangered populations. Approaches to utilize these SSCs could include spermatogonial transplantation or testis tissue grafting into a donor animal of the same or a closely related species, or in vitro spermatogenesis paired with assisted reproduction approaches. The primary roadblock to progress in this field is a lack of fundamental knowledge of SSC biology in non-model species. Herein, we review the current understanding of molecular mechanisms controlling SSC function in laboratory rodents and humans, and given our particular interest in the conservation of Australian marsupials, use a subset of these species as a case-study to demonstrate gaps-in-knowledge that are common to wildlife. Additionally, we review progress in the development and application of SSC technologies in fertility clinics and consider the translation potential of these techniques for species conservation pipelines.
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Affiliation(s)
- Katerina B Damyanova
- Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
- Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia
| | - Brett Nixon
- Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
- Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia
| | - Stephen D Johnston
- School of Environment, The University of Queensland, Gatton, QLD 4343, Australia
- School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia
| | - Andrés Gambini
- School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia
- School of Agriculture and Food Science, The University of Queensland, Gatton, QLD 4343, Australia
| | - Patricio P Benitez
- School of Agriculture and Food Science, The University of Queensland, Gatton, QLD 4343, Australia
| | - Tessa Lord
- Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
- Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia
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Li XX, Zhang DC, Wang Y, Wen J, Wang XJ, Cao YL, Jiang R, Li JR, Li YN, Liu HH, Xie WH, Xu ZF, Hu P, Zou K. Cadherin-18 loss in prospermatogonia and spermatogonial stem cells enhances cell adhesion through a compensatory mechanism. Zool Res 2024; 45:1048-1060. [PMID: 39147719 PMCID: PMC11491781 DOI: 10.24272/j.issn.2095-8137.2023.373] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2024] [Accepted: 04/12/2024] [Indexed: 08/17/2024] Open
Abstract
Extracellular membrane proteins are crucial for mediating cell attachment, recognition, and signal transduction in the testicular microenvironment, particularly germline stem cells. Cadherin 18 (CDH18), a type II classical cadherin, is primarily expressed in the nervous and reproductive systems. Here, we investigated the expression of CDH18 in neonatal porcine prospermatogonia (ProSGs) and murine spermatogonial stem cells (SSCs). Disruption of CDH18 expression did not adversely affect cell morphology, proliferation, self-renewal, or differentiation in cultured porcine ProSGs, but enhanced cell adhesion and prolonged cell maintenance. Transcriptomic analysis indicated that the down-regulation of CDH18 in ProSGs significantly up-regulated genes and signaling pathways associated with cell adhesion. To further elucidate the function of CDH18 in germ cells, Cdh18 knockout mice were generated, which exhibited normal testicular morphology, histology, and spermatogenesis. Transcriptomic analysis showed increased expression of genes associated with adhesion, consistent with the observations in porcine ProSGs. The interaction of CDH18 with β-catenin and JAK2 in both porcine ProSGs and murine SSCs suggested an inhibitory effect on the canonical Wnt and JAK-STAT signaling pathways during CDH18 deficiency. Collectively, these findings highlight the crucial role of CDH18 in regulating cell adhesion in porcine ProSGs and mouse SSCs. Understanding this regulatory mechanism provides significant insights into the testicular niche.
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Affiliation(s)
- Xiao-Xiao Li
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
- Stem Cell Research and Translation Center, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Dan-Chen Zhang
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
- Stem Cell Research and Translation Center, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Yan Wang
- Department of Prenatal Diagnosis, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, Jiangsu 210004, China
| | - Jian Wen
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
- Stem Cell Research and Translation Center, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Xing-Ju Wang
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
- Stem Cell Research and Translation Center, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Yu-Lu Cao
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
- Stem Cell Research and Translation Center, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Ru Jiang
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Jia-Rui Li
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Yi-Nuo Li
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - He-He Liu
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Wen-Hai Xie
- School of Life Sciences and Medicine, Shandong University of Technology, Zibo, Shandong 255000, China
| | - Zheng-Feng Xu
- Department of Prenatal Diagnosis, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, Jiangsu 210004, China. E-mail:
| | - Ping Hu
- Department of Prenatal Diagnosis, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, Jiangsu 210004, China. E-mail:
| | - Kang Zou
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
- Stem Cell Research and Translation Center, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China. E-mail:
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Segunda MN, Díaz C, Torres CG, Parraguez VH, De Los Reyes M, Peralta OA. Bovine Peripheral Blood-Derived Mesenchymal Stem Cells (PB-MSCs) and Spermatogonial Stem Cells (SSCs) Display Contrasting Expression Patterns of Pluripotency and Germ Cell Markers under the Effect of Sertoli Cell Conditioned Medium. Animals (Basel) 2024; 14:803. [PMID: 38473188 DOI: 10.3390/ani14050803] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2023] [Revised: 02/05/2024] [Accepted: 02/16/2024] [Indexed: 03/14/2024] Open
Abstract
In vitro gamete derivation has been proposed as an interesting strategy for treatment of infertility, improvement of genetic traits, and conservation of endangered animals. Spermatogonial stem cells (SSCs) are primary candidates for in vitro gamete derivation; however, recently, mesenchymal stem cells (MSCs) have also been proposed as candidates for germ cell (GCs) differentiation mainly due to their transdifferentiating capacity. The objective of the present study was to compare the potential for GC differentiation of bovine peripheral blood-derived MSCs (PB-MSCs) and SSCs under the effect of conditioned medium (CM) derived from Sertoli cells (SCs/CM). Samples were collected every 7 days for 21 days and analyzed for pluripotent, GC, and MSC marker expression. The absence of OCT4 and the increased (p < 0.05) expression of NANOG seems to play a role in SSC differentiation, whereas the absence of NANOG and the increased expression (p < 0.05) of OCT4 may be required for PB-MSC differentiation into GCs. SSCs cultured with SCs/CM increased (p < 0.05) the expression of PIWIL2 and DAZL, while PB-MSCs cultured under the same condition only increased (p < 0.05) the expression of DAZL. Overall, the patterns of markers expression suggest that PB-MSCs and SSCs activate different signaling pathways after exposure to SCs/CM and during differentiation into GCs.
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Affiliation(s)
- Moisés N Segunda
- Faculty of Veterinary and Animal Sciences, University of Chile, Santiago 8820808, Chile
- Doctorate Program of Forestry, Agriculture, and Veterinary Sciences (DCSAV), University of Chile, Santiago 8820808, Chile
- Faculdade de Medicina Veterinária, Universidade José Eduardo dos Santos, Bairro Santo António-Avenida Nuno Alvarez, Huambo 555, Angola
| | - Carlos Díaz
- Doctorate Program in Sciences, UNED, Bravo Murillo 38, 28015 Madrid, Spain
| | - Cristian G Torres
- Faculty of Veterinary and Animal Sciences, University of Chile, Santiago 8820808, Chile
| | - Víctor H Parraguez
- Faculty of Veterinary and Animal Sciences, University of Chile, Santiago 8820808, Chile
| | - Mónica De Los Reyes
- Faculty of Veterinary and Animal Sciences, University of Chile, Santiago 8820808, Chile
| | - Oscar A Peralta
- Faculty of Veterinary and Animal Sciences, University of Chile, Santiago 8820808, Chile
- Escuela de Medicina Veterinaria, Facultad de Agronomía e Ingeniería Forestal, Facultad de Ciencias Biológicas y Facultad de Medicina, Pontificia Universidad Católica de Chile, Vicuña Mackenna 4860, Macul, Santiago 7820436, Chile
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Aponte PM, Gutierrez-Reinoso MA, Garcia-Herreros M. Bridging the Gap: Animal Models in Next-Generation Reproductive Technologies for Male Fertility Preservation. Life (Basel) 2023; 14:17. [PMID: 38276265 PMCID: PMC10820126 DOI: 10.3390/life14010017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2023] [Revised: 12/12/2023] [Accepted: 12/19/2023] [Indexed: 01/27/2024] Open
Abstract
This review aims to explore advanced reproductive technologies for male fertility preservation, underscoring the essential role that animal models have played in shaping these techniques through historical contexts and into modern applications. Rising infertility concerns have become more prevalent in human populations recently. The surge in male fertility issues has prompted advanced reproductive technologies, with animal models playing a pivotal role in their evolution. Historically, animal models have aided our understanding in the field, from early reproductive basic research to developing techniques like artificial insemination, multiple ovulation, and in vitro fertilization. The contemporary landscape of male fertility preservation encompasses techniques such as sperm cryopreservation, testicular sperm extraction, and intracytoplasmic sperm injection, among others. The relevance of animal models will undoubtedly bridge the gap between traditional methods and revolutionary next-generation reproductive techniques, fortifying our collective efforts in enhancing male fertility preservation strategies. While we possess extensive knowledge about spermatogenesis and its regulation, largely thanks to insights from animal models that paved the way for human infertility treatments, a pressing need remains to further understand specific infertility issues unique to humans. The primary aim of this review is to provide a comprehensive analysis of how animal models have influenced the development and refinement of advanced reproductive technologies for male fertility preservation, and to assess their future potential in bridging the gap between current practices and cutting-edge fertility techniques, particularly in addressing unique human male factor infertility.
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Affiliation(s)
- Pedro M. Aponte
- Colegio de Ciencias Biológicas y Ambientales (COCIBA), Universidad San Francisco de Quito (USFQ), Quito 170901, Ecuador
- Instituto de Investigaciones en Biomedicina “One-Health”, Universidad San Francisco de Quito (USFQ), Campus Cumbayá, Quito 170901, Ecuador
| | - Miguel A. Gutierrez-Reinoso
- Facultad de Ciencias Agropecuarias y Recursos Naturales, Carrera de Medicina Veterinaria, Universidad Técnica de Cotopaxi (UTC), Latacunga 050150, Ecuador;
- Laboratorio de Biotecnología Animal, Departamento de Ciencia Animal, Facultad de Ciencias Veterinarias, Universidad de Concepción (UdeC), Chillán 3780000, Chile
| | - Manuel Garcia-Herreros
- Instituto Nacional de Investigação Agrária e Veterinária (INIAV), 2005-048 Santarém, Portugal
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Tang S, Jones C, Dye J, Coward K. Dissociation, enrichment, and the in vitro formation of gonocyte colonies from cryopreserved neonatal bovine testicular tissues. Theriogenology 2023; 210:143-153. [PMID: 37499372 DOI: 10.1016/j.theriogenology.2023.07.022] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2022] [Revised: 07/11/2023] [Accepted: 07/22/2023] [Indexed: 07/29/2023]
Abstract
Gonocytes play an important role in early development of spermatogonial stem cells and fertility preservation to acquire more high quality gonocytes in vitro for further germ cell-related research and applications, it is necessarily needed to enrich and in vitro propagate gonocytes from cryopreserved bovine testicular tissues. This study aimed to investigate the isolation, enrichment, and colony formation of gonocytes in vitro for germ cell expansion from cryopreserved neonatal bovine testicular tissues. The effects of several different in vitro culture conditions, including seeding density, temperature, serum replacement and extracellular matrices were investigated for the maintenance, proliferation and formation of gonocyte colonies in vitro. Frozen/thawed two-week-old neonatal bovine testicular tissues were digested and gonocytes were enriched using a Percoll density gradient. Cell viability was accessed by trypan blue staining and cell apoptosis was evaluated by TUNEL assays. Gonocytes were identified and confirmed by immunofluorescence with the PGP9.5 germ cell marker and the OCT4 pluripotency marker while Sertoli cells were stained with vimentin. We found that neonatal bovine gonocytes were efficiently enriched by a 30%-40% Percoll density gradient (p < 0.05). No significant differences were detected between neonatal bovine testicular cells cultured at 34 °C or 37 °C. The formation of gonocyte colonies was observed in culture medium supplemented with knockout serum replacement (KSR), but not fetal bovine serum (FBS), at a seeding density higher than 5.0 × 104 cells/well. A greater number of gonocyte colonies were observed in culture plates coated with laminin (38.00 ± 6.24/well) and Matrigel (38.67 ± 3.78/well) when compared to plates coated with collagen IV and fibronectin (p < 0.05). In conclusion, bovine neonatal gonocytes were able to be efficiently isolated, enriched and maintained in gonocyte colonies in vitro; the development of this protocol provides vital information for the clinical translation of this technology and the future restoration of human fertility.
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Affiliation(s)
- Shiyan Tang
- Nuffield Department of Women's and Reproductive Health, University of Oxford, Women's Centre, John Radcliffe Hospital, Oxford, United Kingdom
| | - Celine Jones
- Nuffield Department of Women's and Reproductive Health, University of Oxford, Women's Centre, John Radcliffe Hospital, Oxford, United Kingdom
| | - Julian Dye
- Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, United Kingdom
| | - Kevin Coward
- Nuffield Department of Women's and Reproductive Health, University of Oxford, Women's Centre, John Radcliffe Hospital, Oxford, United Kingdom.
