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1
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Xu Q, Chen H. Applications of spatial transcriptomics in studying spermatogenesis. Andrology 2025. [PMID: 40202007 DOI: 10.1111/andr.70043] [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: 08/20/2024] [Revised: 03/20/2025] [Accepted: 03/27/2025] [Indexed: 04/10/2025]
Abstract
Spermatogenesis is a complex differentiation process that is facilitated by a series of cellular and molecular events. High-throughput genomics approaches, such as single-cell RNA sequencing, have begun to enable the systematic characterization of these events. However, the loss of tissue context because of tissue disassociations in the single-cell isolation protocols limits our ability to understand the regulation of spermatogenesis and how defects in spermatogenesis lead to infertility. The recent advancement of spatial transcriptomics technologies enables the studying of the molecular signatures of various cell types and their interactions in the native tissue context. In this review, we discuss how spatial transcriptomics has been leveraged to identify spatially variable genes, characterize cellular neighborhood, delineate cell‒cell communications, and detect molecular changes under pathological conditions in the mammalian testis. We believe that spatial transcriptomics, along with other emerging spatially resolved omics assays, can be utilized to further our understanding of the underlying causes of male infertility, and to facilitate the development of new treatment approaches.
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Affiliation(s)
- Qianlan Xu
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas, USA
- Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Haiqi Chen
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas, USA
- Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
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2
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Rehder P, Packeiser EM, Körber H, Goericke-Pesch S. Altered Sertoli Cell Function Contributes to Spermatogenic Arrest in Dogs with Chronic Asymptomatic Orchitis. Int J Mol Sci 2025; 26:1108. [PMID: 39940876 PMCID: PMC11817828 DOI: 10.3390/ijms26031108] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2024] [Revised: 01/16/2025] [Accepted: 01/24/2025] [Indexed: 02/16/2025] Open
Abstract
Acquired infertility due to chronic asymptomatic orchitis (CAO) is a common finding in male dogs. It is characterized by spermatogenic arrest, a significant reduction in spermatogonia, immune cell infiltration and a disruption of the blood-testis barrier. Sertoli cells are a key factor for spermatogenesis and the testicular micromilieu. We hypothesize altered Sertoli cell function to be involved in the pathogenesis of canine CAO. Consequently, the aim was to gain further insights into the spermatogonial stem cell niche and Sertoli cell function in CAO-affected dogs. Therefore, the testicular expression of the Sertoli cell-derived factors bFGF, GDNF, WNT5A, BMP4, CXCL12 and LDHC were evaluated in 15 CAO testis tissues and 10 normospermic controls by relative quantitative real-time PCR (qPCR). Additionally, the protein expression patterns of bFGF, GDNF and WNT5A were visualized immunohistochemically (IHC). This study revealed an overexpression of bFGF (IHC, p < 0.0001), GDNF (qPCR, p = 0.0036), WNT5A (IHC, p = 0.0066) and CXCL12 (qPCR, p = 0.0003) and a reduction in BMP4 (qPCR, p = 0.0041) and LDHC (qPCR, p = 0.0003) in CAO-affected testis in dogs, clearly confirming impaired Sertoli cell function in canine CAO. Sertoli cell function is essential for spermatogenesis and must be considered for potential therapeutic approaches.
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Affiliation(s)
| | | | | | - Sandra Goericke-Pesch
- Reproductive Unit—Clinic for Small Animals, University of Veterinary Medicine Hannover, Foundation, 30559 Hannover, Germany; (P.R.); (E.-M.P.); (H.K.)
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3
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Liu W, Du L, Li J, He Y, Tang M. Microenvironment of spermatogonial stem cells: a key factor in the regulation of spermatogenesis. Stem Cell Res Ther 2024; 15:294. [PMID: 39256786 PMCID: PMC11389459 DOI: 10.1186/s13287-024-03893-z] [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: 05/10/2024] [Accepted: 08/25/2024] [Indexed: 09/12/2024] Open
Abstract
Spermatogonial stem cells (SSCs) play a crucial role in the male reproductive system, responsible for maintaining continuous spermatogenesis. The microenvironment or niche of SSCs is a key factor in regulating their self-renewal, differentiation and spermatogenesis. This microenvironment consists of multiple cell types, extracellular matrix, growth factors, hormones and other molecular signals that interact to form a complex regulatory network. This review aims to provide an overview of the main components of the SSCs microenvironment, explore how they regulate the fate decisions of SSCs, and discuss the potential impact of microenvironmental abnormalities on male reproductive health.
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Affiliation(s)
- Wei Liu
- Department of Pathology, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/Hunan Cancer Hospital, Changsha, China
- Department of Pharmacy, The Third Xiangya Hospital, Central South University, Changsha, China
| | - Li Du
- The Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, The Engineering Research Center of Reproduction and Translational Medicine of Hunan Province, Hunan Normal University School of Medicine, Changsha, China
| | - Junjun Li
- Department of Pathology, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/Hunan Cancer Hospital, Changsha, China
| | - Yan He
- Department of Pathology, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/Hunan Cancer Hospital, Changsha, China.
| | - Mengjie Tang
- Department of Pathology, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/Hunan Cancer Hospital, Changsha, China.
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4
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Yan Q, Wang Q, Zhang Y, Yuan L, Hu J, Zhao X. The Novel-m0230-3p miRNA Modulates the CSF1/CSF1R/Ras Pathway to Regulate the Cell Tight Junctions and Blood-Testis Barrier in Yak. Cells 2024; 13:1304. [PMID: 39120333 PMCID: PMC11311379 DOI: 10.3390/cells13151304] [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: 07/25/2024] [Revised: 08/02/2024] [Accepted: 08/02/2024] [Indexed: 08/10/2024] Open
Abstract
The yak (Bos grunniens) is a valuable livestock animal endemic to the Qinghai-Tibet Plateau in China with low reproductive rates. Cryptorchidism is one of the primary causes of infertility in male yaks. Compared with normal testes, the tight junctions (TJs) of Sertoli cells (SCs) and the integrity of the blood-testis barrier (BTB) in cryptorchidism are both disrupted. MicroRNAs are hairpin-derived RNAs of about 19-25 nucleotides in length and are involved in a variety of biological processes. Numerous studies have shown the involvement of microRNAs in the reproductive physiology of yak. In this study, we executed RNA sequencing (RNA-seq) to describe the expression profiles of mRNAs and microRNAs in yaks with normal testes and cryptorchidism to identify differentially expressed genes. GO and KEGG analyses were used to identify the biological processes and signaling pathways which the target genes of the differentially expressed microRNAs primarily engaged. It was found that novel-m0230-3p is an important miRNA that significantly differentiates between cryptorchidism and normal testes, and it is down-regulated in cryptorchidism with p < 0.05. Novel-m0230-3p and its target gene CSF1 both significantly contribute to the regulation of cell adhesion and tight junctions. The binding sites of novel-m0230-3p with CSF1 were validated by a dual luciferase reporter system. Then, mimics and inhibitors of novel-m0230-3p were transfected in vitro into SCs, respectively. A further analysis using qRT-PCR, immunofluorescence (IF), and Western blotting confirmed that the expression of cell adhesion and tight-junction-related proteins Occludin and ZO-1 both showed changes. Specifically, both the mRNA and protein expression levels of Occludin and ZO-1 in SCs decreased after transfection with the novel-m0230-3p mimics, while they increased after transfection with the inhibitors, with p < 0.05. These were achieved via the CSF1/CSF1R/Ras signaling pathway. In summary, our findings indicate a negative miRNA-mRNA regulatory network involving the CSF1/CSF1R/Ras signaling pathway in yak SCs. These results provide new insights into the molecular mechanisms of CSF1 and suggest that novel-m0230-3p and its target protein CSF1 could be used as potential therapeutic targets for yak cryptorchidism.
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Affiliation(s)
- Qiu Yan
- College of Veterinary Medicine, Gansu Agriculture University, Lanzhou 730070, China; (Y.Z.); (L.Y.); (J.H.); (X.Z.)
- Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Lanzhou 730070, China
| | - Qi Wang
- College of Veterinary Medicine, Gansu Agriculture University, Lanzhou 730070, China; (Y.Z.); (L.Y.); (J.H.); (X.Z.)
- Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Lanzhou 730070, China
| | - Yong Zhang
- College of Veterinary Medicine, Gansu Agriculture University, Lanzhou 730070, China; (Y.Z.); (L.Y.); (J.H.); (X.Z.)
- Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Lanzhou 730070, China
| | - Ligang Yuan
- College of Veterinary Medicine, Gansu Agriculture University, Lanzhou 730070, China; (Y.Z.); (L.Y.); (J.H.); (X.Z.)
- Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Lanzhou 730070, China
| | - Junjie Hu
- College of Veterinary Medicine, Gansu Agriculture University, Lanzhou 730070, China; (Y.Z.); (L.Y.); (J.H.); (X.Z.)
- Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Lanzhou 730070, China
| | - Xingxu Zhao
- College of Veterinary Medicine, Gansu Agriculture University, Lanzhou 730070, China; (Y.Z.); (L.Y.); (J.H.); (X.Z.)
- Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Lanzhou 730070, China
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Jing J, Ouyang L, Zhang H, Liang K, Ma R, Ge X, Tang T, Zhao S, Xue T, Shen J, Ma J, Li Z, Wu J, Yang Y, Zhao W, Zheng L, Qian Z, Sun S, Ge Y, Chen L, Li C, Yao B. Omega-3 polyunsaturated fatty acids and its metabolite 12-HEPE rescue busulfan disrupted spermatogenesis via target to GPR120. Cell Prolif 2024; 57:e13551. [PMID: 37743695 PMCID: PMC10849791 DOI: 10.1111/cpr.13551] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2022] [Revised: 08/26/2023] [Accepted: 09/08/2023] [Indexed: 09/26/2023] Open
Abstract
Busulfan is an antineoplastic, which is always accompanied with the abnormal of spermatogonia self-renewal and differentiation. It has been demonstrated that the omega-3 polyunsaturated fatty acids (PUFAs) benefits mature spermatozoa. However, whether omega-3 can protect endogenous spermatogonia and the detailed mechanisms are still unclear. Evaluate of spermatogenesis function (in vivo) were examined by histopathological analysis, immunofluorescence staining, and western blotting. The levels of lipid metabolites in testicular tissue were determined via liquid chromatography. We investigated the effect of lipid metabolites on Sertoli cells provided paracrine factors to regulate spermatogonia proliferation and differentiation using co-culture system. In our study, we showed that omega-3 PUFAs significantly improved the process of sperm production and elevated the quantity of both undifferentiated Lin28+ spermatogonia and differentiated c-kit+ spermatogonia in a mouse model where spermatogenic function was disrupted by busulfan. Mass spectrometry revealed an increase in the levels of several omega-3 metabolites in the testes of mice fed with omega-3 PUFAs. The eicosapentaenoic acid metabolite 12-hydroxyeicosapentaenoic acid (12-HEPE) up-regulated bone morphogenic protein 4 (BMP4) expression through GPR120-ERK1/2 pathway activation in Sertoli cells and restored spermatogonia proliferation and differentiation. Our study provides evidence that omega-3 PUFAs metabolite 12-HEPE effectively protects spermatogonia and reveals that GPR120 might be a tractable pharmacological target for fertility in men received chemotherapy or severe spermatogenesis dysfunction.