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Ibtisham F, Cham TC, Fayaz MA, Honaramooz A. Effects of Growth Factors on In Vitro Culture of Neonatal Piglet Testicular Tissue Fragments. Cells 2023; 12:2234. [PMID: 37759457 PMCID: PMC10526381 DOI: 10.3390/cells12182234] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2023] [Revised: 08/25/2023] [Accepted: 09/05/2023] [Indexed: 09/29/2023] Open
Abstract
In vitro spermatogenesis (IVS) has important applications including fertility preservation of prepubertal cancer patients; however, thus far, IVS has only been achieved using mouse models. To study the effects of growth factors on the maintenance of testicular tissue integrity, germ cell numbers, and potential induction of IVS using a porcine model, we cultured small testicular fragments (~2 mg) from 1-wk-old piglets under six different media conditions (DMEM + 10%KSR alone or supplemented with GDNF, bFGF, SCF, EGF, or a combination of all) for 8 weeks. Overall, tissues supplemented with GDNF and bFGF had the greatest seminiferous tubule integrity and least number of apoptotic cells. GDNF-supplemented tissues had the greatest number of gonocytes per tubule, followed by bFGF-supplemented tissues. There was evidence of gradual Sertoli cell maturation in all groups. Moreover, histological examination and the expression of c-KIT (a marker of differentiating spermatogonia and spermatocytes) and STRA8 (a marker of the pre/meiotic stage germ cells) confirmed the induction of IVS in all groups. However, GDNF- and bFGF-supplemented tissue cultures had greater numbers of seminiferous tubules with spermatocytes compared to other groups. In conclusion, overall, GDNF and bFGF supplementation better maintained the tissue integrity and gonocyte numbers and induced IVS in cultured testicular tissues.
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Affiliation(s)
| | | | | | - Ali Honaramooz
- Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada; (F.I.); (T.-C.C.); (M.A.F.)
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Lee R, Park HJ, Lee WY, Choi Y, Song H. Nanoscale level gelatin-based scaffolds enhance colony formation of porcine testicular germ cells. Theriogenology 2023; 202:125-135. [PMID: 36958136 DOI: 10.1016/j.theriogenology.2023.03.013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2022] [Revised: 02/01/2023] [Accepted: 03/13/2023] [Indexed: 03/16/2023]
Abstract
The extracellular matrix is important in cell growth, proliferation, and differentiation. Gelatin, a support for adhering cells, is used for coating culture plate surfaces of several primary and stem cells. However, gelatin characteristics on culture plates and its cell interactions are not understood. Here, we aimed to identify the effect of gelatin topography on culture plates on the proliferation and colony formation of porcine spermatogonial germ cells (pSGC). To generate different surface topographies, gelatin powder was dissolved in H2O at varying melting temperatures (40, 60, 80, and 120 °C) and coated on the surface of the culture plates. At 40 °C, the pores of the gelatin scaffold were regular ellipses 5-6 μm in diameter and 10-30 nm in thickness. However, at 120 °C, irregular pores 20-30 μm in diameter and 10-20 nm in thickness were obtained. Additionally, the number of attached cells and pSGC colonies were significantly more at 40 °C than at 120 °C after a week of culture. Interestingly, the feeder cells did not settle properly at 120 °C but detached easily from the culture dishes. PSGC colonies were 100 μm in diameter at 40 °C, with small and detached colonies observed at 120 °C. Thus, optimal topography of gelatin was obtained at 40 °C, which was sufficient for the proliferation of feeder cells and the formation of pSGC colonies. Thus, gelatin scaffold conditions at 40 °C and 60 °C were optimal for the derivation and culture of pSGC, and gelatin surface morphology is important for the maintenance of supportive feeder cells for pSGC proliferation and colony formation.
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Affiliation(s)
- Ran Lee
- Department of Stem Cells & Regenerative Technology, Konkuk University, Seoul, 05029, Republic of Korea.
| | - Hyun Jung Park
- Department of Animal Biotechnology, Sangji University, Wonju-si, 26339, Republic of Korea.
| | - Won Young Lee
- Department of Livestock, Korea National University of Agricultures and Fisheries, Jeonju-si, 54874, Republic of Korea.
| | - Youngsok Choi
- Department of Stem Cells & Regenerative Technology, Konkuk University, Seoul, 05029, Republic of Korea.
| | - Hyuk Song
- Department of Stem Cells & Regenerative Technology, Konkuk University, Seoul, 05029, Republic of Korea.
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Yang F, Sun J, Wu X. Primary Cultures of Spermatogonia and Testis Cells. Methods Mol Biol 2023; 2656:127-143. [PMID: 37249869 DOI: 10.1007/978-1-0716-3139-3_7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
Abstract
Spermatogonial stem cells (SSCs) maintain adult spermatogenesis in mammals by undergoing self-renewal and differentiation into spermatozoa. In order to study the biology of SSCs as related to spermatogenesis, an in vitro, long-term expansion system of SSCs constitutes an ideal tool. In this chapter, we describe a robust culture system for mouse and rat SSCs in vitro. In the presence of GDNF, GFRα1, and bFGF, SSCs maintained on STO feeder layers with serum-free medium continuously proliferate for over 6 months. Complete spermatogenesis in infertile recipient mice can be attained following transplantation of the cultured mouse and rat SSCs. Using the in vitro SSC culture systems, elucidation of stem cell biology can be advanced that significantly advances our understanding of spermatogenesis and male fertility.
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Affiliation(s)
- Fan Yang
- State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China
| | - Jiachen Sun
- State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China
| | - Xin Wu
- State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China.
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WARDAK MOHAMMADKAZIM, KULATHUNGA KAUSHALYA, PRIYADARSHANA CHATHURA. Localization and characterization of SSCs from pre-pubertal bovine testes. THE INDIAN JOURNAL OF ANIMAL SCIENCES 2022. [DOI: 10.56093/ijans.v92i10.124617] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Self renewal and proliferation ability of spermatogonial stem cells (SSCs) support spermatogenesis during adult life. Theoretically, these stem cells can be utilized for transmission of genetic information to descendants via testicular transplantation. However, lack of knowledge in methodologies for identification of SSCs limits the application of SSCs transplantation in domestic animals. Accumulated studies have shown that SSCs specific markers (DBA, UCHL1) and stem cell marker (Sox2, Oct4) are useful to screen SSCs that able to be used for transplantation. However, in cattle, less information is available on the expression status of these markers till date. Therefore, a study was carried out in 2019 at Tsukuba University, Japan where testes from 3, 5 and 7 months old calves were utilized to examine testicular localization and in vitro propogation of stem cell markers. SSCs were isolated by enzymatic digestion combined with centrifugal separation on discontinuous Percoll density gradient. Cell propagation and SSCs marker expression were determined at 5, 10 and 15 days post-culture. Immunostaining in conjunction with Western Blot analysis of cultured cells showed that stem cell markers (UCHL1, Oct4 and Sox2) were expressed in SSCs suggesting that differentiation of gonocyte started by 3 months and SSCs differentiation begins after 5 months of age. Taken together, these results demonstrated marker expression and localization of bull SSCs and showed that in vitro culturing of bull SSCs is implementable.
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Voigt AL, Dardari R, Su L, Lara NLM, Sinha S, Jaffer A, Munyoki SK, Alpaugh W, Dufour A, Biernaskie J, Orwig KE, Dobrinski I. Metabolic transitions define spermatogonial stem cell maturation. Hum Reprod 2022; 37:2095-2112. [PMID: 35856882 PMCID: PMC9614685 DOI: 10.1093/humrep/deac157] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2022] [Revised: 06/23/2022] [Indexed: 11/13/2022] Open
Abstract
STUDY QUESTION Do spermatogonia, including spermatogonial stem cells (SSCs), undergo metabolic changes during prepubertal development? SUMMARY ANSWER Here, we show that the metabolic phenotype of prepubertal human spermatogonia is distinct from that of adult spermatogonia and that SSC development is characterized by distinct metabolic transitions from oxidative phosphorylation (OXPHOS) to anaerobic metabolism. WHAT IS KNOWN ALREADY Maintenance of both mouse and human adult SSCs relies on glycolysis, while embryonic SSC precursors, primordial germ cells (PGCs), exhibit an elevated dependence on OXPHOS. Neonatal porcine SSC precursors reportedly initiate a transition to an adult SSC metabolic phenotype at 2 months of development. However, when and if such a metabolic transition occurs in humans is ambiguous. STUDY DESIGN, SIZE, DURATION To address our research questions: (i) we performed a meta-analysis of publicly available and newly generated (current study) single-cell RNA sequencing (scRNA-Seq) datasets in order to establish a roadmap of SSC metabolic development from embryonic stages (embryonic week 6) to adulthood in humans (25 years of age) with a total of ten groups; (ii) in parallel, we analyzed single-cell RNA sequencing datasets of isolated pup (n = 3) and adult (n = 2) murine spermatogonia to determine whether a similar metabolic switch occurs; and (iii) we characterized the mechanisms that regulate these metabolic transitions during SSC maturation by conducting quantitative proteomic analysis using two different ages of prepubertal pig spermatogonia as a model, each with four independently collected cell populations. PARTICIPANTS/MATERIALS, SETTING, METHODS Single testicular cells collected from 1-year, 2-year and 7-year-old human males and sorted spermatogonia isolated from 6- to 8-day (n = 3) and 4-month (n = 2) old mice were subjected to scRNA-Seq. The human sequences were individually processed and then merged with the publicly available datasets for a meta-analysis using Seurat V4 package. We then performed a pairwise differential gene expression analysis between groups of age, followed by pathways enrichment analysis using gene set enrichment analysis (cutoff of false discovery rate < 0.05). The sequences from mice were subjected to a similar workflow as described for humans. Early (1-week-old) and late (8-week-old) prepubertal pig spermatogonia were analyzed to reveal underlying cellular mechanisms of the metabolic shift using immunohistochemistry, western blot, qRT-PCR, quantitative proteomics, and culture experiments. MAIN RESULTS AND THE ROLE OF CHANCE Human PGCs and prepubertal human spermatogonia show an enrichment of OXPHOS-associated genes, which is downregulated at the onset of puberty (P < 0.0001). Furthermore, we demonstrate that similar metabolic changes between pup and adult spermatogonia are detectable in the mouse (P < 0.0001). In humans, the metabolic transition at puberty is also preceded by a drastic change in SSC shape at 11 years of age (P < 0.0001). Using a pig model, we reveal that this metabolic shift could be regulated by an insulin growth factor-1 dependent signaling pathway via mammalian target of rapamycin and proteasome inhibition. LARGE SCALE DATA New single-cell RNA sequencing datasets obtained from this study are freely available through NCBI GEO with accession number GSE196819. LIMITATIONS, REASONS FOR CAUTION Human prepubertal tissue samples are scarce, which led to the investigation of a low number of samples per age. Gene enrichment analysis gives only an indication about the functional state of the cells. Due to limited numbers of prepubertal human spermatogonia, porcine spermatogonia were used for further proteomic and in vitro analyses. WIDER IMPLICATIONS OF THE FINDINGS We show that prepubertal human spermatogonia exhibit high OXHPOS and switch to an adult-like metabolism only after 11 years of age. Prepubescent cancer survivors often suffer from infertility in adulthood. SSC transplantation could provide a powerful tool for the treatment of infertility; however, it requires high cell numbers. This work provides key insight into the dynamic metabolic requirements of human SSCs across development that would be critical in establishing ex vivo systems to support expansion and sustained function of SSCs toward clinical use. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by the NIH/NICHD R01 HD091068 and NIH/ORIP R01 OD016575 to I.D. K.E.O. was supported by R01 HD100197. S.K.M. was supported by T32 HD087194 and F31 HD101323. The authors declare no conflict of interest.
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Affiliation(s)
- A L Voigt
- Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada
| | - R Dardari
- Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada
| | - L Su
- Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada
| | - N L M Lara
- Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada
| | - S Sinha
- Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada
| | - A Jaffer
- Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada
| | - S K Munyoki
- Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - W Alpaugh
- Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada
| | - A Dufour
- Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada
| | - J Biernaskie
- Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada
| | - K E Orwig
- Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - I Dobrinski
- Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada
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11
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Awang-Junaidi AH, Fayaz MA, Goldstein S, Honaramooz A. Using a testis regeneration model, FGF9, LIF, and SCF improve testis cord formation while RA enhances gonocyte survival. Cell Tissue Res 2022; 389:351-370. [PMID: 35596812 DOI: 10.1007/s00441-022-03641-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2021] [Accepted: 05/09/2022] [Indexed: 11/30/2022]
Abstract
Implantation of testis cell aggregates from various donors under the back skin of recipient mice results in de novo formation of testis tissue. We used this implantation model to study the putative in vivo effects of six different growth factors on testis cord development. Recipient mice (n = 7/group) were implanted with eight neonatal porcine testis cell aggregates that were first exposed to a designated growth factor: FGF2 at 1 µg/mL, FGF9 at 5 µg/mL, VEGF at 3.5 µg/mL, LIF at 5 µg/mL, SCF at 3.5 µg/mL, retinoic acid (RA) at 3.5 × 10-5 M, or no growth factors (control). The newly developed seminiferous cords (SC) were classified based on their morphology into regular, irregular, enlarged, or aberrant. Certain treatments enhanced implant weight (LIF), implant cross-sectional area (SCF) or the relative cross-sectional area covered by SC within implants (FGF2). RA promoted the formation of enlarged SC and FGF2 led to the highest ratio of regular SC and the lowest ratio of aberrant SC. Rete testis-like structures appeared earlier in implants treated with FGF2, FGF9, or LIF. These results show that even brief pre-implantation exposure of testis cells to these growth factors can have profound effects on morphogenesis of testis cords using this implantation model.
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Affiliation(s)
- Awang Hazmi Awang-Junaidi
- Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada.,Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
| | - Mohammad Amin Fayaz
- Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada
| | - Savannah Goldstein
- Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada
| | - Ali Honaramooz
- Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada.
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12
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Thiageswaran S, Steele H, Voigt AL, Dobrinski I. A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture. Int J Mol Sci 2022; 23:ijms23094535. [PMID: 35562927 PMCID: PMC9103065 DOI: 10.3390/ijms23094535] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Revised: 04/14/2022] [Accepted: 04/16/2022] [Indexed: 12/22/2022] Open
Abstract
Spermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Alternatively, testicular biopsies taken prior to treatment may be used to restore fertility in adulthood. Testicular SSC populations are limited, and in vitro culture systems are required to increase numbers of SSCs for treatment, demanding culture systems for SSC propagation. Using the pig as a non-rodent model, we developed culture systems to expand spermatogonia from immature testis tissue, comparing different feeders (Sertoli cells, peritubular myoid cells (PMCs) and pig fetal fibroblasts (PFFs)). Spermatogonia co-cultured with Sertoli cells, PMCs and PFFs had comparable rates of proliferation and apoptosis. To elucidate the mechanism behind the beneficial nature of feeder layers, we investigated the role of extracellular vesicles in crosstalk between spermatogonia and feeder cells. Sertoli cell-released exosomes are incorporated by spermatogonia, and inhibition of exosomal release reduces spermatogonial proliferation. Together, these results show that PMCs, PFFs and Sertoli cells promote spermatogonial proliferation in co-culture, with exosomal exchange representing one possible mechanism. Further characterization of exosomal cargo may ultimately allow the development of feeder-free culture systems for clinical use.
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Affiliation(s)
- Shiama Thiageswaran
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada;
| | - Heather Steele
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; (H.S.); (A.L.V.)
| | - Anna Laura Voigt
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; (H.S.); (A.L.V.)
| | - Ina Dobrinski
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada;
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; (H.S.); (A.L.V.)
- Correspondence: ; Tel.: +1-403-210-6532
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13
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Eugeni E, Arato I, Del Sordo R, Sidoni A, Garolla A, Ferlin A, Calafiore R, Brancorsini S, Mancuso F, Luca G. Fertility Preservation and Restoration Options for Pre-Pubertal Male Cancer Patients: Current Approaches. Front Endocrinol (Lausanne) 2022; 13:877537. [PMID: 35784573 PMCID: PMC9244702 DOI: 10.3389/fendo.2022.877537] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/16/2022] [Accepted: 04/25/2022] [Indexed: 12/12/2022] Open
Abstract
Fertility preservation for prepubertal male patients undergoing gonadotoxic therapies, potentially depleting spermatogonial cells, is an expanding necessity, yet most of the feasible options are still in the experimental phase. We present our experience and a summary of current and novel possibilities regarding the different strategies to protect or restore fertility in young male patients, before proceeding with chemotherapy or radiotherapy for malignances or other diseases. Adult oncological patients should always be counselled to cryopreserve the semen before starting treatment, however this approach is not suitable for prepubertal boys, who aren't capable to produce sperm yet. Fortunately, since the survival rate of pediatric cancer patients has skyrocketed in the last decade and it's over 84%, safeguarding their future fertility is becoming a major concern for reproductive medicine. Surgical and medical approaches to personalize treatment or protect the gonads could be a valid first step to take. Testicular tissue autologous grafting or xenografting, and spermatogonial stem cells (SSCs) transplantation, are the main experimental options available, but spermatogenesis in vitro is becoming an intriguing alternative. All of these methods feature both strong and weak prospects. There is also relevant controversy regarding the type of testicular material to preserve and the cryopreservation methods. Since transplanted cells are bound to survive based on SSCs number, many ways to enrich their population in cultures have been proposed, as well as different sites of injection inside the testis. Testicular tissue graft has been experimented on mice, rabbits, rhesus macaques and porcine, allowing the birth of live offspring after performing intracytoplasmic sperm injection (ICSI), however it has never been performed on human males yet. In vitro spermatogenesis remains a mirage, although many steps in the right direction have been performed. The manufacturing of 3D scaffolds and artificial spermatogenetic niche, providing support to stem cells in cultures, seems like the best way to further advance in this field.
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Affiliation(s)
- Elena Eugeni
- Department of Medicine and Surgery, University of Perugia, Perugia, Italy
- Department of Medicine and Medical Specialties, Division of Medical Andrology and Endocrinology of Reproduction, University of Terni, Terni, Italy
- *Correspondence: Elena Eugeni,
| | - Iva Arato
- Department of Medicine and Surgery, University of Perugia, Perugia, Italy
| | - Rachele Del Sordo
- Division of Anatomic Pathology and Histology, Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Angelo Sidoni
- Division of Anatomic Pathology and Histology, Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Andrea Garolla
- Unit of Andrology and Reproductive Medicine, Department of Medicine, School of Medicine and Surgery, University of Padua, Padua, Italy
| | - Alberto Ferlin
- Unit of Andrology and Reproductive Medicine, Department of Medicine, School of Medicine and Surgery, University of Padua, Padua, Italy
| | - Riccardo Calafiore
- Department of Medicine and Surgery, University of Perugia, Perugia, Italy
| | - Stefano Brancorsini
- Section of Pathology (Terni), Department of Medicine and Surgery, University of Perugia, Perugia, Italy
| | - Francesca Mancuso
- Department of Medicine and Surgery, University of Perugia, Perugia, Italy
| | - Giovanni Luca
- Department of Medicine and Surgery, University of Perugia, Perugia, Italy
- Department of Medicine and Medical Specialties, Division of Medical Andrology and Endocrinology of Reproduction, University of Terni, Terni, Italy
- International Biotechnological Center for Endocrine, Metabolic and Embryo-Reproductive Translational Research (CIRTEMER), Department of Medicine and Surgery, University of Perugia, Perugia, Italy
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14
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Diaz EA, Donoso G, Saenz C, Aponte PM. Spermatogenesis in a vulnerable South American cervid, dwarf red brocket (Mazama rufina). Anat Histol Embryol 2021; 51:91-102. [PMID: 34820886 DOI: 10.1111/ahe.12766] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2021] [Revised: 10/12/2021] [Accepted: 11/15/2021] [Indexed: 11/29/2022]
Abstract
The brocket deer (Genus Mazama) is a highly diverse cervid group distributed from Mexico to Argentina, with a downward population trend. However, literature on the basic reproductive biology of the genus is scarce. This work aimed to study biometric, histological and stereological aspects of the testes of Dwarf Red Brocket (Mazama rufina). Testes from free-ranging adult brockets (n = 3) were retrieved from necropsies. Testes were histologically processed. From histological images, several stereological parameters were estimated, and seminiferous epithelium cycle morphology was described. Testes volumes were between 8.2 and 18.4 ml and weights from 8.3 to 19.4 g. Gonadosomatic index (% paired-testes weight to body weight) went from 0.17 to 0.64. The tubular cross-sectional diameter was 179.8 ± 2.8 µm. Estimated volume densities for parenchyma and interstitium were 78.8% and 21.2% respectively. There were (in millions/ml) 96.0 ± 13.1 germ cells and 37.7 ± 6.0 somatic cells. Specific cell densities were (all expressed in millions/ml) as follows: spermatogonia 13.1 ± 4.2; primary spermatocytes 43.1 ± 5.0; round spermatids 36.8 ± 8.0 (lower density near the caudal pole, p < 0.01); sustentacular (Sertoli) cells 16.8 ± 4.1 and interstitial endocrine (Leydig) cells 17.4 ± 3.4. Sertoli cell index (germ cells per Sertoli cell) was 6.72. Eight stages of the cycle were described, and frequencies estimated, resembling those of goats. M. rufina adult testis anatomy is similar to that of other cervids and domestic ruminants, with an apparently lower spermatogenic efficiency. This work is a first approximation to the physiology of the testis of M. rufina. Basic knowledge of the reproductive physiology of vulnerable species may allow biotechnological approaches for the restitution of animal populations.
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Affiliation(s)
- Eduardo A Diaz
- Escuela de Medicina Veterinaria, Colegio de Ciencias de la Salud, Universidad San Francisco de Quito (USFQ), Quito, Ecuador.,Hospital de Fauna Silvestre Tueri, Instituto iBIOTROP, Universidad San Francisco de Quito (USFQ), Quito, Ecuador
| | - Gustavo Donoso
- Hospital de Fauna Silvestre Tueri, Instituto iBIOTROP, Universidad San Francisco de Quito (USFQ), Quito, Ecuador
| | - Carolina Saenz
- Hospital de Fauna Silvestre Tueri, Instituto iBIOTROP, Universidad San Francisco de Quito (USFQ), Quito, Ecuador
| | - Pedro M Aponte
- Escuela de Medicina Veterinaria, Colegio de Ciencias de la Salud, Universidad San Francisco de Quito (USFQ), Quito, Ecuador.,Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de Quito (USFQ), Quito, Ecuador.,Instituto de Investigaciones en Biomedicina iBIOMED, Universidad San Francisco de Quito (USFQ), Quito, Ecuador
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15
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Binsila B, Selvaraju S, Ranjithkumaran R, Archana SS, Krishnappa B, Ghosh SK, Kumar H, Subbarao RB, Arangasamy A, Bhatta R. Current scenario and challenges ahead in application of spermatogonial stem cell technology in livestock. J Assist Reprod Genet 2021; 38:3155-3173. [PMID: 34661801 DOI: 10.1007/s10815-021-02334-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2021] [Accepted: 09/27/2021] [Indexed: 11/28/2022] Open
Abstract
PURPOSE Spermatogonial stem cells (SSCs) are the source for the mature male gamete. SSC technology in humans is mainly focusing on preserving fertility in cancer patients. Whereas in livestock, it is used for mining the factors associated with male fertility. The review discusses the present status of SSC biology, methodologies developed for in vitro culture, and challenges ahead in establishing SSC technology for the propagation of superior germplasm with special reference to livestock. METHOD Published literatures from PubMed and Google Scholar on topics of SSCs isolation, purification, characterization, short and long-term culture of SSCs, stemness maintenance, epigenetic modifications of SSCs, growth factors, and SSC cryopreservation and transplantation were used for the study. RESULT The fine-tuning of SSC isolation and culture conditions with special reference to feeder cells, growth factors, and additives need to be refined for livestock. An insight into the molecular mechanisms involved in maintaining stemness and proliferation of SSCs could facilitate the dissemination of superior germplasm through transplantation and transgenesis. The epigenetic influence on the composition and expression of the biomolecules during in vitro differentiation of cultured cells is essential for sustaining fertility. The development of surrogate males through gene-editing will be historic achievement for the foothold of the SSCs technology. CONCLUSION Detailed studies on the species-specific factors regulating the stemness and differentiation of the SSCs are required for the development of a long-term culture system and in vitro spermatogenesis in livestock. Epigenetic changes in the SSCs during in vitro culture have to be elucidated for the successful application of SSCs for improving the productivity of the animals.
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Affiliation(s)
- Balakrishnan Binsila
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India.
| | - Sellappan Selvaraju
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Rajan Ranjithkumaran
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Santhanahalli Siddalingappa Archana
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Balaganur Krishnappa
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Subrata Kumar Ghosh
- Animal Reproduction Division, Indian Council of Agricultural Research-Indian Veterinary Research Institute, Izatnagar, 243 122, India
| | - Harendra Kumar
- Animal Reproduction Division, Indian Council of Agricultural Research-Indian Veterinary Research Institute, Izatnagar, 243 122, India
| | - Raghavendra B Subbarao
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Arunachalam Arangasamy
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Raghavendra Bhatta
- Indian council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
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16
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Martin-Inaraja M, Ferreira M, Taelman J, Eguizabal C, Chuva De Sousa Lopes SM. Improving In Vitro Culture of Human Male Fetal Germ Cells. Cells 2021; 10:cells10082033. [PMID: 34440801 PMCID: PMC8393746 DOI: 10.3390/cells10082033] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Revised: 07/29/2021] [Accepted: 08/05/2021] [Indexed: 12/14/2022] Open
Abstract
Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising (autologous) source of cells for male fertility preservation; however, in contrast to mouse SSCs, we are still unable to culture them in the long term. Here, we investigated the effect of two different culture media and four substrates (laminin, gelatin, vitronectin and matrigel) in the culture of dissociated second trimester testes, enriched for hFGCs. After 6 days in culture, we quantified the presence of POU5F1 and DDX4 expressing hFGCs. We observed a pronounced difference in hFGC number in different substrates. The combination of gelatin-coated substrate and medium containing GDNF, LIF, FGF2 and EGF resulted in the highest percentage of hFGCs (10% of the total gonadal cells) after 6 days of culture. However, the vitronectin-coated substrate resulted in a comparable percentage of hFGCs regardless of the media used (3.3% of total cells in Zhou-medium and 4.8% of total cells in Shinohara-medium). We provide evidence that not only the choices of culture medium but also choices of the adequate substrate are crucial for optimizing culture protocols for male hFGCs. Optimizing culture conditions in order to improve the expansion of hFGCs will benefit the development of gametogenesis assays in vitro.