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Affiliation(s)
- Jun Jing
- State Key Laboratory of Reproductive Medicine and Offspring HealthNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling HospitalNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Lei Ouyang
- Department of Reproductive Medicine, Affiliated Jinling Hospital, The First School of Clinical MedicineSouthern Medical UniversityNanjingChina
| | - Hong Zhang
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Kuan Liang
- Department of Reproductive Medicine, Affiliated Jinling HospitalNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling Hospital, The First School of Clinical MedicineSouthern Medical UniversityNanjingChina
| | - Rujun Ma
- Department of Reproductive Medicine, Affiliated Jinling HospitalNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Xie Ge
- Department of Reproductive Medicine, Affiliated Jinling HospitalNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Ting Tang
- State Key Laboratory of Reproductive Medicine and Offspring HealthNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling HospitalNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Shanmeizi Zhao
- School of Life ScienceNanjing Normal UniversityNanjingChina
| | - Tongmin Xue
- State Key Laboratory of Reproductive Medicine and Offspring HealthNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling HospitalNanjing Medical UniversityNanjingChina
- Reproductive Medical Center, Clinical Medical College (Northern Jiangsu People's Hospital)Yangzhou UniversityYangzhouChina
| | - Jiaming Shen
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Jinzhao Ma
- Department of Reproductive Medicine, Affiliated Jinling HospitalNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Zhou Li
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Jing Wu
- Core Laboratory, Sir Run Run HospitalNanjing Medical UniversityNanjingChina
| | - Yang Yang
- Basic Medical Laboratory, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Wei Zhao
- Department of Reproductive Medicine, Affiliated Jinling HospitalNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Lu Zheng
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Zhang Qian
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Shanshan Sun
- School of Life ScienceNanjing Normal UniversityNanjingChina
| | - Yifeng Ge
- Department of Reproductive Medicine, Affiliated Jinling HospitalNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Li Chen
- Department of Reproductive Medicine, Affiliated Jinling HospitalNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
| | - Chaojun Li
- State Key Laboratory of Reproductive Medicine and Offspring HealthNanjing Medical UniversityNanjingChina
| | - Bing Yao
- State Key Laboratory of Reproductive Medicine and Offspring HealthNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling HospitalNanjing Medical UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling Hospital, Clinical School of Medical CollegeNanjing UniversityNanjingChina
- Department of Reproductive Medicine, Affiliated Jinling Hospital, The First School of Clinical MedicineSouthern Medical UniversityNanjingChina
- School of Life ScienceNanjing Normal UniversityNanjingChina
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6
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Klees C, Alexandri C, Demeestere I, Lybaert P. The Role of microRNA in Spermatogenesis: Is There a Place for Fertility Preservation Innovation? Int J Mol Sci 2023; 25:460. [PMID: 38203631 PMCID: PMC10778981 DOI: 10.3390/ijms25010460] [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: 11/15/2023] [Revised: 12/24/2023] [Accepted: 12/27/2023] [Indexed: 01/12/2024] Open
Abstract
Oncological treatments have dramatically improved over the last decade, and as a result, survival rates for cancer patients have also improved. Quality of life, including concerns about fertility, has become a major focus for both oncologists and patients. While oncologic treatments are often highly effective at suppressing neoplastic growth, they are frequently associated with severe gonadotoxicity, leading to infertility. For male patients, the therapeutic option to preserve fertility is semen cryopreservation. In prepubertal patients, immature testicular tissue can be sampled and stored to allow post-cure transplantation of the tissue, immature germ cells, or in vitro spermatogenesis. However, experimental techniques have not yet been proven effective for restoring sperm production for these patients. MicroRNAs (miRNAs) have emerged as promising molecular markers and therapeutic tools in various diseases. These small regulatory RNAs possess the unique characteristic of having multiple gene targets. MiRNA-based therapeutics can, therefore, be used to modulate the expression of different genes involved in signaling pathways dysregulated by changes in the physiological environment (disease, temperature, ex vivo culture, pharmacological agents). This review discusses the possible role of miRNA as an innovative treatment option in male fertility preservation-restoration strategies and describes the diverse applications where these new therapeutic tools could serve as fertility protection agents.
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Affiliation(s)
- Charlotte Klees
- Research Laboratory on Human Reproduction, Faculty of Medicine, Université Libre de Bruxelles (ULB), 1070 Brussels, Belgium; (C.K.); (C.A.); (I.D.)
| | - Chrysanthi Alexandri
- Research Laboratory on Human Reproduction, Faculty of Medicine, Université Libre de Bruxelles (ULB), 1070 Brussels, Belgium; (C.K.); (C.A.); (I.D.)
| | - Isabelle Demeestere
- Research Laboratory on Human Reproduction, Faculty of Medicine, Université Libre de Bruxelles (ULB), 1070 Brussels, Belgium; (C.K.); (C.A.); (I.D.)
- Fertility Clinic, HUB-Erasme Hospital, 1070 Brussels, Belgium
| | - Pascale Lybaert
- Research Laboratory on Human Reproduction, Faculty of Medicine, Université Libre de Bruxelles (ULB), 1070 Brussels, Belgium; (C.K.); (C.A.); (I.D.)
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Jokar J, Abdulabbas HT, Alipanah H, Ghasemian A, Ai J, Rahimian N, Mohammadisoleimani E, Najafipour S. Tissue engineering studies in male infertility disorder. HUM FERTIL 2023; 26:1617-1635. [PMID: 37791451 DOI: 10.1080/14647273.2023.2251678] [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: 06/11/2022] [Accepted: 07/06/2023] [Indexed: 10/05/2023]
Abstract
Infertility is an important issue among couples worldwide which is caused by a variety of complex diseases. Male infertility is a problem in 7% of all men. In vitro spermatogenesis (IVS) is the experimental approach that has been developed for mimicking seminiferous tubules-like functional structures in vitro. Currently, various researchers are interested in finding and developing a microenvironmental condition or a bioartificial testis applied for fertility restoration via gamete production in vitro. The tissue engineering (TE) has developed new approaches to treat male fertility preservation through development of functional male germ cells. This makes TE a possible future strategy for restoration of male fertility. Although 3D culture systems supply the perception of the effect of cellular interactions in the process of spermatogenesis, formation of a native gradient of autocrine/paracrine factors in 3D culture systems have not been considered. These results collectively suggest that maintaining the microenvironment of testicular cells even in the form of a 3D-culture system is crucial in achieving spermatogenesis ex vivo. It is also possible to engineer the testicular structures using biomaterials to provide a supporting scaffold for somatic and stem cells. The insemination of these cells with GFs is possible for temporally and spatially adjusted release to mimic the microenvironment of the in situ seminiferous epithelium. This review focuses on recent studies and advances in the application of TE strategies to cell-tissue culture on synthetic or natural scaffolds supplemented with growth factors.
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Affiliation(s)
- Javad Jokar
- Department of Tissue Engineering, Faculty of Medicine, Fasa University of Medical Science, Fasa, Iran
| | | | - Hiva Alipanah
- Department of Physiology, School of Medicine, Fasa University of Medical Science, Fasa, Iran
| | - Abdolmajid Ghasemian
- Noncommunicable Diseases Research Center, Fasa University of Medical Sciences, Fasa, Iran
| | - Jafar Ai
- Tissue Engineering and Applied Cell Sciences Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Niloofar Rahimian
- Department of Biotechnology, Faculty of Medicine, Fasa University of Medical Sciences, Fasa, Iran
| | - Elham Mohammadisoleimani
- Department of Biotechnology, Faculty of Medicine, Fasa University of Medical Sciences, Fasa, Iran
| | - Sohrab Najafipour
- Department of Microbiology, Faculty of Medicine, Fasa University of Medical Sciences, Fasa, Iran
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8
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Legrand JMD, Hobbs RM. Defining Gene Function in Spermatogonial Stem Cells Through Conditional Knockout Approaches. Methods Mol Biol 2023; 2656:261-307. [PMID: 37249877 DOI: 10.1007/978-1-0716-3139-3_15] [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
Mammalian male fertility is maintained throughout life by a population of self-renewing mitotic germ cells known as spermatogonial stem cells (SSCs). Much of our current understanding regarding the molecular mechanisms underlying SSC activity is derived from studies using conditional knockout mouse models. Here, we provide a guide for the selection and use of mouse strains to develop conditional knockout models for the study of SSCs, as well as their precursors and differentiation-committed progeny. We describe Cre recombinase-expressing strains, breeding strategies to generate experimental groups, and treatment regimens for inducible knockout models and provide advice for verifying and improving conditional knockout efficiency. This resource can be beneficial to those aiming to develop conditional knockout models for the study of SSC development and postnatal function.
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Affiliation(s)
- Julien M D Legrand
- Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, VIC, Australia
- Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia
| | - Robin M Hobbs
- Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, VIC, Australia.
- Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia.
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9
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Munyoki SK, Orwig KE. Perspectives: Methods for Evaluating Primate Spermatogonial Stem Cells. Methods Mol Biol 2023; 2656:341-364. [PMID: 37249880 DOI: 10.1007/978-1-0716-3139-3_18] [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] [Indexed: 05/31/2023]
Abstract
Mammalian spermatogenesis is a complex, highly productive process generating millions of sperm per day. Spermatogonial stem cells (SSCs) are at the foundation of spermatogenesis and can either self-renew, producing more SSCs, or differentiate to initiate spermatogenesis and produce sperm. The biological potential of SSCs to produce and maintain spermatogenesis makes them a promising tool for the treatment of male infertility. However, translating knowledge from rodents to higher primates (monkeys and humans) is challenged by different vocabularies that are used to describe stem cells and spermatogenic lineage development in those species. Furthermore, while rodent SSCs are defined by their biological potential to produce and maintain spermatogenesis in a transplant assay, there is no equivalent routine and accessible bioassay to test monkey and human SSCs or replicate their functions in vitro. This chapter describes progress characterizing, isolating, culturing, and transplanting SSCs in higher primates.
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Affiliation(s)
- Sarah K Munyoki
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Integrative Systems Biology Graduate Program, Magee-Women's Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Kyle E Orwig
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Integrative Systems Biology Graduate Program, Magee-Women's Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
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10
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Huang L, Zhang J, Zhang P, Huang X, Yang W, Liu R, Sun Q, Lu Y, Zhang M, Fu Q. Single-cell RNA sequencing uncovers dynamic roadmap and cell-cell communication during buffalo spermatogenesis. iScience 2022; 26:105733. [PMID: 36582818 PMCID: PMC9793287 DOI: 10.1016/j.isci.2022.105733] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2022] [Revised: 10/24/2022] [Accepted: 12/01/2022] [Indexed: 12/12/2022] Open
Abstract
Spermatogenesis carries the task of precise intergenerational transmission of genetic information from the paternal genome and involves complex developmental processes regulated by the testicular microenvironment. Studies performed mainly in mouse models have established the theoretical basis for spermatogenesis, yet the wide interspecies differences preclude direct translation of the findings, and farm animal studies are progressing slowly. More than 32,000 cells from prepubertal (3-month-old) and pubertal (24-month-old) buffalo testes were analyzed by using single-cell RNA sequencing (scRNA-seq), and dynamic gene expression roadmaps of germ and somatic cell development were generated. In addition to identifying the dynamic processes of sequential cell fate transitions, the global cell-cell communication essential to maintain regular spermatogenesis in the buffalo testicular microenvironment was uncovered. The findings provide the theoretical basis for establishing buffalo germline stem cells in vitro or culturing organoids and facilitating the expansion of superior livestock breeding.
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Affiliation(s)
- Liangfeng Huang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China
| | - Junjun Zhang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China
| | - Pengfei Zhang
- Institute of Medical and Health, Guangxi Academy of Sciences, Nanning 530007, China
| | - Xingchen Huang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China
| | - Weihan Yang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China
| | - Runfeng Liu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China
| | - Qinqiang Sun
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China
| | - Yangqing Lu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China,Corresponding author
| | - Ming Zhang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China,Corresponding author
| | - Qiang Fu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China,Corresponding author
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Abofoul‐Azab M, Lunenfeld E, Kleiman S, Barda S, Hauser R, Huleihel M. Determining the expression levels of CSF-1 and OCT4, CREM-1, and protamine in testicular biopsies of adult Klinefelter patients: Their possible correlation with spermatogenesis. Andrologia 2022; 54:e14558. [PMID: 36177809 PMCID: PMC9786270 DOI: 10.1111/and.14558] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2021] [Revised: 06/24/2022] [Accepted: 08/01/2022] [Indexed: 12/30/2022] Open
Abstract
Klinefelter syndrome (KS) is the most prevalent genetic disorder of infertile males. This study aimed to determine in Klinefelter patients (KS) the expression levels of spermatogenic markers and testicular growth factors that might predict spermatogenesis based on conventional testicular sperm extraction (TESE). The expression levels of the pre-meiotic (OCT4, CD9, GFR-α1, α-6-INTEGRIN, SALL4, C-KIT), meiotic (CREM-1), and post-meiotic (protamine) markers, as well as the colony stimulating factor-1 (CSF-1) were examined in testicular biopsies with and without mature sperm of KS and normal karyotype of azoospermic patients (AZO) with complete spermatogenesis. In the biopsies of AZO, the expression levels (fold of expression compared to the PPI of the same sample) of OCT4 were 9.68± 7.93, CREM 42.78± 28.22, CSF-1 3.07 ± 3.19, and protamine 78498.12 ± 73214.40. Biopsies from KS included 7 with sperm and 17 without sperm. Among the biopsies with sperm, the expression levels of OCT4 were 7.27± 9.29, CREM 3.13± 7.89, CSF-1 35.5 ± 48.01, and protamine 902.97 ± 2365.92. In 14 biopsies without sperm, we found low expression levels of OCT4, CREM and CSF-1, and no expression of protamine. However, in three of the biopsies without sperm that highly expressed OCT4 and CSF-1, the expression levels of CREM-1 and protamine were high. These results may be used for further consulting with patients considering repeating conventional TESE or micro TESE and cryopreservation for possible future in-vitro spermatogenesis.
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Affiliation(s)
- Maram Abofoul‐Azab
- The Shraga Segal Department of Microbiology, Immunology, and GeneticsBen‐Gurion University of the NegevBeer‐ShevaIsrael,The Center of Advanced Research and Education in Reproduction (CARER), Faculty of Health SciencesBen‐Gurion University of the NegevBeer‐ShevaIsrael,Faculty of Health SciencesBen‐Gurion University of the NegevBeer‐ShevaIsrael
| | | | - Sandra Kleiman
- Male Fertility Clinic and Sperm BankLis Maternity HospitalTel AvivIsrael,Sourasky Medical CenterTel‐AvivIsrael,Sackler School of Medicine Tel Aviv UniversityTel AvivIsrael
| | - Shimi Barda
- Male Fertility Clinic and Sperm BankLis Maternity HospitalTel AvivIsrael
| | - Ron Hauser
- Male Fertility Clinic and Sperm BankLis Maternity HospitalTel AvivIsrael,Sourasky Medical CenterTel‐AvivIsrael,Sackler School of Medicine Tel Aviv UniversityTel AvivIsrael
| | - Mahmoud Huleihel
- The Shraga Segal Department of Microbiology, Immunology, and GeneticsBen‐Gurion University of the NegevBeer‐ShevaIsrael,The Center of Advanced Research and Education in Reproduction (CARER), Faculty of Health SciencesBen‐Gurion University of the NegevBeer‐ShevaIsrael,Faculty of Health SciencesBen‐Gurion University of the NegevBeer‐ShevaIsrael
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12
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Sperm DNA methylation alterations from cannabis extract exposure are evident in offspring. Epigenetics Chromatin 2022; 15:33. [PMID: 36085240 PMCID: PMC9463823 DOI: 10.1186/s13072-022-00466-3] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Accepted: 08/26/2022] [Indexed: 11/10/2022] Open
Abstract
Abstract
Background
Cannabis legalization is expanding and men are the predominant users. We have limited knowledge about how cannabis impacts sperm and whether the effects are heritable.
Results
Whole genome bisulfite sequencing (WGBS) data were generated for sperm of rats exposed to: (1) cannabis extract (CE) for 28 days, then 56 days of vehicle only (~ one spermatogenic cycle); (2) vehicle for 56 days, then 28 days of CE; or (3) vehicle only. Males were then mated with drug-naïve females to produce F1 offspring from which heart, brain, and sperm tissues underwent analyses. There were 3321 nominally significant differentially methylated CpGs in F0 sperm identified via WGBS with select methylation changes validated via bisulfite pyrosequencing. Significant methylation changes validated in F0 sperm of the exposed males at the gene 2-Phosphoxylose Phosphatase 1 (Pxylp1) were also detectable in their F1 sperm but not in controls. Changes validated in exposed F0 sperm at Metastasis Suppressor 1-Like Protein (Mtss1l) were also present in F1 hippocampal and nucleus accumbens (NAc) of the exposed group compared to controls. For Mtss1l, a significant sex-specific relationship between DNA methylation and gene expression was demonstrated in the F1 NAc. Phenotypically, rats born to CSE-exposed fathers exhibited significant cardiomegaly relative to those born to control fathers.
Conclusions
This is the first characterization of the effect of cannabis exposure on the entirety of the rat sperm methylome. We identified CE-associated methylation changes across the sperm methylome, some of which persisted despite a “washout” period. Select methylation changes validated via bisulfite pyrosequencing, and genes associated with methylation changes were involved in early developmental processes. Preconception CE exposure is associated with detectable changes in offspring DNA methylation that are functionally related to changes in gene expression and cardiomegaly.
These results support that paternal preconception exposure to cannabis can influence offspring outcomes.
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13
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Youjing granules ameliorate spermatogenesis in rats through regulating the prolifereation of spermatogonial stem cells. Chin J Nat Med 2022; 20:580-588. [DOI: 10.1016/s1875-5364(22)60209-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2022] [Indexed: 11/20/2022]
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14
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Chen X, Zheng Y, Han Y, He H, Lv J, Yu J, Li H, Hou S, Shen C, Zheng B. SAT2 regulates Sertoli cell-germline interactions via STIM1-mediated ROS/WNT/β-catenin signaling pathway. Cell Biol Int 2022; 46:1704-1713. [PMID: 35819096 DOI: 10.1002/cbin.11857] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2022] [Revised: 06/15/2022] [Accepted: 06/30/2022] [Indexed: 01/01/2023]
Abstract
As the main component of seminiferous tubules, Sertoli cells are in close contact with germ cells and generate niche signals, which exhibit pivotal functions in spermatogenesis and male fertility. However, the regulatory mechanisms of Sertoli cell-germline interactions (SGIs) in the testes of neonatal mice (NM) remain largely unclear. Previously, we identified spermidine/spermine N1-acetyl transferase 2 (SAT2) and stromal interaction molecule 1 (STIM1) to be potential regulators of testicular cord formation via comparative proteomics analysis. Here, we demonstrated a novel role of SAT2 for SGIs during testicular development in NM. Testicular explants lacking SAT2 affected the mislocation, but not the quantity, of Sertoli cells, which led to maintenance defects in spermatogonial stem cells (SSCs). Interestingly, SAT2 was essential for the migration of TM4 cells, a Sertoli cell line. Mechanistically, SAT2 was able to bind STIM1, repress its expression, and regulate homeostasis of a reactive oxygen species/wingless type (WNT)/β-catenin pathway in NM testes. Collectively, our study identified that SAT2 was able to regulate SGIs via a STIM1-mediated WNT signaling pathway.
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Affiliation(s)
- Xia Chen
- Department of Obstetrics and Gynecology, Affiliated Hospital 2 of Nantong University and First People's Hospital of Nantong City, Nantong, Jiangsu, China
| | - Yanli Zheng
- Department of Obstetrics and Gynecology, Affiliated Hospital 2 of Nantong University and First People's Hospital of Nantong City, Nantong, Jiangsu, China
| | - Yun Han
- Department of Obstetrics and Gynecology, Affiliated Hospital 2 of Nantong University and First People's Hospital of Nantong City, Nantong, Jiangsu, China
| | - Hui He
- Department of Human Anatomy, School of Medicine, Nantong University, Nantong, China
| | - Jinxing Lv
- Suzhou Dushu Lake Hospital (Dushu Lake Hospital Affiliated to Soochow University), Suzhou, China
| | - Jun Yu
- Institute of Reproductive Medicine, School of Medicine, Nantong University, Nantong, China
| | - Hong Li
- State Key Laboratory of Reproductive Medicine, Center for Reproduction and Genetics, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou, China
| | - Shunyu Hou
- Department of Gynaecology, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou, China
| | - Cong Shen
- State Key Laboratory of Reproductive Medicine, Center for Reproduction and Genetics, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou, China
| | - Bo Zheng
- State Key Laboratory of Reproductive Medicine, Center for Reproduction and Genetics, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou, China
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15
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Bhaskar R, Kumar Gupta M, Soon Han S. Tissue engineering approaches for the in vitro production of spermatids to treat male infertility: A review. Eur Polym J 2022; 174:111318. [DOI: 10.1016/j.eurpolymj.2022.111318] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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16
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Syntaxin binding protein 2 in sertoli cells regulates spermatogonial stem cell maintenance through directly interacting with connexin 43 in the testes of neonatal mice. Mol Biol Rep 2022; 49:7557-7566. [DOI: 10.1007/s11033-022-07564-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Accepted: 05/04/2022] [Indexed: 11/26/2022]
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17
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Singh SP, Kharche SD, Pathak M, Soni YK, Ranjan R, Singh MK, Chauhan MS. Reproductive stage- and season-dependent culture characteristics of enriched caprine male germline stem cells. Cytotechnology 2022; 74:123-140. [PMID: 35185290 PMCID: PMC8816984 DOI: 10.1007/s10616-021-00515-x] [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: 05/19/2021] [Accepted: 12/10/2021] [Indexed: 02/03/2023] Open
Abstract
The present study aims to evaluate season- and reproductive-stage dependent variation in culture characteristics and expression of pluripotency and adhesion markers in caprine-male germline stem cells (cmGSCs). For this, testes from pre-pubertal (4-6 months) and adult (~ 2 years) bucks during non-breeding (July-August; n = 4 each) and breeding (October-November; n = 4 each) seasons were used to isolated testicular cells by two-step enzymatic digestion. After cmGSCs enrichment by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), cell viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer up to 3rd-passage (P-3). The culture characteristics of cmGSCs were compared during primary culture (P-0) and P-3 with different assays [BrdU-assay (proliferation), MTT-assay (senescence), and Cluster-forming activity-assay] and transcript expression analyses by qRT-PCR. Moreover, the co-localization of UCHL-1, CD90, and DBA was examined by a double-immunofluorescence method. In adult bucks, significantly (p < 0.05) higher cell numbers with the ability to proliferate faster and form a greater number of cell clusters, besides up-regulation of pluripotency and adhesion markers expression were observed during the breeding season than the non-breeding season. In contrast, such season-dependent variation was lacking in pre-pubertal bucks. The expression of transcripts during non-breeding seasons was significantly (p < 0.05) higher in pre-pubertal cmGSCs than in adult cells (UCHL-1 = 2.38-folds; CD-90 = 6.66-folds; PLZF = 20.87-folds; ID-4 = 4.75-folds; E-cadherin = 3.89-folds and β1-integrin = 5.70-folds). Overall, the reproductive stage and season affect the population, culture characteristics, and expression of pluripotency and adhesion specific markers in buck testis. These results provide an insight to develop an efficient system for successful cell culture processes targeting cmGSCs. SUPPLEMENTARY INFORMATION The online version contains supplementary material available at 10.1007/s10616-021-00515-x.