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Affiliation(s)
- Myriam Martin-Inaraja
- Cell Therapy, Stem Cells and Tissues Group, Basque Centre for Blood Transfusion and Human Tissues, 48960 Galdakao, Spain; (M.M.-I.); (C.E.)
- Biocruces Bizkaia Health Research Institute, Cell Therapy, Stem Cells and Tissues Group, 48903 Barakaldo, Spain
| | - Monica Ferreira
- Department of Anatomy and Embryology, Leiden University Medical Centre, Einthovenweg 20, 2333 ZC Leiden, The Netherlands; (M.F.); (J.T.)
| | - Jasin Taelman
- Department of Anatomy and Embryology, Leiden University Medical Centre, Einthovenweg 20, 2333 ZC Leiden, The Netherlands; (M.F.); (J.T.)
| | - Cristina Eguizabal
- Cell Therapy, Stem Cells and Tissues Group, Basque Centre for Blood Transfusion and Human Tissues, 48960 Galdakao, Spain; (M.M.-I.); (C.E.)
- Biocruces Bizkaia Health Research Institute, Cell Therapy, Stem Cells and Tissues Group, 48903 Barakaldo, Spain
| | - Susana M. Chuva De Sousa Lopes
- Department of Anatomy and Embryology, Leiden University Medical Centre, Einthovenweg 20, 2333 ZC Leiden, The Netherlands; (M.F.); (J.T.)
- Ghent-Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium
- Correspondence: ; Tel.: +31-71-526-9350
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17
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Effect of Epidermal Growth Factor on the Colony-formation Ability of Porcine Spermatogonial Germ Cells. BIOTECHNOL BIOPROC E 2021. [DOI: 10.1007/s12257-020-0372-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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18
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Survivable potential of germ cells after trehalose cryopreservation of bovine testicular tissues. Cryobiology 2021; 101:105-114. [PMID: 33989617 DOI: 10.1016/j.cryobiol.2021.05.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2021] [Revised: 03/23/2021] [Accepted: 05/01/2021] [Indexed: 12/26/2022]
Abstract
Germplasm preservation of livestock or endangered animals and expansion of germline stem cells are important. The purpose of this study is to investigate whether supplementation of trehalose to the freezing medium (FM) reduces tissular damage and improves the quality of testicular cells in the cryopreserved bovine testicular tissues. We herein established an optimized protocol for the cryopreservation of bovine testicular tissues, and the isolation as well as culture of bovine germ cells containing spermatogonial stem cells (SSCs) from these tissues. The results showed that FM containing 10% dimethyl sulfoxide (Me2SO/DMSO), 10% knockout serum replacement (KSR) and 20% trehalose (FM5) combined with the uncontrolled slow freezing (USF) procedures has the optimized cryoprotective effect on bovine testicular tissues. The FM5 + USF protocol reduced the cell apoptosis, maintained high cell viability, supported the structural integrity and seminiferous epithelial cohesion similar to that in the fresh tissues. Viable germ cells containing SSCs were effectively isolated from these tissues and they maintained germline marker expressions in the co-testicular cells and co-mouse embryonic fibroblasts (MEF) feeder culture systems respectively, during the short-term culture. Additionally, upregulated transcriptions of spermatogenic differentiation marker C-KIT and meiotic marker SYCP3 were detected in these cells after retinoic acid-induced differentiation. Together, FM5 + USF is suitable for the cryopreservation of bovine testicular tissues, with benefits of reducing the apoptosis, maintaining the cell viability, supporting the testicular structure integrity, and sustaining the survival and differentiation potential of bovine germ cells containing SSCs.
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19
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Cai H, Jiang Y, Zhang S, Cai NN, Zhu WQ, Yang R, Tang B, Li ZY, Zhang XM. Culture bovine prospermatogonia with 2i medium. Andrologia 2021; 53:e14056. [PMID: 33763906 DOI: 10.1111/and.14056] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2020] [Revised: 01/28/2021] [Accepted: 03/09/2021] [Indexed: 12/31/2022] Open
Abstract
Germplasm cryopreservation and expansion of gonocytes/prospermatogonia or spermatogonial stem cells (SSCs) are important; however, it's difficult in cattle. Since inhibitors of Mek1/2 and Gsk3β (2i) can enhance pluripotency maintenance, effects of 2i-based medium on the cultivation of bovine prospermatogonia from the cryopreserved tissues were examined. The testicular tissues of newborn bulls were well cryopreserved. High mRNA levels of prospermatogonium/SSC markers (PLZF, GFRα-1) and pluripotency markers (Oct4/Pouf5, Sox2, Nanog) were detected and the PLZF+ /GFRα-1+ prospermatogonia were consistently identified immunohistochemically in the seminiferous cords. Using differential plating and Percoll-based centrifugation, 41.59% prospermatogonia were enriched and they proliferated robustly in 2i medium. The 2i medium boosted mRNA abundances of Pouf5, Sox2, Nanog, GFRα-1, PLZF, anti-apoptosis gene Bcl2, LIF receptor gene LIFR and enhanced PLZF protein expression, but suppressed mRNA expressions of spermatogonial differentiation marker c-kit and pro-apoptotic gene Bax, in the cultured prospermatogonia. It also alleviated H2 O2 -induced apoptosis of the enriched cells and decreased histone H3 lysine (K9) trimethylation (H3K9me3) and its methylase Suv39h1/2 mRNA level in the cultured seminiferous cords. Overall, 2i medium improves the cultivation of bovine prospermatogonia isolated from the cryopreserved testes, by inhibiting Suv39h1/2-mediated H3K9me3 through Mek1/2 and Gsk3β signalling, evidencing successful cryopreservation and expansion of bovine germplasm.
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Affiliation(s)
- Huan Cai
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Yu Jiang
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Sheng Zhang
- First Bethune Hospital, Jilin University, Changchun, China
| | - Ning-Ning Cai
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Wen-Qian Zhu
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Rui Yang
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Bo Tang
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Zi-Yi Li
- First Bethune Hospital, Jilin University, Changchun, China
| | - Xue-Ming Zhang
- College of Veterinary Medicine, Jilin University, Changchun, China
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20
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Application of platelet-rich plasma (PRP) improves self-renewal of human spermatogonial stem cells in two-dimensional and three-dimensional culture systems. Acta Histochem 2020; 122:151627. [PMID: 33002788 DOI: 10.1016/j.acthis.2020.151627] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2020] [Revised: 08/17/2020] [Accepted: 09/14/2020] [Indexed: 12/20/2022]
Abstract
Spermatogonial stem cells (SSCs) are very sensitive to chemotherapy and radiotherapy, so male infertility is a great challenge for prepubertal cancer survivors. Cryoconservation of testicular cells before cancer treatment can preserve SSCs from treatment side effects. Different two-dimensional (2D) and three-dimensional (3D) culture systems of SSCs have been used in many species as a useful technique to in vitro spermatogenesis. We evaluated the proliferation of SSCs in 2D and 3D culture systems of platelet-rich plasma (PRP). testicular cells of four brain-dead patients cultivated in 2D pre-culture system, characterization of SSCs performed by RT-PCR, flow cytometry, immunocytochemistry and their functionality assessed by xenotransplantation to azoospermia mice. PRP prepared and dosimetry carried out to determine the optimized dose of PRP. After preparation of PRP scaffold, cytotoxic and histological evaluation performed and SSCs cultivated into three groups: control, 2D culture by optimized dose of PRP and PRP scaffold. The diameter and number of colonies measured and relative expression of GFRa1 and c-KIT evaluated by real-time PCR. Results indicated the expression of PLZF, VASA, OCT4, GFRa1 and vimentin in colonies after 2D pre-culture, xenotransplantation demonstrated proliferated SSCs have proper functionality to homing in mouse testes. The relative expression of c-KIT showed a significant increase as compared to the control group (*: p < 0.05) in PRP- 2D group, expression of GFRa1 and c-KIT in PRP scaffold group revealed a significant increase as compared to other groups (***: p < 0.001). The number and diameter of colonies in the PRP-2D group showed a considerable increase (p < 0.01) as compared to the control group. In PRP- scaffold group, a significant increase (p < 0.01) was seen only in the number of colonies related to the control group. Our results suggested that PRP scaffold can reconstruct a suitable structure to the in vitro proliferation of SSCs.
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Deebel NA, Galdon G, Zarandi NP, Stogner-Underwood K, Howards S, Lovato J, Kogan S, Atala A, Lue Y, Sadri-Ardekani H. Age-related presence of spermatogonia in patients with Klinefelter syndrome: a systematic review and meta-analysis. Hum Reprod Update 2020; 26:58-72. [PMID: 31822886 DOI: 10.1093/humupd/dmz038] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2019] [Revised: 09/15/2019] [Accepted: 09/30/2019] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND Klinefelter syndrome (KS) has been defined by sex chromosome aneuploidies (classically 47, XXY) in the male patient. The peripubertal timeframe in KS patients has been associated with the initiation of progressive testicular fibrosis, loss of spermatogonial stem cells (SSC), hypogonadism and impaired fertility. Less than half of KS patients are positive for spermatozoa in the ejaculate or testis via semen analysis or testicular sperm extraction, respectively. However, the chance of finding spermatogonia including a sub-population of SSCs in KS testes has not been well defined. Given the recent demonstration of successful cell culture for mouse and human SSCs, it could be feasible to isolate and propagate SSCs and transplant the cells back to the patient or to differentiate them in vitro to haploid cells. OBJECTIVE AND RATIONALE The main objective of this study was to meta-analyse the currently available data from KS patients to identify the prevalence of KS patients with spermatogonia on testicular biopsy across four age groups (year): fetal/infantile (age ≤ 1), prepubertal (age 1 ≤ x ≤ 10), peripubertal/adolescent (age 10 < x < 18) and adult (age ≥ 18) ages. Additionally, the association of endocrine parameters with presence or absence of spermatogonia was tested to obtain a more powered analysis of whether FSH, LH, testosterone and inhibin B can serve as predictive markers for successful spermatogonia retrieval. SEARCH METHODS A thorough Medline/PubMed search was conducted using the following search terms: 'Klinefelter, germ cells, spermatogenesis and spermatogonia', yielding results from 1 October 1965 to 3 February 2019. Relevant articles were added from the bibliographies of selected articles. Exclusion criteria included non-English language, abstracts only, non-human data and review papers. OUTCOMES A total of 751 papers were identified with independent review returning 36 papers with relevant information for meta-analysis on 386 patients. For the most part, articles were case reports, case-controlled series and cohort studies (level IV-VI evidence). Spermatogonial cells were present in all of the fetal/infantile and 83% of the prepubertal patients' testes, and in 42.7% and 48.5% of the peripubertal and adult groups, respectively were positive for spermatogonia. Additionally, 26 of the 56 (46.4%) peripubertal/adolescent and 37 of the 152 (24.3%) adult patients negative for spermatozoa were positive for spermatogonia (P < 0.05). In peripubertal/adolescent patients, the mean ± SEM level for FSH was 12.88 ± 3.13 IU/L for spermatogonia positive patients and 30.42 ± 4.05 IU/L for spermatogonia negative patients (P = 0.001); the mean ± SEM level LH levels were 4.36 ± 1.31 and 11.43 ± 1.68 IU/L for spermatogonia positive and negative, respectively (P < 0.01); the mean ± SEM level for testosterone levels were 5.04 ± 1.37 and 9.05 ± 0.94 nmol/L (equal to 145 ± 40 and 261 ± 27 and ng/dl) for the spermatogonia positive and negative groups, respectively (P < 0.05), while the difference in means for inhibin B was not statistically significant (P > 0.05). A similar analysis in the adult group showed the FSH levels in spermatogonia positive and negative patients to be 25.77 ± 2.78 and 36.12 ± 2.90 IU/L, respectively (mean ± SEM level, P < 0.05). All other hormone measurements were not statistically significantly different between groups. WIDER IMPLICATIONS While azoospermia is a common finding in the KS patient population, many patients are positive for spermatogonia. Recent advances in SSC in vitro propagation, transplantation and differentiation open new avenues for these patients for fertility preservation. This would offer a new subset of KS patients a chance of biological paternity. Data surrounding the hormonal profiles of KS patients and their relation to fertility should be interpreted with caution as a paucity of adequately powered data exists. Future work is needed to clarify the utility of FSH, LH, testosterone and inhibin B as biomarkers for successful retrieval of spermatogonia.
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Affiliation(s)
- Nicholas A Deebel
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA.,Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - Guillermo Galdon
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - Nima Pourhabibi Zarandi
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | | | - Stuart Howards
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - James Lovato
- Department of Biostatistics and Data Science, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - Stanley Kogan
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA.,Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - Anthony Atala
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA.,Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - Yanhe Lue
- Division of Endocrinology, Department of Medicine, Los Angeles Biomedical Research Institute and Harbor-UCLA Medical Center, Torrance, CA, USA
| | - Hooman Sadri-Ardekani
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA.,Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
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22
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Binsila BK, Selvaraju S, Ghosh SK, Ramya L, Arangasamy A, Ranjithkumaran R, Bhatta R. EGF, GDNF, and IGF-1 influence the proliferation and stemness of ovine spermatogonial stem cells in vitro. J Assist Reprod Genet 2020; 37:2615-2630. [PMID: 32821972 DOI: 10.1007/s10815-020-01912-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Accepted: 08/03/2020] [Indexed: 11/25/2022] Open
Abstract
PURPOSE The objective of the present study was to purify sheep spermatogonial stem cells (SSCs) from testicular isolate using combined enrichment methods and to study the effect of growth factors on SSC stemness during culture. METHODS The testicular cells from prepubertal male sheep were isolated, and SSCs were purified using Ficoll gradients (10 and 12%) followed by differential plating (laminin with BSA). SSCs were cultured with StemPro®-34 SFM, additives, and FBS for 7 days. The various doses (ng/ml) of growth factors, EGF at 10, 15, and 20, GDNF at 40, 70, and 100 and IGF-1 at 50, 100, and 150 were tested for the proliferation and stemness of SSCs in vitro. The stemness in cultured cells was assessed using SSC markers PLZF, ITGA6, and GFRα1. RESULTS Ficoll density gradient separation significantly (p < 0.05) increased the percentage of SSCs in 12% fraction (35.1 ± 3.8 vs 11.2 ± 3.7). Subsequently, purification using laminin with BSA plating further enriched SSCs to 61.7 ± 4.7%. GDNF at 40 ng/ml, EGF at 15 and 20 ng/ml and IGF1 at 100 and 150 ng/ml significantly (p < 0.05) improved proliferation and stemness of SSCs up to 7 days in culture. GDNF at 40 ng/ml outperformed other growth factors tested and could maintain the ovine SSCs proliferation and stemness for 36 days. CONCLUSIONS The combined enrichment method employing density gradient centrifugation and laminin with BSA plating improves the purification efficiency of ovine SSCs. GDNF at 40 ng/ml is essential for optimal proliferation and sustenance of stemness of ovine SSCs in vitro.
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Affiliation(s)
- B K Binsila
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India.
| | - S Selvaraju
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - S K Ghosh
- Animal Reproduction Division, Indian Council of Agricultural Research-Indian Veterinary Research Institute, Izatnagar, 243 122, India
| | - L Ramya
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - A Arangasamy
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - R Ranjithkumaran
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - R Bhatta
- Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
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23
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Goossens E, Jahnukainen K, Mitchell RT, van Pelt A, Pennings G, Rives N, Poels J, Wyns C, Lane S, Rodriguez-Wallberg KA, Rives A, Valli-Pulaski H, Steimer S, Kliesch S, Braye A, Andres MM, Medrano J, Ramos L, Kristensen SG, Andersen CY, Bjarnason R, Orwig KE, Neuhaus N, Stukenborg JB. Fertility preservation in boys: recent developments and new insights †. Hum Reprod Open 2020; 2020:hoaa016. [PMID: 32529047 PMCID: PMC7275639 DOI: 10.1093/hropen/hoaa016] [Citation(s) in RCA: 108] [Impact Index Per Article: 21.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Revised: 01/22/2020] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS The work was funded by ESHRE. None of the authors has a conflict of interest.
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Affiliation(s)
- E Goossens
- Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), 1090 Brussels, Belgium
| | - K Jahnukainen
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet and Karolinska University Hospital, Solna, Sweden.,Division of Haematology-Oncology and Stem Cell Transplantation, New Children's Hospital, Pediatric Research Center, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
| | - R T Mitchell
- MRC Centre for Reproductive Health, The Queen's Medical Research Institute, The University of Edinburgh; and the Edinburgh Royal Hospital for Sick Children, Edinburgh, UK
| | - Amm van Pelt
- Center for Reproductive Medicine, Amsterdam UMC, Amsterdam Reproduction and Development Research Institute, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
| | - G Pennings
- Bioethics Institute Ghent, Ghent University, 9000 Ghent, Belgium
| | - N Rives
- Normandie Univ, UNIROUEN, EA 4308 "Gametogenesis and Gamete Quality", Rouen University Hospital, Biology of Reproduction-CECOS Laboratory, F 76000, Rouen, France
| | - J Poels
- Department of Gynecology and Andrology, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium
| | - C Wyns
- Department of Gynecology and Andrology, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium
| | - S Lane
- Department of Paediatric Oncology and Haematology, Children's Hospital Oxford, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
| | - K A Rodriguez-Wallberg
- Department of Oncology Pathology, Karolinska Institutet, Solna, Sweden.,Section of Reproductive Medicine, Division of Gynecology and Reproduction, Karolinska University Hospital, Stockholm, Sweden
| | - A Rives
- Normandie Univ, UNIROUEN, EA 4308 "Gametogenesis and Gamete Quality", Rouen University Hospital, Biology of Reproduction-CECOS Laboratory, F 76000, Rouen, France
| | - H Valli-Pulaski
- Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - S Steimer
- Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - S Kliesch
- Centre of Reproductive Medicine and Andrology, Institute of Reproductive and Regenerative Biology, University of Münster, Münster, Germany
| | - A Braye
- Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), 1090 Brussels, Belgium
| | - M M Andres
- Reproductive Medicine Unit, Hospital Universitario y Politécnico La Fe, Valencia, Spain
| | - J Medrano
- Reproductive Medicine Unit, Hospital Universitario y Politécnico La Fe, Valencia, Spain
| | - L Ramos
- Departement of Obstetrics and Gynacology, Division Reproductive Medicine, Radboud University Medical Center, Nijmegen, The Netherlands
| | - S G Kristensen
- Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, Denmark
| | - C Y Andersen
- Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, Denmark
| | - R Bjarnason
- Children's Medical Center, Landspítali University Hospital, Reykjavik, Iceland and Faculty of Medicine, University of Iceland, Reykjavik, Iceland
| | - K E Orwig
- Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - N Neuhaus
- Centre of Reproductive Medicine and Andrology, Institute of Reproductive and Regenerative Biology, University of Münster, Münster, Germany
| | - J B Stukenborg
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet and Karolinska University Hospital, Solna, Sweden
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Lara NDLEM, Costa GMJ, Figueiredo AFA, de França LR. The Sertoli cell: what can we learn from different vertebrate models? Anim Reprod 2020; 16:81-92. [PMID: 33299481 PMCID: PMC7720927 DOI: 10.21451/1984-3143-ar2018-125] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Besides having medical applications, comparative studies on reproductive biology are very useful, providing, for instance, essential knowledge for basic, conservation and biotechnological research. In order to maintain the reproductive potential and the survival of all vertebrate species, both sperm and steroid production need to occur inside the testis. From the approximately fifty thousand vertebrate species still alive, very few species are already investigated; however, our knowledge regarding Sertoli cell biology is quite good. In this regard, it is already known that since testis differentiation the Sertoli cells are the somatic cells in charge of supporting and orchestrating germ cells during development and full spermatogenesis in adult animals. In the present review, we highlight key aspects related to Sertoli cell biology in vertebrates and show that this key testis somatic cell presents huge and intrinsic plasticity, particularly when cystic (fish and amphibians) and non-cystic (reptiles, birds and mammals) spermatogenesis is compared. In particular, we briefly discuss the main aspects related to Sertoli cells functions, interactions with germ cells, Sertoli cells proliferation and efficiency, as well as those regarding spermatogonial stem cell niche regulation, which are crucial aspects responsible for the magnitude of sperm production. Most importantly, we show that we could greatly benefit from investigations using different vertebrate experimental models, mainly now that there is a big concern regarding the decline in human sperm counts caused by a multitude of factors.
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Affiliation(s)
| | - Guilherme Mattos Jardim Costa
- Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
| | | | - Luiz Renato de França
- Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
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25
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Sharma A, Shah SM, Tiwari M, Roshan M, Singh MK, Singla SK, Palta P, Manik RS, Chauhan MS. Propagation of goat putative spermatogonial stem cells under growth factors defined serum-free culture conditions. Cytotechnology 2020; 72:489-497. [PMID: 32124159 DOI: 10.1007/s10616-020-00386-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2019] [Accepted: 02/24/2020] [Indexed: 12/22/2022] Open
Abstract
In the present study, we used a serum-free culture media to propagate goat putative spermatogonial stem cells (SSCs) and evaluated the effect of crucial growth factors on relative expression of some SSC markers and self-renewal related genes. The enriched SSCs were cultured on a homologous Sertoli cell feeder layer in KO-DMEM supplemented with 10% KOSR. Putative SSC colonies emerged between day 6 and 10 which were then characterized by the expression of numerous spermatogonial and pluripotency related markers. After 15 days of subculture, the relative mRNA expression study revealed that 40 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of BCL6B, ID4, PLZF, and UCHL1. Moreover, the supplementation of GDNF + bFGF up-regulated the expression of PLZF and BCL6B. UCHL1 expression was higher after addition of GDNF + LIF while, THY1 overexpressed in response to the addition of GDNF + CSF1. These results demonstrated that the goat SSCs were efficiently propagated using a KOSR based serum-free media and the growth factor supplementation markedly influences their gene expression profile.
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Affiliation(s)
- Ankur Sharma
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India.
| | - Syed Mohmad Shah
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Manish Tiwari
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Mayank Roshan
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Manoj Kumar Singh
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Suresh Kumar Singla
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Prabhat Palta
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Radhay Sham Manik
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Manmohan Singh Chauhan
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
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26
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Aponte PM. Isolation, Culture, Cryopreservation, and Identification of Bovine, Murine, and Human Spermatogonial Stem Cells. Methods Mol Biol 2020; 2155:151-164. [PMID: 32474875 DOI: 10.1007/978-1-0716-0655-1_13] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Spermatogonial stem cells (SSCs) are the germ cells at the basis of spermatogenesis in adult mammals. SSCs offer many biotechnological possibilities and are fundamental cells in the study of spermatogenesis (Aponte, World J Stem Cells 7:669-680, 2015). This chapter describes detailed procedures for SSC isolation, culture, cryopreservation, and characterization in bovine, murine, and human models.
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Affiliation(s)
- Pedro M Aponte
- Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de Quito (USFQ), Quito, Ecuador. .,Colegio de Ciencias de la Salud, Escuela de Medicina Veterinaria, Universidad San Francisco de Quito (USFQ), Quito, Ecuador. .,Instituto de Investigaciones en Biomedicina "One-health", Universidad San Francisco de Quito (USFQ), Quito, Ecuador.
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27
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Jalali SS, Talebi J, Allymehr M, Soleimanzadeh A, Razi M. Effects of nano-selenium on mRNA expression of markers for spermatogonial stem cells in the testis of broiler breeder males. VETERINARY RESEARCH FORUM : AN INTERNATIONAL QUARTERLY JOURNAL 2019; 10:139-144. [PMID: 31338147 PMCID: PMC6626653 DOI: 10.30466/vrf.2018.86992.2128] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/03/2018] [Accepted: 07/24/2018] [Indexed: 01/22/2023]
Abstract
Fertility is one of the most important parameters in breeder farms and cockerels play an outstanding role in the fertility of eggs in broiler breeder farms. Todays, supplementation of chicken diet with additives such as organic selenium is used to increase fertility. The aim of this study was to evaluate the effects of different levels of nano-selenium (Nano-Se) on the expression of molecular markers of spermatogonial stem cells (SSCs) in the testis of broiler breeder males. A total of 30 roosters of 40 weeks of age were randomly divided into five groups. Groups were as follows: 1) control (normal diet) group, 2) diet supplemented with 0.30 mg kg-1 sodium selenite, 3) diet supplemented with 0.15 mg kg-1 Nano-Se, 4) diet supplemented with 0.30 mg kg-1 Nano-Se, and 5) diet supplemented with 0.60 mg kg-1 Nano-Se. At the end of the experimental period (5th week), birds were autopsied and samples from testis of all birds were collected. The testis samples were used to examine the β1-integrin (CD29), thy-1 (CD90) and NANOG mRNA expression by real-time PCR. The results showed that testis of the groups fed with the diets supplemented with 0.60 mg kg-1 and 0.15 mg kg-1 of Nano-Se had the highest and lowest mRNA expression of SSCs markers, respectively. In conclusion, the present study indicated that Nano-Se had advantages over sodium selenite. Diet supplemented with 0.60 mg kg-1 of Nano-Se may contribute to optimal fertility via increasing the mRNA expression of SSCs markers of roosters’ testis and could be used to delay the reduction of fertility caused by aging in broiler breeder males.