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Affiliation(s)
- Shiva Pratap Singh
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281122 India
| | - Suresh Dinkar Kharche
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281122 India
| | - Manisha Pathak
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281122 India
| | - Yogesh Kumar Soni
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281122 India
| | - Ravi Ranjan
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281122 India
| | - Manoj Kumar Singh
- Animal Genetics and Breeding Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281122 India
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18
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Liu R, Liu Z, Guo M, Zeng W, Zheng Y. SETDB1 Regulates Porcine Spermatogonial Adhesion and Proliferation through Modulating MMP3/10 Transcription. Cells 2022; 11:cells11030370. [PMID: 35159180 PMCID: PMC8834347 DOI: 10.3390/cells11030370] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2021] [Revised: 12/30/2021] [Accepted: 01/20/2022] [Indexed: 12/16/2022] Open
Abstract
The transition from gonocytes into spermatogonia takes place during the homing process. A subpopulation of undifferentiated spermatogonia in niche then shifts to spermatogonial stem cells (SSCs), accompanied by the self-renewal ability to maintain life-long fertility in males. Enormous changes in cell morphology, gene expression, and epigenetic features have been reported during spermatogenesis. However, little is known about the difference of these features in SSCs during aging. Here, we examined the dynamics of SET domain bifurcated 1 (SETDB1) expression in porcine testes. SETDB1 was expressed in postnatal undifferentiated spermatogonia, while gradually disappeared after being packed within the basal compartment of seminiferous tubules. In addition, the cell-adhesion ability, proliferative activity, and trimethylation of the histone H3 lysine 9 (H3K9me3) level were significantly altered in SETDB1-deficient porcine SSCs. Moreover, the matrix metalloproteinases 3/10 (MMP3/10) was upregulated at both mRNA and protein levels. These results illustrate the significance of SETDB1 in modulating early male germ cell development.
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19
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Jensen CFS, Wang D, Mamsen LS, Giwercman A, Jørgensen N, Fode M, Ohl D, Dong L, Hildorf SE, Pors SE, Fedder J, Ntemou E, Andersen CY, Sønksen J. Sertoli and Germ Cells Within Atrophic Seminiferous Tubules of Men With Non-Obstructive Azoospermia. Front Endocrinol (Lausanne) 2022; 13:825904. [PMID: 35721721 PMCID: PMC9201000 DOI: 10.3389/fendo.2022.825904] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Accepted: 04/06/2022] [Indexed: 11/13/2022] Open
Abstract
BACKGROUND Infertile men with non-obstructive azoospermia (NOA) have impaired spermatogenesis. Dilated and un-dilated atrophic seminiferous tubules are often present in the testes of these patients, with the highest likelihood of active spermatogenesis in the dilated tubules. Little is known about the un-dilated tubules, which in NOA patients constitute the majority. To advance therapeutic strategies for men with NOA who fail surgical sperm retrieval we aimed to characterize the spermatogonial stem cell microenvironment in atrophic un-dilated tubules. METHODS Testis biopsies approximately 3x3x3 mm3 were obtained from un-dilated areas from 34 patients. They were classified as hypospermatogenesis (HS) (n=5), maturation arrest (MA) (n=14), and Sertoli cell only (SCO) (n= 15). Testis samples from five fertile men were included as controls. Biopsies were used for histological analysis, RT-PCR analysis and immunofluorescence of germ and Sertoli cell markers. RESULTS Anti-Müllerian hormone mRNA and protein expression was increased in un-dilated tubules in all three NOA subtypes, compared to the control, showing an immature state of Sertoli cells (p<0.05). The GDNF mRNA expression was significantly increased in MA (P=0.0003). The BMP4 mRNA expression showed a significant increase in HS, MA, and SCO (P=0.02, P=0.0005, P=0.02, respectively). The thickness of the tubule wall was increased 2.2-fold in the SCO-NOA compared to the control (p<0.05). In germ cells, we found the DEAD-box helicase 4 (DDX4) and melanoma-associated antigen A4 (MAGE-A4) mRNA and protein expression reduced in NOA (MAGE-A: 46% decrease in HS, 53% decrease in MA, absent in SCO). In HS-NOA, the number of androgen receptor positive Sertoli cells was reduced 30% with a similar pattern in mRNA expression. The γH2AX expression was increased in SCO as compared to HS and MA. However, none of these differences reached statistical significance probably due to low number of samples. CONCLUSIONS Sertoli cells were shown to be immature in un-dilated tubules of three NOA subtypes. The increased DNA damage in Sertoli cells and thicker tubule wall in SCO suggested a different mechanism for the absence of spermatogenesis from SCO to HS and MA. These results expand insight into the differences in un-dilated tubules from the different types of NOA patients.
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Affiliation(s)
- Christian Fuglesang Skjødt Jensen
- Department of Urology, Herlev and Gentofte Hospital, Herlev, Denmark
- Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
| | - Danyang Wang
- Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Linn Salto Mamsen
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Aleksander Giwercman
- Department of Translational Medicine and Reproductive Medicine Centre, Lunds University and Skane University Hospital, Malmö, Sweden
| | - Niels Jørgensen
- Department of Growth and Reproduction, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Mikkel Fode
- Department of Urology, Herlev and Gentofte Hospital, Herlev, Denmark
- Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
| | - Dana Ohl
- Department of Urology, University of Michigan, Ann Arbor, MI, United States
| | - Lihua Dong
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Simone Engmann Hildorf
- Centre of Andrology & Fertility Clinic, Department D, Odense University Hospital, Odense C, Denmark
| | - Susanne Elisabeth Pors
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Jens Fedder
- Centre of Andrology & Fertility Clinic, Department D, Odense University Hospital, Odense C, Denmark
| | - Elissavet Ntemou
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
- *Correspondence: Elissavet Ntemou,
| | - Claus Yding Andersen
- Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Jens Sønksen
- Department of Urology, Herlev and Gentofte Hospital, Herlev, Denmark
- Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
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20
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Schrott R, Murphy SK, Modliszewski JL, King DE, Hill B, Itchon-Ramos N, Raburn D, Price T, Levin ED, Vandrey R, Corcoran DL, Kollins SH, Mitchell JT. Refraining from use diminishes cannabis-associated epigenetic changes in human sperm. ENVIRONMENTAL EPIGENETICS 2021; 7:dvab009. [PMID: 34557312 PMCID: PMC8455898 DOI: 10.1093/eep/dvab009] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/12/2021] [Revised: 08/04/2021] [Accepted: 08/19/2021] [Indexed: 05/20/2023]
Abstract
Cannabis use alters sperm DNA methylation, but the potential reversibility of these changes is unknown. Semen samples from cannabis users and non-user controls were collected at baseline and again following a 77-day period of cannabis abstinence (one spermatogenic cycle). Users and controls did not significantly differ by demographics or semen analyses. Whole-genome bisulfite sequencing identified 163 CpG sites with significantly different DNA methylation in sperm between groups (P < 2.94 × 10-9). Genes associated with altered CpG sites were enriched with those involved in development, including cardiogenesis and neurodevelopment. Many of the differences in sperm DNA methylation between groups were diminished after cannabis abstinence. These results indicate that sustained cannabis abstinence significantly reduces the number of sperm showing cannabis-associated alterations at genes important for early development.
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Affiliation(s)
- Rose Schrott
- Duke University Program in Environmental Health, Nicholas School of the Environment, Duke University, 9 Circuit Drive, Durham, NC, USA
| | - Susan K Murphy
- Duke University Program in Environmental Health, Nicholas School of the Environment, Duke University, 9 Circuit Drive, Durham, NC, USA
- Department of Obstetrics and Gynecology, Division of Reproductive Sciences, Duke University Medical Center, 701 W. Main Street, Durham, NC, USA
| | - Jennifer L Modliszewski
- Duke Center for Genomic and Computational Biology, Duke University Medical Center, 101 Science Drive, Durham, NC, USA
| | - Dillon E King
- Duke University Program in Environmental Health, Nicholas School of the Environment, Duke University, 9 Circuit Drive, Durham, NC, USA
| | - Bendu Hill
- Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, 2608 Erwin Road, Durham, NC, USA
| | - Nilda Itchon-Ramos
- Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, 2608 Erwin Road, Durham, NC, USA
| | - Douglas Raburn
- Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Duke University Medical Center, 5704 Fayetteville Road, Durham, NC, USA
| | - Thomas Price
- Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Duke University Medical Center, 5704 Fayetteville Road, Durham, NC, USA
| | - Edward D Levin
- Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, 2608 Erwin Road, Durham, NC, USA
| | - Ryan Vandrey
- Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 5510 Nathan Shock Drive, Baltimore, MD, USA
| | - David L Corcoran
- Duke Center for Genomic and Computational Biology, Duke University Medical Center, 101 Science Drive, Durham, NC, USA
| | - Scott H Kollins
- Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, 2608 Erwin Road, Durham, NC, USA
| | - John T Mitchell
- Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, 2608 Erwin Road, Durham, NC, USA
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21
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Cellular Therapy via Spermatogonial Stem Cells for Treating Impaired Spermatogenesis, Non-Obstructive Azoospermia. Cells 2021; 10:cells10071779. [PMID: 34359947 PMCID: PMC8304133 DOI: 10.3390/cells10071779] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Revised: 07/04/2021] [Accepted: 07/12/2021] [Indexed: 12/18/2022] Open
Abstract
Male infertility is a major health problem affecting about 8–12% of couples worldwide. Spermatogenesis starts in the early fetus and completes after puberty, passing through different stages. Male infertility can result from primary or congenital, acquired, or idiopathic causes. The absence of sperm in semen, or azoospermia, results from non-obstructive causes (pretesticular and testicular), and post-testicular obstructive causes. Several medications such as antihypertensive drugs, antidepressants, chemotherapy, and radiotherapy could lead to impaired spermatogenesis and lead to a non-obstructive azoospermia. Spermatogonial stem cells (SSCs) are the basis for spermatogenesis and fertility in men. SSCs are characterized by their capacity to maintain the self-renewal process and differentiation into spermatozoa throughout the male reproductive life and transmit genetic information to the next generation. SSCs originate from gonocytes in the postnatal testis, which originate from long-lived primordial germ cells during embryonic development. The treatment of infertility in males has a poor prognosis. However, SSCs are viewed as a promising alternative for the regeneration of the impaired or damaged spermatogenesis. SSC transplantation is a promising technique for male infertility treatment and restoration of spermatogenesis in the case of degenerative diseases such as cancer, radiotherapy, and chemotherapy. The process involves isolation of SSCs and cryopreservation from a testicular biopsy before starting cancer treatment, followed by intra-testicular stem cell transplantation. In general, treatment for male infertility, even with SSC transplantation, still has several obstacles. The efficiency of cryopreservation, exclusion of malignant cells contamination in cancer patients, and socio-cultural attitudes remain major challenges to the wider application of SSCs as alternatives. Furthermore, there are limitations in experience and knowledge regarding cryopreservation of SSCs. However, the level of infrastructure or availability of regulatory approval to process and preserve testicular tissue makes them tangible and accurate therapy options for male infertility caused by non-obstructive azoospermia, though in their infancy, at least to date.