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Affiliation(s)
- Seyed Sattar Jalali
- Department of Poultry Health and Diseases, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
| | - Jalali Talebi
- Department of Poultry Health and Diseases, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
| | - Manoochehr Allymehr
- Department of Poultry Health and Diseases, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
| | - Ali Soleimanzadeh
- Department of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
| | - Mazdak Razi
- Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
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28
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Valdivia M, Castañeda-Zegarra S, Lévano G, Lazo J, Reyes J, Bravo Z, Santiani A, Mujica F, Ruíz J, Gonzales GF. Spermatogonial stem cells identified by molecular expression of PLZF, integrin β1 and reactivity to Dolichos biflorus agglutinin in alpaca adult testes. Andrologia 2019; 51:e13283. [PMID: 30957907 DOI: 10.1111/and.13283] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2018] [Revised: 02/21/2019] [Accepted: 03/08/2019] [Indexed: 12/13/2022] Open
Abstract
The identification system of spermatogonial stem cell (SSC) was established in alpaca using the molecular expression as well as the reactivity pattern to Dolichos biflorus agglutinin (DBA) by flow cytometry. Twenty-four testicles with their epididymis were recovered from adult alpacas at the slaughterhouse of Huancavelica-Perú. Samples were transported to the Laboratory of Reproductive Physiology at Universidad Nacional Mayor de San Marcos. Testes were selected for our study when the progressive motility of epididymal spermatozoa (ESPM) was above 30%. Isolation of SSC was performed with two enzymatic digestions. Finally, sperm viability was evaluated by means of the trypan blue vital stain in spermatogonial round cells. Samples with more than 80% viability were selected. Isolated cells cultured for 2 days were used for identifying the presence of SSCs by the expression of integrin β1 (116 bp) and PLZF (206 bp) genes. Spermatogonia were classified according to the DBA reactivity. Spermatogonia with a strong positive to DBA (sDBA+ ) were classified as SSC (Mean ± SEM=4.44 ± 0.68%). Spermatogonia in early differentiation stages stained weakly positive with DBA (wDBA+ ) (Mean ± SEM=37.44 ± 3.07%) and differentiated round cells as DBA negative (Mean ± SEM=54.12 ± 3.18%). With the use of molecular and DBA markers, it is possible to identify easily the spermatogonial stem cells in alpaca.
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Affiliation(s)
- Martha Valdivia
- Laboratory of Animal Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú.,Endocrine and Reproductive Laboratory, Department of Biological and Physiological Science, Laboratory of Investigation and Development (LID), Faculty of Sciences and Philosophy, Universidad Peruana Cayetano Heredia, Lima, Perú
| | - Sergio Castañeda-Zegarra
- Laboratory of Animal Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Gloria Lévano
- Laboratory of Animal Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Jorge Lazo
- Laboratory of Animal Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Jhakelin Reyes
- Laboratory of Animal Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Zezé Bravo
- Laboratory of Animal Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Alexei Santiani
- Laboratory of Animal Reproduction, Faculty of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Fidel Mujica
- Biological Sciences Faculty, Universidad Nacional San Cristóbal de Huamanga, Ayacucho, Perú
| | - Jaime Ruíz
- Laboratory of Reproductive Biotechnology, Faculty of Engineering Sciences, Universidad Nacional de Huancavelica, Huancavelica, Perú
| | - Gustavo F Gonzales
- Endocrine and Reproductive Laboratory, Department of Biological and Physiological Science, Laboratory of Investigation and Development (LID), Faculty of Sciences and Philosophy, Universidad Peruana Cayetano Heredia, Lima, Perú
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Valdivia M, Reyes J, Bravo Z, Cancho C, Castañeda S, Limaymanta O, Woll P, Santiani A, Gonzales GF. In vitro culture of spermatogonial stem cells isolated from adult alpaca (Vicugna pacos) testes analysed with Dolichos biflorus by flow cytometry. Andrologia 2019; 51:e13269. [PMID: 30900308 DOI: 10.1111/and.13269] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2019] [Revised: 02/11/2019] [Accepted: 02/18/2019] [Indexed: 02/06/2023] Open
Abstract
Spermatogonial stem cell (SSC) is known for its self-renewal capacity. We have studied the in vitro proliferation of isolated SSC from adult alpaca (Vicugna pacos) testes. A total of 107 samples were evaluated of which 31 were evaluated at baseline, 36 were cultivated in DMEM and 40 in STEMPRO media. Half of the cultivated samples was analysed after 14 days, and the rest after 21 days. Round cell subpopulations were identified with FITC-DBA by flow cytometry: strongly positive DBA (sDBA+) as SSC, weakly positive DBA (wDBA+) as in early differentiation and negative DBA as differentiated. At the beginning, 4.16% of the cells were SSC, 37.61% wDBA+ while 54.12% were DBA-. After 14 days, 42.28% of SSC, 44.68% wDBA+ and 11.07% DBA- were found in DMEM while 47.09% of SSC, 32.57% wDBA+ and 18.48% DBA- in STEMPRO. After 21 days 38.66% were SSC, 52.78% wDBA and 7.65% DBA- in DMEM and on STEMPRO media 47.92% SSC, 44.20% wDBA+, 4.93% DBA-. There is a significant difference between the number of initial and SSC cultivated, as well as between DBA- (p < 0.05), while there is no significant difference between the wDBA+ (p > 0.05). Our results suggest that both culture media are appropriate for the in vitro proliferation of alpacas SSC.
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Affiliation(s)
- Martha Valdivia
- Laboratory of Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú.,Endocrine and Reproductive Laboratory, Department of Biological and Physiological Science, Laboratory of Investigation and Development (LID), Faculty of Sciences and Philosophy, Universidad Peruana Cayetano Heredia, Lima, Perú
| | - Jhakelin Reyes
- Laboratory of Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Zezé Bravo
- Laboratory of Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Christian Cancho
- Laboratory of Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Sergio Castañeda
- Laboratory of Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Orlando Limaymanta
- Laboratory of Reproductive Physiology, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Patricia Woll
- Biochemical Laboratory, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Alexei Santiani
- Laboratory of Animal Reproduction, Faculty of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Perú
| | - Gustavo F Gonzales
- Endocrine and Reproductive Laboratory, Department of Biological and Physiological Science, Laboratory of Investigation and Development (LID), Faculty of Sciences and Philosophy, Universidad Peruana Cayetano Heredia, Lima, Perú
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Giassetti MI, Ciccarelli M, Oatley JM. Spermatogonial Stem Cell Transplantation: Insights and Outlook for Domestic Animals. Annu Rev Anim Biosci 2019; 7:385-401. [PMID: 30762440 DOI: 10.1146/annurev-animal-020518-115239] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The demand for food will increase to an unprecedented level over the next 30 years owing to human population expansion, thus necessitating an evolution that improves the efficiency of livestock production. Genetic gain to improve production traits of domestic animal populations is most effectively achieved via selective use of gametes from animals deemed to be elite, and this principle has been the basis of selective breeding strategies employed by humans for thousands of years. In modern-day animal agriculture, artificial insemination (AI) has been the staple of selective breeding programs, but it has inherent limitations for applications in beef cattle and pig production systems. In this review, we discuss the potential and current state of development for a concept termed Surrogate Sires as a next-generation breeding tool in livestock production. The scheme capitalizes on the capacity of spermatogonial stem cells to regenerate sperm production after isolation from donor testicular tissue and transfer into the testes of a recipient male that lacks endogenous germline, thereby allowing the surrogate male to produce offspring with the donor haplotype via natural mating. This concept provides an effective selective breeding tool to achieve genetic gain that is conducive for livestock production systems in which AI is difficult to implement.
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Affiliation(s)
- Mariana I Giassetti
- School of Molecular Biosciences, Center for Reproductive Biology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164, USA;
| | - Michela Ciccarelli
- School of Molecular Biosciences, Center for Reproductive Biology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164, USA;
| | - Jon M Oatley
- School of Molecular Biosciences, Center for Reproductive Biology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164, USA;
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Sharma A, Shah SM, Saini N, Mehta P, Kumar BB, Dua D, Singh MK, Singla SK, Palta P, Manik RS, Chauhan MS. Optimization of Serum-Free Culture Conditions for Propagation of Putative Buffalo (Bubalus bubalis) Spermatogonial Stem Cells. Cell Reprogram 2019; 21:1-10. [DOI: 10.1089/cell.2018.0018] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Affiliation(s)
- Ankur Sharma
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Syed Mohmad Shah
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Neha Saini
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Parul Mehta
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - B.S. Bharath Kumar
- Animal Physiology Division, ICAR-National Dairy Research Institute, Karnal, India
| | - Diksha Dua
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Manoj Kumar Singh
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Suresh Kumar Singla
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Prabhat Palta
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Radhay Sham Manik
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Manmohan Singh Chauhan
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
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Sharma A, Lagah SV, Nagoorvali D, Kumar BB, Singh MK, Singla SK, Manik RS, Palta P, Chauhan MS. Supplementation of Glial Cell Line-Derived Neurotrophic Factor, Fibroblast Growth Factor 2, and Epidermal Growth Factor Promotes Self-Renewal of Putative Buffalo (Bubalus bubalis) Spermatogonial Stem Cells by Upregulating the Expression of miR-20b, miR-21, and miR-106a. Cell Reprogram 2019; 21:11-17. [DOI: 10.1089/cell.2018.0034] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Affiliation(s)
- Ankur Sharma
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Swati Viviyan Lagah
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Dudekula Nagoorvali
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - B.S. Bharath Kumar
- Animal Physiology Division, ICAR-National Dairy Research Institute, Karnal, India
| | - Manoj Kumar Singh
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Suresh Kumar Singla
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Radhay Sham Manik
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Prabhat Palta
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
| | - Manmohan Singh Chauhan
- Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India
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Takashima S. Biology and manipulation technologies of male germline stem cells in mammals. Reprod Med Biol 2018; 17:398-406. [PMID: 30377393 PMCID: PMC6194257 DOI: 10.1002/rmb2.12220] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2018] [Accepted: 06/24/2018] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND Spermatogonial stem cells (SSCs) are the origin of sperm and defined by their functions of "colonization in the testis" and "spermatogenesis". In vitro manipulation techniques of SSCs contribute to a wide variety of fields including reproductive medicine and molecular breeding. This review presents the recent progress of the biology and manipulation technologies of SSCs. METHODS Research articles regarding SSC biology and technologies were collected and summarized. MAIN FINDINGS Dr. Ralph Brinster developed the spermatogonial transplantation technique that enables SSC detection by functional markers. Using this technique, cultured SSCs, termed germline stem (GS) cells, were established from the mouse. GS cells provide the opportunity to produce genome-edited animals without using zygotes. In vitro spermatogenesis allows production of haploid germ cells from GS cells without spermatogonial transplantation. The recent advancement of pluripotent stem cell culture techniques has also achieved production of functional GS-like cells in addition to male/female germ cells. CONCLUSION Although in vitro manipulation techniques of GS cells have been developed for the mouse, it appears to be difficult to apply these techniques to other species. Understanding and control of interspecies barriers are required to extend this technology to nonrodent mammals.
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Affiliation(s)
- Seiji Takashima
- Faculty of Textile Science and TechnologyShinshu UniversityUedaJapan
- Graduate school of Science and TechnologyShinshu UniversityUedaJapan
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Lei Q, Pan Q, Li N, Zhou Z, Zhang J, He X, Peng S, Li G, Sidhu K, Chen S, Hua J. H19 regulates the proliferation of bovine male germline stem cells via IGF-1 signaling pathway. J Cell Physiol 2018; 234:915-926. [PMID: 30069947 DOI: 10.1002/jcp.26920] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2018] [Accepted: 06/13/2018] [Indexed: 01/12/2023]
Abstract
Self-renewal and differentiation of male germline stem cells (mGSCs) provide the basic function for continual spermatogenesis. Studies of in vitro culture of germline stem cells are important and meaningful for basic biological research and practical application. Growth factors, such as GDNF, bFGF, CSF1, and EGF, could maintain the self-renewal of mGSCs. Insulin-like growth factor 1 (IGF-1), an important growth factor, and its pathway have been reported to maintain the survival of several types of stem cells and play important roles in male reproduction. However, the mechanism through which the IGF-1 pathway acts to regulate the self-renewal of mGSCs remains unclear. We analyzed the effect of IGF-1 on the proliferation and apoptosis of bovine mGSCs. We evaluated the expression profile of long noncoding RNA (LncRNA) H19 in bovine and mouse tissues. Moreover, we investigated whether LncRNA H19 could regulate the IGF-1 pathway. Results showed that IGF-1 could activate the phosphorylation of AKT and ERK signaling pathways, and the IGF-1 pathway played an important role in regulating the proliferation and apoptosis of bovine mGSCs. The proliferation rate of mGSCs decreased, whereas the apoptosis rate of mGSCs increased when the IGF-1 receptor (IGF-1R) was blocked using the IGF-1R-specific inhibitor (picropodophyllin). LncRNA H19 could regulate the IGF-1 signaling pathway and, consequently, the proliferation and apoptosis of mGSCs. The number of cells in the seminiferous tubule decreased when H19 was interfered by injecting a virus-containing supernatant. Hence, LncRNA H19 participated in the regulation of the proliferation and apoptosis of mGSCs via the IGF-1 signaling pathway.
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Affiliation(s)
- Qijing Lei
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, China
| | - Qin Pan
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, China
| | - Na Li
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, China
| | - Zhe Zhou
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, China
| | - Juqing Zhang
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, China
| | - Xin He
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, China
| | - Sha Peng
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, China
| | - Guangpeng Li
- Key Laboratory of Mammalian Reproductive Biology and Biotechnology of the Ministry of Education, College of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Kuldip Sidhu
- Centre for Healthy Brain Ageing, UNSW Medicine, High St Randwick, NSW, Australia
| | - Shulin Chen
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, China
| | - Jinlian Hua
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, China
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35
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Sahare MG, Suyatno, Imai H. Recent advances of in vitro culture systems for spermatogonial stem cells in mammals. Reprod Med Biol 2018; 17:134-142. [PMID: 29692670 PMCID: PMC5902468 DOI: 10.1002/rmb2.12087] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2017] [Accepted: 12/23/2017] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Spermatogonial stem cells (SSCs) in the mammalian testis are unipotent stem cells for spermatozoa. They show unique cell characteristics as stem cells and germ cells after being isolated from the testis and cultured in vitro. This review introduces recent progress in the development of culture systems for the establishment of SSC lines in mammalian species, including humans. METHODS Based on the published reports, the isolation and purification of SSCs, identification and characteristics of SSCs, and culture system for mice, humans, and domestic animals have been summarized. RESULTS In mice, cell lines from SSCs are established and can be reprogrammed to show pluripotent stem cell potency that is similar to embryonic stem cells. However, it is difficult to establish cell lines for animals other than mice because of the dearth of understanding about species-specific requirements for growth factors and mechanisms supporting the self-renewal of cultured SSCs. Among the factors that are associated with the development of culture systems, the enrichment of SSCs that are isolated from the testis and the combination of growth factors are essential. CONCLUSION Providing an example of SSC culture in cattle, a rational consideration was made about how it can be possible to establish cell lines from neonatal and immature testes.
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Affiliation(s)
- Mahesh G Sahare
- National Facility for Gene Function in Health and Disease Indian Institute of Science, Education and Research Pune India
| | - Suyatno
- Indonesian Agency for Agricultural Research and Development Jakarta Indonesia
- Laboratory of Reproductive Biology Graduate School of Agriculture Kyoto University Kyoto Japan
| | - Hiroshi Imai
- Laboratory of Reproductive Biology Graduate School of Agriculture Kyoto University Kyoto Japan
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Tang L, Bondareva A, González R, Rodriguez-Sosa JR, Carlson DF, Webster D, Fahrenkrug S, Dobrinski I. TALEN-mediated gene targeting in porcine spermatogonia. Mol Reprod Dev 2018; 85:250-261. [PMID: 29393557 DOI: 10.1002/mrd.22961] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Revised: 01/22/2018] [Accepted: 01/25/2018] [Indexed: 01/05/2023]
Abstract
Spermatogonia represent a diploid germ cell population that includes spermatogonial stem cells. In this report, we describe new methods for isolation of highly enriched porcine spermatogonia based on light scatter properties, and for targeted mutagenesis in porcine spermatogonia using nucleofection and TALENs. We optimized a nucleofection protocol to deliver TALENs specifically targeting the DMD locus in porcine spermatogonia. We also validated specific sorting of porcine spermatogonia based on light scatter properties. We were able to obtain a highly enriched germ cell population with over 90% of cells being UCH-L1 positive undifferentiated spermatogonia. After gene targeting in porcine spermatogonia, indel (insertion or deletion) mutations as a result of non-homologous end joining (NHEJ) were detected in up to 18% of transfected cells. Our report demonstrates for the first time an approach to obtain a live cell population highly enriched in undifferentiated spermatogonia from immature porcine testes, and that gene targeting can be achieved in porcine spermatogonia which will enable germ line modification.
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Affiliation(s)
- Lin Tang
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | - Alla Bondareva
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | - Raquel González
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | - Jose R Rodriguez-Sosa
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | | | | | | | - Ina Dobrinski
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
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Jabarpour M. Evaluation of the effect of follicular stimulating hormone on the in vitro bovine spermatogonial stem cells self-renewal: An experimental study. Int J Reprod Biomed 2017. [DOI: 10.29252/ijrm.15.12.795] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
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Navid S, Abbasi M, Hoshino Y. The effects of melatonin on colonization of neonate spermatogonial mouse stem cells in a three-dimensional soft agar culture system. Stem Cell Res Ther 2017; 8:233. [PMID: 29041987 PMCID: PMC5646105 DOI: 10.1186/s13287-017-0687-y] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2017] [Revised: 09/18/2017] [Accepted: 10/02/2017] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND Melatonin is a pleiotropic hormone with powerful antioxidant activity both in vivo and in vitro. The present study aimed to investigate the effects of melatonin on the proliferation efficiency of neonatal mouse spermatogonial stem cells (SSCs) using a three-dimensional soft agar culture system (SACS) which has the capacity to induce development of SSCs similar to in vivo conditions. METHODS SSCs were isolated from testes of neonate mice and their purities were assessed by flow cytometry using PLZF antibody. Isolated testicular cells were cultured in the upper layer of the SACS in αMEM medium in the absence or presence of melatonin extract for 4 weeks. RESULTS The identity of colonies was confirmed by alkaline phosphatase staining and immunocytochemistry using PLZF and α6 integrin antibodies. The number and diameter of colonies of SSCs in the upper layer were evaluated at days 14 and 28 of culture. The number and diameter of colonies of SSCs were significantly higher in the melatonin group compared with the control group. The levels of expression of ID-4 and Plzf, unlike c-kit, were significantly higher in the melatonin group than in the control group. CONCLUSIONS Results of the present study show that supplementation of the culture medium (SACS) with 100 μM melatonin significantly decreased reactive oxygen species (ROS) production in the treated group compared with the control group, and increased SSC proliferation.
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Affiliation(s)
- Shadan Navid
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Mehdi Abbasi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Yumi Hoshino
- Laboratory of Reproductive Endocrinology, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Kagamiyama 1-4-4, Hiroshima 739-8528 Japan
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Abstract
Spermatogonial stem cells (SSCs) are crucial for maintaining spermatogenesis throughout life, and understanding how these cells function has important implications for understanding male infertility. Recently, various populations of cells harbouring stem cell-like properties have been identified in rodent seminiferous tubules, but deciphering how these cells might fuel spermatogenesis has been difficult, and various models to explain SSC dynamics have been put forward. This Review provides an overview of the organization and timing of spermatogenesis and then discusses these models in light of recent studies of SSC markers, heterogeneity and cell division dynamics, highlighting the evidence for and against each model.
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Affiliation(s)
- Dirk G. de Rooij
- Reproductive Biology Group, Division of Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht 3584 CH, The Netherlands
- Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Amsterdam 1105 AZ, The Netherlands
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Zhang P, Chen X, Zheng Y, Zhu J, Qin Y, Lv Y, Zeng W. Long-Term Propagation of Porcine Undifferentiated Spermatogonia. Stem Cells Dev 2017; 26:1121-1131. [PMID: 28474535 DOI: 10.1089/scd.2017.0018] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis and fertility throughout the adult life of a male. Genetic manipulations of SSCs combined with germ cell transplantation present a novel approach for gene therapy and production of genetically modified animals. However, the rarity of SSCs within mammalian testes remains an impediment to related applications, making in vitro expansion of SSCs a prerequisite. Nevertheless, long-term culture systems of SSCs from large animals have not been established yet. In this study, we developed an optimized in vitro culture condition for porcine undifferentiated spermatogonia. The germ cells were isolated and enriched from 7-day-old porcine testes by an optimized differential planting. We tested different feeder layers and found that neonatal autologous Sertoli cells acted better than the SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cell line and adult Sertoli cells. The effects of several growth factors were also investigated. Using neonatal Sertoli cells as feeder and Dulbecco's modified eagle medium: nutrient mixture F-12 (DMEM/F12) culture medium supplemented with 10% KSR and four cytokines, the undifferentiated spermatogonia can proliferate in vitro for at least 2 months without loss of stemness. The expression of SSC markers indicated that the cultured cells maintained SSC expression profiles. Moreover, xenotransplantation and in vitro induction showed that the long-term cultured cells preserved the capacity to colonize in vivo and differentiate in vitro, respectively, demonstrating the presence of SSCs in the cultured cells. In conclusion, the conditions described in this study can support the normal proliferation of porcine undifferentiated spermatogonia with stemness and normal karyotype for at least 2 months. This culture system will serve as a basic refinement in the future studies and facilitate studies on SSC biology and genetic manipulation of male germ cells.
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Affiliation(s)
- Pengfei Zhang
- 1 College of Animal Science and Technology, Northwest A&F University , Shaanxi, China
| | - Xiaoxu Chen
- 1 College of Animal Science and Technology, Northwest A&F University , Shaanxi, China
| | - Yi Zheng
- 2 Center for Reproductive Medicine, Amsterdam Research Institute Reproduction and Development, Academic Medical Centre, University of Amsterdam , Amsterdam, the Netherlands
| | - Jinshen Zhu
- 1 College of Animal Science and Technology, Northwest A&F University , Shaanxi, China
| | - Yuwei Qin
- 1 College of Animal Science and Technology, Northwest A&F University , Shaanxi, China
| | - Yinghua Lv
- 1 College of Animal Science and Technology, Northwest A&F University , Shaanxi, China
| | - Wenxian Zeng
- 1 College of Animal Science and Technology, Northwest A&F University , Shaanxi, China
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Gadella BM, Ferraz MA. A Review of New Technologies that may Become Useful for in vitro Production of Boar Sperm. Reprod Domest Anim 2016; 50 Suppl 2:61-70. [PMID: 26174921 DOI: 10.1111/rda.12571] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2015] [Accepted: 05/20/2015] [Indexed: 01/07/2023]
Abstract
Making sperm cells outside the original testicular environment in a culture dish has been considered for a long time as impossible due to the very complicated process of spermatogenesis and sperm maturation, which altogether, encompasses a 2-month period. However, new approaches in complex three-dimensional co-cell cultures, micro-perfusion and micro-fluidics technologies, new knowledge in the functioning, culturing and differentiation of spermatogonial stem cells (SSC) and their precursor cells have revolutionized this field. Furthermore, the use of better molecular markers as well as stimulatory factors has led to successful in vitro culture of stem cells either derived from germ line stem cells, from induced pluripotent stem cells (iPSC) or from embryonic stem cells (ESC). These stem cells when placed into small seminiferous tubule fragments are able to become SSC. The SSC beyond self-renewal can also be induced into haploid sperm-like cells under in vitro conditions. In mouse, this in vitro produced sperm can be injected into a mature oocyte and allow post-fertilization development into an early embryo in vitro. After transferring such obtained embryos into the uterus of a recipient mouse, they can further develop into healthy offspring. Recently, a similar approach has been performed with combining selected cells from testicular cell suspensions followed by a complete in vitro culture of seminiferous cords producing sperm-like cells. However, most of the techniques developed are laborious, time-consuming and have low efficiency, placing questionable that it will become useful used for setting up an efficient in vitro sperm production system for the boar. The benefits and drawbacks as well as the likeliness of in vitro pig sperm production to become applied in assisted technologies for swine reproduction are critically discussed. In this contribution, also the process of sperm production in the testis and sperm maturation is reviewed.
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Affiliation(s)
- B M Gadella
- Department of Farm Animal Health, Faculty of Veterinary Sciences, Utrecht University, Utrecht, The Netherlands.,Department of Biochemistry and Cell Biology, Faculty of Veterinary Sciences, Utrecht University, Utrecht, The Netherlands
| | - M A Ferraz
- Department of Farm Animal Health, Faculty of Veterinary Sciences, Utrecht University, Utrecht, The Netherlands
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42
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González R, Dobrinski I. Beyond the mouse monopoly: studying the male germ line in domestic animal models. ILAR J 2016; 56:83-98. [PMID: 25991701 DOI: 10.1093/ilar/ilv004] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and essential to maintain the continuous production of spermatozoa after the onset of puberty in the male. The study of the male germ line is important for understanding the process of spermatogenesis, unravelling mechanisms of stemness maintenance, cell differentiation, and cell-to-cell interactions. The transplantation of SSCs can contribute to the preservation of the genome of valuable individuals in assisted reproduction programs. In addition to the importance of SSCs for male fertility, their study has recently stimulated interest in the generation of genetically modified animals because manipulations of the male germ line at the SSC stage will be maintained in the long term and transmitted to the offspring. Studies performed mainly in the mouse model have laid the groundwork for facilitating advancements in the field of male germ line biology, but more progress is needed in nonrodent species in order to translate the technology to the agricultural and biomedical fields. The lack of reliable markers for isolating germ cells from testicular somatic cells and the lack of knowledge of the requirements for germ cell maintenance have precluded their long-term maintenance in domestic animals. Nevertheless, some progress has been made. In this review, we will focus on the state of the art in the isolation, characterization, culture, and manipulation of SSCs and the use of germ cell transplantation in domestic animals.