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22
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Tao K, Sun Y, Chao Y, Xing L, Leng L, Zhou D, Zhu W, Fan L. β-estradiol promotes the growth of primary human fetal spermatogonial stem cells via the induction of stem cell factor in Sertoli cells. J Assist Reprod Genet 2021; 38:2481-2490. [PMID: 34050447 DOI: 10.1007/s10815-021-02240-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2021] [Accepted: 05/17/2021] [Indexed: 11/29/2022] Open
Abstract
BACKGROUND Mammalian spermatogenesis is responsible for male fertility and is supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs). Sertoli cells provide a supportive microenvironment for SSCs, in part by the production of stem cell factor (SCF), which is a potent regulator of spermatogonia proliferation and survival. METHODS We investigated the novel role of β-estradiol in modulating the proliferation and apoptosis of fetal SSCs via the regulation of SCF secretion in Sertoli cells isolated from human fetal testes. The proliferation of SSCs in the co-culture system was determined by colony formation and BrdU incorporation assays. TUNEL assay was used to measure SSC apoptosis in co-culture in response to treatment with control, β-estradiol, or the combination of β-estradiol and the estrogen receptor inhibitor ICI 182780. RESULTS In the system with purified human fetal Sertoli cells (MIS+/c-Kit-/AP-), β-estradiol upregulated the production of SCF in a dose- and time-dependent manner. In the co-culture system of primary human fetal SSCs (c-Kit+/SSEA-4+/Oct-4+/AP+) and Sertoli cells (MIS+), β-estradiol markedly increased the proliferation of SSCs. Moreover, SSC apoptosis was significantly inhibited by β-estradiol and was completely reversed by the combination of β-estradiol and ICI 182780. CONCLUSION Here we report, for the first time, that β-estradiol can induce the increase of SCF expression in human fetal Sertoli cells and regulates the growth and survival of human fetal SSCs. These novel findings provide new perspectives on the current understanding of the role of estrogen in human spermatogenesis.
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Affiliation(s)
- Ke Tao
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, 410078, China.,Department of Medical Laboratory, School of Medicine, Hunan Normal University, Changsha, 410013, China
| | - Yuan Sun
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, 410078, China
| | - Yuanchi Chao
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, 410078, China
| | - Liu Xing
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, 410078, China.,Reproductive & Genetic Hospital of CITIC-Xiangya, Changsha, 410078, China
| | - Lizhi Leng
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, 410078, China.,Reproductive & Genetic Hospital of CITIC-Xiangya, Changsha, 410078, China
| | - Dai Zhou
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, 410078, China.,Reproductive & Genetic Hospital of CITIC-Xiangya, Changsha, 410078, China
| | - Wenbing Zhu
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, 410078, China.,Reproductive & Genetic Hospital of CITIC-Xiangya, Changsha, 410078, China
| | - Liqing Fan
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, 410078, China. .,Reproductive & Genetic Hospital of CITIC-Xiangya, Changsha, 410078, China.
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23
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Bu T, Wang L, Wu X, Li L, Mao B, Wong CKC, Perrotta A, Silvestrini B, Sun F, Cheng CY. A laminin-based local regulatory network in the testis that supports spermatogenesis. Semin Cell Dev Biol 2021; 121:40-52. [PMID: 33879391 DOI: 10.1016/j.semcdb.2021.03.025] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2020] [Revised: 03/24/2021] [Accepted: 03/29/2021] [Indexed: 12/13/2022]
Abstract
In adult rat testes, the basement membrane is structurally constituted by laminin and collagen chains that lay adjacent to the blood-testis barrier (BTB). It plays a crucial scaffolding role to support spermatogenesis. On the other hand, laminin-333 comprised of laminin-α3/ß3/γ3 at the apical ES (ectoplasmic specialization, a testis-specific cell-cell adherens junction at the Sertoli cell-step 8-19 spermatid interface) expressed by spermatids serves as a unique cell adhesion protein that forms an adhesion complex with α6ß1-integrin expressed by Sertoli cells to support spermiogenesis. Emerging evidence has shown that biologically active fragments are derived from basement membrane and apical ES laminin chains through proteolytic cleavage mediated by matrix metalloproteinase 9 (MMP9) and MMP2, respectively. Two of these laminin bioactive fragments: one from the basement membrane laminin-α2 chain called LG3/4/5-peptide, and one from the apical ES laminin-γ3 chain known as F5-peptide, are potent regulators that modify cell adhesion function at the Sertoli-spermatid interface (i.e., apical ES) but also at the Sertoli cell-cell interface designated basal ES at the blood-testis barrier (BTB) with contrasting effects. These findings not only highlight the physiological significance of these bioactive peptides that create a local regulatory network to support spermatogenesis, they also open a unique area of research. For instance, it is likely that several other bioactive peptides remain to be identified. These bioactive peptides including their downstream signaling proteins and cascades should be studied collectively in future investigations to elucidate the underlying mechanism(s) by which they coordinate with each other to maintain spermatogenesis. This is the goal of this review.
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Affiliation(s)
- Tiao Bu
- The Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China; Institute of Reproductive Medicine, Nantong University School of Medicine, Nantong, Jiangsu 226001, China
| | - Lingling Wang
- The Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China; Institute of Reproductive Medicine, Nantong University School of Medicine, Nantong, Jiangsu 226001, China
| | - Xiaolong Wu
- Institute of Reproductive Medicine, Nantong University School of Medicine, Nantong, Jiangsu 226001, China
| | - Linxi Li
- The Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China
| | - Baiping Mao
- The Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China
| | - Chris K C Wong
- Department of Biology, Croucher Institute for Environmental Sciences, Hong Kong Baptist University, Kowloon, Hong Kong, China
| | - Adolfo Perrotta
- Department of Translational & Precision Medicine, La Sapienza University of Rome, 00185 Rome, Italy
| | | | - Fei Sun
- Institute of Reproductive Medicine, Nantong University School of Medicine, Nantong, Jiangsu 226001, China
| | - C Yan Cheng
- The Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China; Institute of Reproductive Medicine, Nantong University School of Medicine, Nantong, Jiangsu 226001, China.
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24
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Sênos Demarco R, Jones DL. Redox signaling as a modulator of germline stem cell behavior: Implications for regenerative medicine. Free Radic Biol Med 2021; 166:67-72. [PMID: 33592309 PMCID: PMC8021480 DOI: 10.1016/j.freeradbiomed.2021.02.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2020] [Revised: 01/30/2021] [Accepted: 02/01/2021] [Indexed: 10/22/2022]
Abstract
Germline stem cells (GSCs) are crucial for the generation of gametes and propagation of the species. Both intrinsic signaling pathways and environmental cues are employed in order to tightly control GSC behavior, including mitotic divisions, the choice between self-renewal or onset of differentiation, and survival. Recently, oxidation-reduction (redox) signaling has emerged as an important regulator of GSC and gamete behavior across species. In this review, we will highlight the primary mechanisms through which redox signaling acts to influence GSC behavior in different model organisms (Caenorhabditis elegans, Drosophila melanogaster and Mus musculus). In addition, we will summarize the latest research on the use of antioxidants to support mammalian spermatogenesis and discuss potential strategies for regenerative medicine in humans to enhance reproductive fitness.
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Affiliation(s)
- Rafael Sênos Demarco
- Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, USA
| | - D Leanne Jones
- Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, USA; Molecular Biology Institute, University of California, Los Angeles, CA, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA, USA.
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25
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Li Q, Li H, Liang J, Mei J, Cao Z, Zhang L, Luo J, Tang Y, Huang R, Xia H, Zhang Q, Xiang Q, Yang Y, Huang Y. Sertoli cell-derived exosomal MicroRNA-486-5p regulates differentiation of spermatogonial stem cell through PTEN in mice. J Cell Mol Med 2021; 25:3950-3962. [PMID: 33608983 PMCID: PMC8051706 DOI: 10.1111/jcmm.16347] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Revised: 11/24/2020] [Accepted: 01/12/2021] [Indexed: 12/14/2022] Open
Abstract
Self‐renewal and differentiation of spermatogonial stem cell (SSC) are critical for male fertility and reproduction, both of which are highly regulated by testicular microenvironment. Exosomal miRNAs have emerged as new components in intercellular communication. However, their roles in the differentiation of SSC remain unclear. Here, we observed miR‐486‐5p enriched in Sertoli cell and Sertoli cell‐derived exosomes. The exosomes mediate the transfer of miR‐486‐5p from Sertoli cells to SSCs. Exosomes release miR‐486‐5p, thus up‐regulate expression of Stra8 (stimulated by retinoic acid 8) and promote differentiation of SSC. And PTEN was identified as a target of miR‐486‐5p. Overexpression of miR‐486‐5p in SSCs down‐regulates PTEN expression, which up‐regulates the expression of STRA8 and SYCP3, promotes SSCs differentiation. In addition, blocking the exosome‐mediated transfer of miR‐486‐5p inhibits differentiation of SSC. Our findings demonstrate that miR‐486‐5p acts as a communication molecule between Sertoli cells and SSCs in modulating differentiation of SSCs. This provides a new insight on molecular mechanisms that regulates SSC differentiation and a basis for the diagnosis, treatment, and prevention of male infertility.