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Affiliation(s)
- Raquel González
- Raquel González, DVM, PhD, is a postdoctoral research fellow at the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada. Ina Dobrinski, DVM, MVSc, PhD, Dipl ACT, is a professor and the head of the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
| | - Ina Dobrinski
- Raquel González, DVM, PhD, is a postdoctoral research fellow at the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada. Ina Dobrinski, DVM, MVSc, PhD, Dipl ACT, is a professor and the head of the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
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França LR, Hess RA, Dufour JM, Hofmann MC, Griswold MD. The Sertoli cell: one hundred fifty years of beauty and plasticity. Andrology 2016; 4:189-212. [PMID: 26846984 DOI: 10.1111/andr.12165] [Citation(s) in RCA: 277] [Impact Index Per Article: 30.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2015] [Revised: 12/30/2015] [Accepted: 01/04/2016] [Indexed: 12/18/2022]
Abstract
It has been one and a half centuries since Enrico Sertoli published the seminal discovery of the testicular 'nurse cell', not only a key cell in the testis, but indeed one of the most amazing cells in the vertebrate body. In this review, we begin by examining the three phases of morphological research that have occurred in the study of Sertoli cells, because microscopic anatomy was essentially the only scientific discipline available for about the first 75 years after the discovery. Biochemistry and molecular biology then changed all of biological sciences, including our understanding of the functions of Sertoli cells. Immunology and stem cell biology were not even topics of science in 1865, but they have now become major issues in our appreciation of Sertoli cell's role in spermatogenesis. We end with the universal importance and plasticity of function by comparing Sertoli cells in fish, amphibians, and mammals. In these various classes of vertebrates, Sertoli cells have quite different modes of proliferation and epithelial maintenance, cystic vs. tubular formation, yet accomplish essentially the same function but in strikingly different ways.
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Affiliation(s)
- L R França
- Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.,National Institute for Amazonian Research (INPA), Manaus, Amazonas, Brazil
| | - R A Hess
- Reproductive Biology and Toxicology, Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, IL, USA
| | - J M Dufour
- Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, USA
| | - M C Hofmann
- Department of Endocrine Neoplasia and Hormonal Disorders, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - M D Griswold
- Center for Reproductive Biology, School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA
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Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes. Stem Cells Int 2016; 2016:6029271. [PMID: 26880974 PMCID: PMC4736562 DOI: 10.1155/2016/6029271] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2015] [Accepted: 11/25/2015] [Indexed: 01/15/2023] Open
Abstract
Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs) have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months) limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs) and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4), which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells.
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Sahare M, Kim SM, Otomo A, Komatsu K, Minami N, Yamada M, Imai H. Factors supporting long-term culture of bovine male germ cells. Reprod Fertil Dev 2016; 28:2039-2050. [DOI: 10.1071/rd15003] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2015] [Accepted: 06/11/2015] [Indexed: 01/15/2023] Open
Abstract
Spermatogonial stem cells (SSCs) are unipotent in nature, but mouse SSCs acquire pluripotency under the appropriate culture conditions. Although culture systems are available for rodent and human germ-cell lines, no proven culture system is yet available for livestock species. Here, we examined growth factors, matrix substrates and serum-free supplements to develop a defined system for culturing primitive germ cells (gonocytes) from neonatal bovine testis. Poly-L-lysine was a suitable substrate for selective inhibition of the growth of somatic cells and made it possible to maintain a higher gonocyte : somatic cell ratio than those maintained with gelatin, collagen or Dolichos biflorus agglutinin (DBA) substrates. Among the serum-free supplements tested in our culture medium, knockout serum replacement (KSR) supported the proliferation and survival of gonocytes better than the supplements B-27 and StemPro-SFM after sequential passages of colonies. Under our optimised culture conditions consisting of 15% KSR supplement on poly-L-lysine-coated dishes, the stem-cell and germ-cell potentials of the cultured gonocytes were maintained with normal karyotype for more than 2 months (over 13 passages). The proposed culture system, which can maintain a population of proliferating bovine germ stem cells, could be useful for studying SSC biology and germline modifications in livestock animals.
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Abbasi H, Hosseini SM, Hajian M, Nasiri Z, Bahadorani M, Tahmoorespur M, Nasiri MR, Nasr-Esfahani MH. Lentiviral vector-mediated transduction of goat undifferentiated spermatogonia. Anim Reprod Sci 2015; 163:10-7. [DOI: 10.1016/j.anireprosci.2015.09.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2015] [Revised: 08/05/2015] [Accepted: 09/02/2015] [Indexed: 11/26/2022]
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Feng W, Chen S, Do D, Liu Q, Deng Y, Lei X, Luo C, Huang B, Shi D. Isolation and Identification of Prepubertal Buffalo (Bubalus bubalis) Spermatogonial Stem Cells. ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 2015; 29:1407-15. [PMID: 26954139 PMCID: PMC5003965 DOI: 10.5713/ajas.15.0592] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/13/2015] [Revised: 09/30/2015] [Accepted: 11/14/2015] [Indexed: 12/13/2022]
Abstract
Isolation and culture of spermatogonial stem cells (SSCs) are attractive for production of genetic modified offspring. In the present study, buffalo spermatogonial stem-like cells were isolated, cultured and expression pattern of different germ cell marker genes were determined. To recover spermatogonia, testes from age 3 to 7 months of buffalo were decapsulated, and seminiferous tubules were enzymatically dissociated. Two types of cells, immature sertoli cell and type A spermatogonia were observed in buffalo testes in this stage. Germ cell marker genes, OCT3/4 (Pou5f1), THY-1, c-kit, PGP9.5 (UCHL-1) and Dolichos biflorus agglutinin, were determined to be expressed both in mRNA and protein level by reverse transcription polymerase chain reaction and immunostaining in buffalo testes and buffalo spermatogonial stem-like cells, respectively. In the following, when the isolated buffalo buffalo spermatogonial stem-like cells were cultured in the medium supplemented 2.5% fetal bovine serum and 40 ng/mL glial cell-derived neurotrophic factor medium, SSCs proliferation efficiency and colony number were significantly improved than those of other groups (p<0.05). These findings may help in isolation and establishing long term in vitro culture system for buffalo spermatogonial stem-like cells, and accelerating the generation of genetic modified buffaloes.
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Affiliation(s)
- Wanyou Feng
- State Key Laboratory of Conservation and Untilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530005, China
| | - Shibei Chen
- State Key Laboratory of Conservation and Untilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530005, China.,Center of Reproduction of Nanxishan Hospital, Guilin 541002, China
| | - Dagiang Do
- State Key Laboratory of Conservation and Untilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530005, China.,Bacgiang Agriculture and Forestry University, Bacgiang 220000 Vietnam
| | - Qinyou Liu
- State Key Laboratory of Conservation and Untilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530005, China
| | - Yanfei Deng
- State Key Laboratory of Conservation and Untilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530005, China
| | - Xiaocan Lei
- State Key Laboratory of Conservation and Untilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530005, China
| | - Chan Luo
- State Key Laboratory of Conservation and Untilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530005, China
| | - Ben Huang
- State Key Laboratory of Conservation and Untilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530005, China
| | - Deshun Shi
- State Key Laboratory of Conservation and Untilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530005, China
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Wang X, Chen T, Zhang Y, Li B, Xu Q, Song C. Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes. Int J Mol Sci 2015; 16:26333-46. [PMID: 26556335 PMCID: PMC4661817 DOI: 10.3390/ijms161125958] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2015] [Revised: 10/21/2015] [Accepted: 10/23/2015] [Indexed: 12/14/2022] Open
Abstract
Spermatogonial stem cells (SSCs) renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like cells and adipocytes. SSCs and Sertoli cells were isolated from neonatal piglet testis by differential adhesion and SSCs were cultured on a feeder layer of Sertoli cells. Third-generation SSCs were induced to differentiate into neuron-like cells by addition of retinoic acid, β-mercaptoethanol, and 3-isobutyl-1-methylxanthine (IBMX) to the induction media and into adipocytes by the addition of hexadecadrol, insulin, and IBMX to the induction media. The differentiated cells were characterized by biochemical staining, qRT-PCR, and immunocytochemistry. The cells were positive for SSC markers, including alkaline phosphatase and SSC-specific genes, consistent with the cells being undifferentiated. The isolated SSCs survived on the Sertoli cells for 15 generations. Karyotyping confirmed that the chromosomal number of the SSCs were normal for pig (2n = 38, n = 19). Pig SSCs were successfully induced into neuron-like cells eight days after induction and into adipocytes 22 days after induction as determined by biochemical and immunocytochemical staining. qPCR results also support this conclusion. The nervous tissue markers genes, Nestin and β-tubulin, were expressed in the neuron-like cells and the adipocyte marker genes, PPARγ and C/EBPα, were expressed in the adipocytes.
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Affiliation(s)
- Xiaoyan Wang
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
| | - Tingfeng Chen
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
| | - Yani Zhang
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
| | - Bichun Li
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
| | - Qi Xu
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
| | - Chengyi Song
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
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49
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Zhu H, Ma J, Du R, Zheng L, Wu J, Song W, Niu Z, He X, Du E, Zhao S, Hua J. Characterization of immortalized dairy goat male germline stem cells (mGSCs). J Cell Biochem 2015; 115:1549-60. [PMID: 24692210 DOI: 10.1002/jcb.24812] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2013] [Accepted: 03/27/2014] [Indexed: 12/19/2022]
Abstract
Male germline stem cells (mGSCs), in charge for the fertility in male testis, are the only kind of adult stem cells that transmit genetic information to next generation, with promising prospects in germplasm resources preservation and optimization, and production of transgenic animals. Mouse male germline stem cell lines have been established and are valuable for studying the mechanisms of spermatogenesis. However, there is a lack of stable mGSC cell lines in livestock, which restricts the progress of transgenic research and related biotechnology. Here, we firstly established an immortalized dairy goat mGSC cell line to study the biological properties and the signaling pathways associated with mGSCs self-renewal and differentiation. The ectopic factors SV40 large T antigen and Bmi1 genes were transduced into dairy goat mGSCs, and the results showed that the proliferation of these cells that were named mGSCs-I-SB was improved significantly. They maintained the typical characteristics including the expression of mGSC markers, and the potential to differentiate into all three germ layers, sperm-like cells in vitro. Additionally, mGSCs-I-SB survived and differentiated into three germ layer cell types when they were transplanted into chicken embryos. Importantly, the cells also survived in mouse spermatogenesis deficiency model testis which seemed to be the golden standard to examine mGSCs. Conclusively, our results demonstrate that mGSCs-I-SB present the characteristics of mGSCs and may promote the future study on goat mGSCs.
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Affiliation(s)
- Haijing Zhu
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Key Lab for Animal Biotechnology of Agriculture Ministry of China, Northwest A&F University, Yangling, Shaanxi, 712100, China
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50
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Saito S, Lin YC, Murayama Y, Nakamura Y, Eckner R, Niemann H, Yokoyama KK. Retracted article: In vitro derivation of mammalian germ cells from stem cells and their potential therapeutic application. Cell Mol Life Sci 2015; 72:4545-60. [PMID: 26439925 PMCID: PMC4628088 DOI: 10.1007/s00018-015-2020-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2015] [Revised: 07/27/2015] [Accepted: 08/07/2015] [Indexed: 01/12/2023]
Abstract
Pluripotent stem cells (PSCs) are a unique type of cells because they
exhibit the characteristics of self-renewal and pluripotency. PSCs may be induced to
differentiate into any cell type, even male and female germ cells, suggesting their
potential as novel cell-based therapeutic treatment for infertility problems.
Spermatogenesis is an intricate biological process that starts from self-renewal of
spermatogonial stem cells (SSCs) and leads to differentiated haploid spermatozoa.
Errors at any stage in spermatogenesis may result in male infertility. During the
past decade, much progress has been made in the derivation of male germ cells from
various types of progenitor stem cells. Currently, there are two main approaches for
the derivation of functional germ cells from PSCs, either the induction of in vitro
differentiation to produce haploid cell products, or combination of in vitro
differentiation and in vivo transplantation. The production of mature and fertile
spermatozoa from stem cells might provide an unlimited source of autologous gametes
for treatment of male infertility. Here, we discuss the current state of the art
regarding the differentiation potential of SSCs, embryonic stem cells, and induced
pluripotent stem cells to produce functional male germ cells. We also discuss the
possible use of livestock-derived PSCs as a novel option for animal reproduction and
infertility treatment.
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Affiliation(s)
- Shigeo Saito
- Saito Laboratory of Cell Technology, Yaita, Tochigi, 329-1571, Japan. .,SPK Co., Ltd., Aizuwakamatsu, Fukushima, 965-0025, Japan. .,College of Engineering, Nihon University, Koriyama, Fukushima, 963-8642, Japan.
| | - Ying-Chu Lin
- School of Dentistry, College of Dental Medicine, Kaoshiung Medical University, 100 Shin-Chuan 1st Road, Kaohsiung, 807, Taiwan
| | - Yoshinobu Murayama
- College of Engineering, Nihon University, Koriyama, Fukushima, 963-8642, Japan
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Center, Tsukuba, 3050074, Japan
| | - Richard Eckner
- Department of Biochemistry and Molecular Biology, Rutgers New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, 07101, USA
| | - Heiner Niemann
- Institute of Farm Animal Genetics, Friedrich-Löffler-Institut, Mariensee, 31535, Neustadt, Germany.
| | - Kazunari K Yokoyama
- Graduate Institute of Medicine, Center of Stem Cell Research, Center of Environmental Medicine, Kaohsiung Medical University, 100 Shih-Chuan 1st Rd, San Ming District, Kaohsiung, 807, Taiwan. .,Faculty of Science and Engineering, Tokushima Bunri University, Sanuki, 763-2193, Japan. .,Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-0033, Japan.
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