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Affiliation(s)
- Quan Li
- Department of Cell Biology & Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, China
| | - Hanhao Li
- Department of Pharmacology, Jinan University, Guangzhou, China
| | - Jinlian Liang
- Department of Cell Biology & Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, China
| | - Jiaxin Mei
- Department of Cell Biology & Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, China
| | - Zhen Cao
- Department of Cell Biology & Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, China
| | - Lei Zhang
- Guangdong Provincial Institute of Biological Products and Materia Medica, Guangzhou, China
| | - Jiao Luo
- Institute for Translational Medicine, Shenzhen Second People's Hospital / The First Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen, China
| | - Yan Tang
- Department of Cell Biology & Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, China
| | - Rufei Huang
- Department of Pharmacology, Jinan University, Guangzhou, China
| | - Huan Xia
- Department of Cell Biology & Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, China
| | - Qihao Zhang
- Department of Cell Biology & Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, China.,Guangdong Provincial Biotechnology Drug & Engineering Technology Research Center, Guangzhou, China
| | - Qi Xiang
- Department of Cell Biology & Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, China.,Biopharmaceutical Research & Development Center of Jinan University, Guangzhou, China
| | - Yan Yang
- Department of Cell Biology & Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, China.,Guangdong Provincial Biotechnology Drug & Engineering Technology Research Center, Guangzhou, China
| | - Yadong Huang
- Department of Cell Biology & Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, China.,Department of Pharmacology, Jinan University, Guangzhou, China.,Guangdong Provincial Biotechnology Drug & Engineering Technology Research Center, Guangzhou, China
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26
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Kurek M, Åkesson E, Yoshihara M, Oliver E, Cui Y, Becker M, Alves-Lopes JP, Bjarnason R, Romerius P, Sundin M, Norén Nyström U, Langenskiöld C, Vogt H, Henningsohn L, Petersen C, Söder O, Guo J, Mitchell RT, Jahnukainen K, Stukenborg JB. Spermatogonia Loss Correlates with LAMA 1 Expression in Human Prepubertal Testes Stored for Fertility Preservation. Cells 2021; 10:241. [PMID: 33513766 PMCID: PMC7911157 DOI: 10.3390/cells10020241] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Revised: 12/23/2020] [Accepted: 01/21/2021] [Indexed: 02/07/2023] Open
Abstract
Fertility preservation for male childhood cancer survivors not yet capable of producing mature spermatozoa, relies on experimental approaches such as testicular explant culture. Although the first steps in somatic maturation can be observed in human testicular explant cultures, germ cell depletion is a common obstacle. Hence, understanding the spermatogonial stem cell (SSC) niche environment and in particular, specific components such as the seminiferous basement membrane (BM) will allow progression of testicular explant cultures. Here, we revealed that the seminiferous BM is established from 6 weeks post conception with the expression of laminin alpha 1 (LAMA 1) and type IV collagen, which persist as key components throughout development. With prepubertal testicular explant culture we found that seminiferous LAMA 1 expression is disrupted and depleted with culture time correlating with germ cell loss. These findings highlight the importance of LAMA 1 for the human SSC niche and its sensitivity to culture conditions.
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Affiliation(s)
- Magdalena Kurek
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, and Karolinska University Hospital, 171 64 Solna, Sweden; (E.O.); (Y.C.); (J.P.A.-L.); (C.P.); (O.S.); (K.J.)
| | - Elisabet Åkesson
- Division of Neurogeriatrics, Department of Neurobiology Care Sciences & Society, Karolinska Institutet, 141 83 Huddinge, Sweden;
- The R & D Unit, Stockholms Sjukhem, 112 19 Stockholm, Sweden
| | - Masahito Yoshihara
- Department of Biosciences and Nutrition, Karolinska Institutet, 141 83 Huddinge, Sweden;
| | - Elizabeth Oliver
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, and Karolinska University Hospital, 171 64 Solna, Sweden; (E.O.); (Y.C.); (J.P.A.-L.); (C.P.); (O.S.); (K.J.)
| | - Yanhua Cui
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, and Karolinska University Hospital, 171 64 Solna, Sweden; (E.O.); (Y.C.); (J.P.A.-L.); (C.P.); (O.S.); (K.J.)
| | - Martin Becker
- Center of Neurodevelopmental Disorders (KIND), Department of Women’s and Children’s Health, Karolinska Institutet, Centre for Psychiatry Research, Region Stockholm and Astrid Lindgren Children’s Hospital, Karolinska University Hospital, 171 64 Solna, Sweden;
| | - João Pedro Alves-Lopes
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, and Karolinska University Hospital, 171 64 Solna, Sweden; (E.O.); (Y.C.); (J.P.A.-L.); (C.P.); (O.S.); (K.J.)
| | - Ragnar Bjarnason
- Children’s Medical Center, Landspítali University Hospital, 101 Reykjavik, Iceland;
- Department of Paediatrics Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland
| | - Patrik Romerius
- Department of Paediatric Oncology and Haematology, Clinical Sciences, Lund University, Barn-och Ungdomssjukhuset Lund, Skånes Universitetssjukhus, 221 85 Lund, Sweden;
| | - Mikael Sundin
- Division of Paediatrics, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, 141 52 Huddinge, Sweden;
- Pediatric Blood Disorders, Immunodeficiency and Stem Cell Transplantation Unit, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, 141 86 Huddinge, Sweden
| | - Ulrika Norén Nyström
- Division of Paediatrics, Department of Clinical Science, Umeå University, 901 87 Umeå, Sweden;
| | - Cecilia Langenskiöld
- Department of Paediatric Oncology, The Queen Silvia Children’s Hospital, 416 50 Gothenburg, Sweden;
| | - Hartmut Vogt
- Crown Princess Victoria’s Child and Youth Hospital, and Department of Biomedical and Clinical Sciences, Linköping University, 581 83 Linköping, Sweden;
| | - Lars Henningsohn
- Division of Urology, Institution for Clinical Science Intervention and Technology, Karolinska Institutet, 141 52 Huddinge, Sweden;
| | - Cecilia Petersen
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, and Karolinska University Hospital, 171 64 Solna, Sweden; (E.O.); (Y.C.); (J.P.A.-L.); (C.P.); (O.S.); (K.J.)
| | - Olle Söder
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, and Karolinska University Hospital, 171 64 Solna, Sweden; (E.O.); (Y.C.); (J.P.A.-L.); (C.P.); (O.S.); (K.J.)
| | - Jingtao Guo
- Division of Urology, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT 84112, USA;
| | - Rod T. Mitchell
- MRC Centre for Reproductive Health, The Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh EH16 4TJ, UK;
- Edinburgh Royal Hospital for Sick Children, Edinburgh EH9 1LF, UK
| | - Kirsi Jahnukainen
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, and Karolinska University Hospital, 171 64 Solna, Sweden; (E.O.); (Y.C.); (J.P.A.-L.); (C.P.); (O.S.); (K.J.)
- Division of Haematology-Oncology and Stem Cell Transplantation, Children’s Hospital, University of Helsinki, Helsinki University Central Hospital, 00029 Helsinki, Finland
| | - Jan-Bernd Stukenborg
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, and Karolinska University Hospital, 171 64 Solna, Sweden; (E.O.); (Y.C.); (J.P.A.-L.); (C.P.); (O.S.); (K.J.)
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27
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Khanehzad M, Abbaszadeh R, Holakuyee M, Modarressi MH, Nourashrafeddin SM. FSH regulates RA signaling to commit spermatogonia into differentiation pathway and meiosis. Reprod Biol Endocrinol 2021; 19:4. [PMID: 33407539 PMCID: PMC7789255 DOI: 10.1186/s12958-020-00686-w] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Accepted: 12/17/2020] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND Spermatogenesis is a complex process that is controlled by interactions between germ cells and somatic cells. The commitment of undifferentiated spermatogonia to differentiating spermatogonia and normal spermatogenesis requires the action of gonadotropins. Additionally, numerous studies revealed the role of retinoic acid signaling in induction of germ cell differentiation and meiosis entry. MAIN TEXT Recent studies have shown that expression of several RA signaling molecules including Rdh10, Aldh1a2, Crabp1/2 are influenced by changes in gonadotropin levels. Components of signaling pathways that are regulated by FSH signaling such as GDNF, Sohlh1/2, c-Kit, DMRT, BMP4 and NRGs along with transcription factors that are important for proliferation and differentiation of spermatogonia are also affected by retinoic acid signaling. CONCLUSION According to all studies that demonstrate the interface between FSH and RA signaling, we suggest that RA may trigger spermatogonia differentiation and initiation of meiosis through regulation by FSH signaling in testis. Therefore, to the best of our knowledge, this is the first time that the correlation between FSH and RA signaling in spermatogenesis is highlighted.
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Affiliation(s)
- Maryam Khanehzad
- Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Roya Abbaszadeh
- Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | | | | | - Seyed Mehdi Nourashrafeddin
- Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pittsburgh, USA.
- School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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28
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Qin W, Wang B, Yang L, Yuan Y, Xiong X, Li J, Yin S. Molecular cloning, characterization, and function analysis of the AMH gene in Yak (Bos grunniens) Sertoli cells. Theriogenology 2021; 163:1-9. [PMID: 33476894 DOI: 10.1016/j.theriogenology.2021.01.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2020] [Revised: 01/03/2021] [Accepted: 01/04/2021] [Indexed: 10/22/2022]
Abstract
Sertoli cells (SCs) are important testicular somatic cells that carry out various functions in spermatogenesis. Understanding the biological mechanisms underlying SC development may facilitate the understanding of animal reproduction. Anti-Mullerian hormone (AMH) is a dimeric glycoprotein produced by SCs and plays essential roles in spermatogenesis. In this study, we cloned the coding sequence of the yak AMH, predicated the structure of AMH protein, analyzed AMH expression in the testis at different stages, and studied the functions of AMH in yak SCs. The open reading frame (ORF) of the yak AMH contained 1728 bp and encoded 575 amino acids. Structural analysis revealed that the yak AMH protein had a highly conserved transforming growth factor-β (TGF-β) domain. The mRNA expression level for the AMH gene in yak testis increased significantly from the fetal stage to calf stage, then decreased with the increase of age. The highest expression was found in calf stage. Cell proliferation was depressed in AMH-deficient SCs. Expression of several genes involved in SC proliferation and development, including PCNA, BCL-2, BAX, CASP3, AR and AMHR2 were altered after knockdown of AMH. Also, three SC-secreted factors essential for spermatogenesis, SCF, GDNF and ABP, were repressed at the transcription level after AMH knockdown in yak SCs. Moreover, supplementation with exogenous AMH protein partially rescued SC proliferation, and the expression of PCNA, BCL-2, AR and AMHR2 after AMH gene interference. This research provided theoretical basis for understanding the mechanism by which AMH regulates yak spermatogenesis and might give new insights in improving yak reproductive performance in the future.
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Affiliation(s)
- Wenchang Qin
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education, Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Southwest Minzu University, Chengdu, Sichuan, 610041, China; College of Animal and Veterinary Sciences, Southwest Minzu University, Chengdu, Sichuan, 610041, China
| | - Bin Wang
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education, Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Southwest Minzu University, Chengdu, Sichuan, 610041, China; College of Animal and Veterinary Sciences, Southwest Minzu University, Chengdu, Sichuan, 610041, China
| | - Liuqing Yang
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education, Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Southwest Minzu University, Chengdu, Sichuan, 610041, China; College of Animal and Veterinary Sciences, Southwest Minzu University, Chengdu, Sichuan, 610041, China
| | - YuJie Yuan
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education, Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Southwest Minzu University, Chengdu, Sichuan, 610041, China; College of Animal and Veterinary Sciences, Southwest Minzu University, Chengdu, Sichuan, 610041, China
| | - Xianrong Xiong
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education, Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Southwest Minzu University, Chengdu, Sichuan, 610041, China; College of Animal and Veterinary Sciences, Southwest Minzu University, Chengdu, Sichuan, 610041, China
| | - Jian Li
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education, Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Southwest Minzu University, Chengdu, Sichuan, 610041, China; College of Animal and Veterinary Sciences, Southwest Minzu University, Chengdu, Sichuan, 610041, China
| | - Shi Yin
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education, Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Southwest Minzu University, Chengdu, Sichuan, 610041, China; College of Animal and Veterinary Sciences, Southwest Minzu University, Chengdu, Sichuan, 610041, China; Key Laboratory of Modem Technology (Southwest Minzu University), State Ethnic Affairs Commission, Chengdu, Sichuan, 610041, China.
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29
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Medrano JV, Acimovic I, Navarro-Gomezlechon A, Noguera I, Pellicer A, Andrés MM, Novella-Maestre E. Timing of spermatogonial stem cell transplantation affects the spermatogenic recovery outcome in mice. In Vitro Cell Dev Biol Anim 2021; 57:21-29. [PMID: 33420579 DOI: 10.1007/s11626-020-00531-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2020] [Accepted: 11/12/2020] [Indexed: 11/29/2022]
Abstract
Spermatogonial stem cell transplantation (SSCT) is a strategy that has demonstrated to be feasible to restore spermatogenesis in animal models when it is performed shortly after the gonadotoxic onset to destroy their endogenous germ cells. However, in the case of boys subjected to fertility preservation, future transplantations will be performed with a delay of many years. In order to study how timing of SSCT affects donor-derived spermatogenic recovery in mice, we compared the percentage of spermatogenic tubule cross-sections within testes of 59 C57BL/6NCrl mice distributed in 6 groups: group 1, untreated mice controls (n = 9); group 2, mice that received a single dose of busulfan 40 mg/kg (n = 10); group 3, mice that received two additional doses of busulfan 10 mg/kg every 5 weeks (n = 10); group 4 (SSCT-A), mice subjected to a standard SSCT performed 5 weeks after a single injection of busulfan 40 mg/kg (n = 10); group 5 (SSCT-B), mice subjected to a delayed SSCT performed 15 weeks after a single injection of busulfan 40 mg/kg (n = 10); and group 6 (SSCT-C), mice subjected to a delayed SSCT with two additional doses of busulfan 10 mg/kg every 5 weeks (n = 10). Spermatogenic recovery in standard SSCT-A and SSCT-C groups ranged between 22.29 and 22.65%, compared with a lower recovery rate of 11.54% showed in the SSCT-B group. However, donor contribution resulted higher in standard SSCT-A, representing a 69.71% of cross-sections, compared with the rest of conditions ranging from 34.69 to 35.42%. Overall, we concluded that a delay in the SSCT from the gonadotoxic onset decreases the efficiency of donor-derived spermatogenic recovery in mice.
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Affiliation(s)
- J V Medrano
- Unidad de Medicina Reproductiva, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Av. Fernando Abril Martorell, 106. Torre A, Lab. 6.22, 46026, Valencia, Spain.
| | - I Acimovic
- Department of Biology, Faculty of Medicine, Masaryk University, 62500, Brno, Czech Republic
| | - A Navarro-Gomezlechon
- Unidad de Medicina Reproductiva, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Av. Fernando Abril Martorell, 106. Torre A, Lab. 6.22, 46026, Valencia, Spain
| | - I Noguera
- Animal Facility, Faculty of Pharmacy, Valencia University, 46015, Valencia, Spain
| | - A Pellicer
- Unidad de Medicina Reproductiva, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Av. Fernando Abril Martorell, 106. Torre A, Lab. 6.22, 46026, Valencia, Spain
- Fundación IVI, 46026, Valencia, Spain
| | - M M Andrés
- Hospital Universitario y Politécnico La Fe, 46026, Valencia, Spain
| | - E Novella-Maestre
- Unidad de Medicina Reproductiva, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Av. Fernando Abril Martorell, 106. Torre A, Lab. 6.22, 46026, Valencia, Spain
- Hospital Universitario y Politécnico La Fe, 46026, Valencia, Spain
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30
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31
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Histologic Analysis of Testes from Prepubertal Patients Treated with Chemotherapy Associates Impaired Germ Cell Counts with Cumulative Doses of Cyclophosphamide, Ifosfamide, Cytarabine, and Asparaginase. Reprod Sci 2020; 28:603-613. [PMID: 33150486 DOI: 10.1007/s43032-020-00357-6] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2020] [Accepted: 10/11/2020] [Indexed: 01/15/2023]
Abstract
Cryopreservation of immature testicular tissue is an experimental strategy for the preservation of fertility in prepubertal boys that will be subjected to a gonadotoxic onset, as is the case of oncologic patients. Therefore, the objective of this study was to assess the impact of chemotherapeutic treatments on the testicular histologic phenotype in prepubertal patients. A total of 56 testicular tissue samples from pediatric patients between 0 and 16 years old (28 with at least one previous chemotherapeutic onset and 28 untreated controls) were histologically analyzed and age-matched compared. At least two 5-μm sections from testis per patient separated by a distance of 100 μm were immunostained for the germ cell marker VASA, the spermatogonial markers UTF1, PLZF, UCHL1, and SALL4, the marker for proliferative cells KI67, and the Sertoli cell marker SOX9. The percentage of tubule cross-sections positive for each marker and the number of positive cells per tubule cross-section were determined and association with the cumulative dose received of each chemotherapeutic drug was statistically assessed. Results indicated that alkylating agents, cyclophosphamide and ifosfamide, but also the antimetabolite cytarabine and asparaginase were associated with a decreased percentage of positive tubules and a lower number of positive cells per tubule for the analyzed markers. Our results provide new evidences of the potential of chemotherapeutic agents previously considered to have low gonadotoxic effects such as cytarabine and asparaginase to trigger a severe testicular phenotype, hampering the potential success of future fertility restoration in experimental programs of fertility preservation in prepubertal boys.
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McClusky LM. Simple, once-off mapping of various, recurrent immunostaining patterns of proliferating cell nuclear antigen in spermatogonia at the immature pole of the testis of adult wild-caught blue shark, Prionace glauca: Correlations with changes in testicular status. Mol Reprod Dev 2020; 87:1111-1123. [PMID: 33104292 DOI: 10.1002/mrd.23429] [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: 02/25/2020] [Revised: 09/11/2020] [Accepted: 10/05/2020] [Indexed: 12/28/2022]
Abstract
This study was a single time-point mapping of various immunostaining patterns revealed with the proliferating cell nuclear antigen (PCNA) PC10 antibody in spermatogonia at the immature pole of the testis of the Blue shark (Prionace glauca). Scattered in the stroma of the germinal ridge that demarcates the immature pole's outer boundary were nests of variously immunoreactive A-spermatogonia, each flanked by a fusiform cell. Spermatocysts were assembled from niche-derived stromal cells, displaced A-progenitors, and their progeny, which showed one of two main immunostaining patterns (i.e., an uneven light brown/globular and homogeneous dark [hod] brown appearance). The testes of wild-caught Prionace showed two conditions, namely, extensive multinucleate cell death (MNC) near the mitosis-meiosis transition or an early recovery phase from the latter showing vacuolated areas. Both the proportion of cysts with immature Bhod -spermatogonia and the frequency of mitotic figures in such cysts in the early recovery testis condition were significantly higher than the comparable parameters in MNC testis condition. Moreover, the post-MNC recovery phase revealed a decrease in the proportion of immature cysts with uneven light brown/globular-like spermatogonia. The protracted spread of a cell cycle signal in an anatomically discrete, syncytially connected spermatogonial clone manifests as different PCNA immunoreactivities.
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Affiliation(s)
- Leon M McClusky
- Department of Health and Care, UiT The Arctic University of Norway, Narvik, Norway
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Liu HC, Xie Y, Deng CH, Liu GH. Stem cell-based therapies for fertility preservation in males: Current status and future prospects. World J Stem Cells 2020; 12:1097-1112. [PMID: 33178394 PMCID: PMC7596443 DOI: 10.4252/wjsc.v12.i10.1097] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Revised: 05/13/2020] [Accepted: 08/26/2020] [Indexed: 02/06/2023] Open
Abstract
With the decline in male fertility in recent years, strategies for male fertility preservation have received increasing attention. In this study, by reviewing current treatments and recent publications, we describe research progress in and the future directions of stem cell-based therapies for male fertility preservation, focusing on the use of spermatogonial stem cells (SSCs), SSC niches, SSC-based testicular organoids, other stem cell types such as mesenchymal stem cells, and stem cell-derived extracellular vesicles. In conclusion, a more comprehensive understanding of the germ cell microenvironment, stem cell-derived extracellular vesicles, and testicular organoids will play an important role in achieving male fertility preservation.
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Affiliation(s)
- Han-Chao Liu
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Yun Xie
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Chun-Hua Deng
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Gui-Hua Liu
- Reproductive Medicine Research Center, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, Guangdong Province, China
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E4 Transcription Factor 1 (E4F1) Regulates Sertoli Cell Proliferation and Fertility in Mice. Animals (Basel) 2020; 10:ani10091691. [PMID: 32962114 PMCID: PMC7552733 DOI: 10.3390/ani10091691] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2020] [Revised: 09/13/2020] [Accepted: 09/16/2020] [Indexed: 12/13/2022] Open
Abstract
Simple Summary Male fertility relies on the generation of functional sperm in seminiferous tubules of the testis. In mammals, Sertoli cells are the only somatic cells that directly interact with spermatogenic cells. Compelling evidences suggest that the number of Sertoli cells determines testis size and sperm output, however, molecular mechanisms regulating Sertoli cell proliferation and maturation are not well-understood. Using a Sertoli cell specific loss-of-function approach, here we showed that transcription factor E4F1 played an important role in murine Sertoli cell proliferation. Compared with their littermate control, E4f1 conditional knockout male mice sired a significantly low number of pups. E4f1 deletion resulted in reduced Sertoli cell number and testis size. Further analyses revealed that E4f1 deletion affected Sertoli cell proliferation in the neonatal testis and caused an increase in apoptosis of spermatogenic cells without affecting normal development of spermatogonia, meiotic and post-meiotic germ cells. These findings have shed new light on molecular controlling of spermatogenesis in mice and a similar mechanism likely exists in other animals. Abstract In the mammalian testes, Sertoli cells are the only somatic cells in the seminiferous tubules that provide structural, nutritional and regulatory support for developing spermatogenic cells. Sertoli cells only proliferate during the fetal and neonatal periods and enter a quiescent state after puberty. Functional evidences suggest that the size of Sertoli cell population determines sperm production and fertility. However, factors that direct Sertoli cell proliferation and maturation are not fully understood. Transcription factor E4F1 is a multifunctional protein that serves essential roles in cell fate decisions and because it interacts with pRB, a master regulator of Sertoli cell function, we hypothesized that E4F1 may have a functional role in Sertoli cells. E4f1 mRNA was present in murine testis and immunohistochemical staining confirmed that E4F1 was enriched in mature Sertoli cells. We generated a conditional knockout mouse model using Amh-cre and E4f1flox/flox lines to study E4F1 fucntion in Sertoli cells and the results showed that E4f1 deletion caused a significant reduction in testis size and fertility. Further analyses revealed that meiosis progression and spermiogenesis were normal, however, Sertoli cell proliferation was impaired and germ cell apoptosis was elevated in the testis of E4f1 conditional knockout mice. On the basis of these findings, we concluded that E4F1 was expressed in murine Sertoli cells and served important functions in regulating Sertoli cell proliferation and fertility.
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Idrees M, Oh SH, Muhammad T, El-Sheikh M, Song SH, Lee KL, Kong IK. Growth Factors, and Cytokines; Understanding the Role of Tyrosine Phosphatase SHP2 in Gametogenesis and Early Embryo Development. Cells 2020; 9:cells9081798. [PMID: 32751109 PMCID: PMC7465981 DOI: 10.3390/cells9081798] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Revised: 07/28/2020] [Accepted: 07/28/2020] [Indexed: 12/19/2022] Open
Abstract
Growth factors and cytokines have vital roles in germ cell development, gamete maturation, and early embryo development. Cell surface receptors are present for growth factors and cytokines to integrate with and trigger protein signaling in the germ and embryo intracellular milieu. Src-homology-2-containing phosphotyrosine phosphatase (SHP2) is a ubiquitously expressed, multifunctional protein that plays a central role in the signaling pathways involved in growth factor receptors, cytokine receptors, integrins, and G protein-coupled receptors. Over recent decades, researchers have recapitulated the protein signaling networks that influence gamete progenitor specification as well as gamete differentiation and maturation. SHP2 plays an indispensable role in cellular growth, survival, proliferation, differentiation, and migration, as well as the basic events in gametogenesis and early embryo development. SHP2, a classic cytosolic protein and a key regulator of signal transduction, displays unconventional nuclear expression in the genital organs. Several observations provided shreds of evidence that this behavior is essential for fertility. The growth factor and cytokine-dependent roles of SHP2 and its nuclear/cytoplasmic presence during gamete maturation, early embryonic development and embryo implantation are fascinating and complex subjects. This review is intended to summarize the previous and recent knowledge about the SHP2 functions in gametogenesis and early embryo development.
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Affiliation(s)
- Muhammad Idrees
- Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Korea; (M.I.); (S.-H.O.); (M.E.-S.)
| | - Seon-Hwa Oh
- Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Korea; (M.I.); (S.-H.O.); (M.E.-S.)
| | - Tahir Muhammad
- Institute of Medical Science, University of Toronto, Toronto, ON M5S 1A8, Canada;
| | - Marwa El-Sheikh
- Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Korea; (M.I.); (S.-H.O.); (M.E.-S.)
- Department of Microbial Biotechnology, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, Cairo 12622, Egypt
| | - Seok-Hwan Song
- The King Kong Ltd., Gyeongsang National University, Jinju 52828, Korea; (S.-H.S.); (K.-L.L.)
| | - Kyeong-Lim Lee
- The King Kong Ltd., Gyeongsang National University, Jinju 52828, Korea; (S.-H.S.); (K.-L.L.)
| | - Il-Keun Kong
- Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Korea; (M.I.); (S.-H.O.); (M.E.-S.)
- The King Kong Ltd., Gyeongsang National University, Jinju 52828, Korea; (S.-H.S.); (K.-L.L.)
- Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea
- Correspondence: ; Tel.: +82-55-772-1942
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Zhang PY, Fan Y, Tan T, Yu Y. Generation of Artificial Gamete and Embryo From Stem Cells in Reproductive Medicine. Front Bioeng Biotechnol 2020; 8:781. [PMID: 32793569 PMCID: PMC7387433 DOI: 10.3389/fbioe.2020.00781] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2020] [Accepted: 06/19/2020] [Indexed: 12/20/2022] Open
Abstract
In addition to the great growing need for assisted reproduction technologies (ART), additional solutions for patients without functional gametes are strongly needed. Due to ethical restrictions, limited studies can be performed on human gametes and embryos; however, artificial gametes and embryos represent a new hope for clinical application and basic research in the field of reproductive medicine. Here, we provide a review of the research progress and possible application of artificial gametes and embryos from different species, including mice, monkeys and humans. Gametes specification from adult stem cells and embryonic stem cells (ESCs) as well as propagation of stem cells from the reproductive system and from organized embryos, which are similar to blastocysts, have been realized in some nonhuman mammals, but not all achievements can be replicated in humans. This area of research remains noteworthy and requires further study and effort to achieve the reconstitution of the entire cycle of gametogenesis and embryo development in vitro.
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Affiliation(s)
- Pu-Yao Zhang
- Clinical Stem Cell Research Center, Peking University Third Hospital, Beijing, China.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology and Key Laboratory of Assisted Reproduction, Ministry of Education, Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China
| | - Yong Fan
- Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Tao Tan
- Clinical Stem Cell Research Center, Peking University Third Hospital, Beijing, China.,Yunnan Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, China
| | - Yang Yu
- Clinical Stem Cell Research Center, Peking University Third Hospital, Beijing, China.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology and Key Laboratory of Assisted Reproduction, Ministry of Education, Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China
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37
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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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Yang Y, Lin Q, Zhou C, Li Q, Li Z, Cao Z, Liang J, Li H, Mei J, Zhang Q, Xiang Q, Xue W, Huang Y. A Testis-Derived Hydrogel as an Efficient Feeder-Free Culture Platform to Promote Mouse Spermatogonial Stem Cell Proliferation and Differentiation. Front Cell Dev Biol 2020; 8:250. [PMID: 32509769 PMCID: PMC7248195 DOI: 10.3389/fcell.2020.00250] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2020] [Accepted: 03/24/2020] [Indexed: 12/15/2022] Open
Abstract
Fertility preservation and assisted reproductive medicine require effective culture systems for the successful proliferation and differentiation of spermatogonial stem cells (SSCs). Many SSC culture systems require the addition of feeder cells at each subculture, which is tedious and inefficient. Here, we prepared decellularized testicular matrix (DTM) from testicular tissue, which preserved essential structural proteins of testis. The DTM was then solubilized and induced to form a porous hydrogel scaffold with randomly oriented fibrillar structures that exhibited good cytocompatibility. The viability of SSCs inoculated onto DTM hydrogel scaffolds was significantly higher than those inoculated on Matrigel or laminin, and intracellular gene expression and DNA imprinting patterns were similar to that of native SSCs. Additionally, DTM promoted SSC differentiation into round spermatids. More importantly, the DTM hydrogel supported SSC proliferation and differentiation without requiring additional somatic cells. The DTM hydrogel scaffold culture system provided an alternative and simple method for culturing SSCs that eliminates potential variability and contamination caused by feeder cells. It might be a valuable tool for reproductive medicine.
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Affiliation(s)
- Yan Yang
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology, Jinan University, Guangzhou, China.,Department of Biomedical Engineering, Jinan University, Guangzhou, China
| | - Qilian Lin
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology, Jinan University, Guangzhou, China
| | - Chengxing Zhou
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology, Jinan University, Guangzhou, China
| | - Quan Li
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology, Jinan University, Guangzhou, China
| | - Ziyi Li
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology, Jinan University, Guangzhou, China
| | - Zhen Cao
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology, Jinan University, Guangzhou, China
| | - Jinlian Liang
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology, Jinan University, Guangzhou, China
| | - Hanhao Li
- Department of Pharmacology, Jinan University, Guangzhou, China
| | - Jiaxin Mei
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology, Jinan University, Guangzhou, China
| | - Qihao Zhang
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology, Jinan University, Guangzhou, China
| | - Qi Xiang
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology, Jinan University, Guangzhou, China.,Biopharmaceutical Research & Development Center of Jinan University, Guangzhou, China
| | - Wei Xue
- Department of Biomedical Engineering, Jinan University, Guangzhou, China
| | - Yadong Huang
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology, Jinan University, Guangzhou, China.,Department of Pharmacology, Jinan University, Guangzhou, China
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Sagaradze G, Basalova N, Kirpatovsky V, Ohobotov D, Nimiritsky P, Grigorieva O, Popov V, Kamalov A, Tkachuk V, Efimenko A. A magic kick for regeneration: role of mesenchymal stromal cell secretome in spermatogonial stem cell niche recovery. Stem Cell Res Ther 2019; 10:342. [PMID: 31753023 PMCID: PMC6873442 DOI: 10.1186/s13287-019-1479-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2019] [Revised: 10/28/2019] [Accepted: 10/30/2019] [Indexed: 01/24/2023] Open
Abstract
Background Injury of stem cell niches may disturb tissue homeostasis and regeneration coordinated by specific niche components. Yet, the mechanisms of stem cell niche restoration remain poorly understood. Herein, we examined the role of mesenchymal stromal cells (MSCs) as pivotal regulators of stem cell niche recovery focusing on the effects of their secretome. Methods The spermatogonial stem cell (SSC) niche was selected as a model. SSC niches were injured by inducing abdominal cryptorchidism in rats. Briefly, testes of anesthetized rats were elevated into the abdominal cavity through the inguinal canal for 14 days. After descent of testes, MSC or MSC secretome treatment was applied to the animals by local subtunical injections. Results Local administration of MSC or MSC secretome was sufficient to recover spermatogenesis and production of functional germ cells. The effects of MSC and their secreted components were comparable, leading to restoration of Sertoli cell pools and recovery of Leydig cell secretory functions. Conclusion Our data suggest that MSCs mimic the functions of lost supportive cells within the stem cell niche, transiently providing paracrine stimuli for target cells and triggering tissue regenerative processes after damage.
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Affiliation(s)
- Georgy Sagaradze
- Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Nataliya Basalova
- Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russian Federation.,Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Vladimir Kirpatovsky
- Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russian Federation.,Research Institute of Urology and Interventional Radiology named N.A. Lopatkin - branch FSBI National Medical Research Radiological Center of the Ministry of Health of the Russian Federation, Moscow, Russian Federation
| | - Dmitry Ohobotov
- Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russian Federation.,Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Peter Nimiritsky
- Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Olga Grigorieva
- Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Vladimir Popov
- Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Armais Kamalov
- Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russian Federation.,Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Vsevolod Tkachuk
- Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russian Federation.,Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Anastasia Efimenko
- Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russian Federation. .,Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russian Federation.
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40
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Oncofertility: Pharmacological Protection and Immature Testicular Tissue (ITT)-Based Strategies for Prepubertal and Adolescent Male Cancer Patients. Int J Mol Sci 2019; 20:ijms20205223. [PMID: 31640294 PMCID: PMC6834329 DOI: 10.3390/ijms20205223] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2019] [Revised: 10/12/2019] [Accepted: 10/18/2019] [Indexed: 01/15/2023] Open
Abstract
While the incidence of cancer in children and adolescents has significantly increased over the last decades, improvements made in the field of cancer therapy have led to an increased life expectancy for childhood cancer survivors. However, the gonadotoxic effect of the treatments may lead to infertility. Although semen cryopreservation represents the most efficient and safe fertility preservation method for males producing sperm, it is not feasible for prepubertal boys. The development of an effective strategy based on the pharmacological protection of the germ cells and testicular function during gonadotoxic exposure is a non-invasive preventive approach that prepubertal boys could benefit from. However, the progress in this field is slow. Currently, cryopreservation of immature testicular tissue (ITT) containing spermatogonial stem cells is offered to prepubertal boys as an experimental fertility preservation strategy by a number of medical centers. Several in vitro and in vivo fertility restoration approaches based on the use of ITT have been developed so far with autotransplantation of ITT appearing more promising. In this review, we discuss the pharmacological approaches for fertility protection in prepubertal and adolescent boys and the fertility restoration approaches developed on the utilization of ITT.
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41
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La HM, Hobbs RM. Mechanisms regulating mammalian spermatogenesis and fertility recovery following germ cell depletion. Cell Mol Life Sci 2019; 76:4071-4102. [PMID: 31254043 PMCID: PMC11105665 DOI: 10.1007/s00018-019-03201-6] [Citation(s) in RCA: 39] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2018] [Revised: 06/07/2019] [Accepted: 06/19/2019] [Indexed: 12/19/2022]
Abstract
Mammalian spermatogenesis is a highly complex multi-step process sustained by a population of mitotic germ cells with self-renewal potential known as spermatogonial stem cells (SSCs). The maintenance and regulation of SSC function are strictly dependent on a supportive niche that is composed of multiple cell types. A detailed appreciation of the molecular mechanisms underpinning SSC activity and fate is of fundamental importance for spermatogenesis and male fertility. However, different models of SSC identity and spermatogonial hierarchy have been proposed and recent studies indicate that cell populations supporting steady-state germline maintenance and regeneration following damage are distinct. Importantly, dynamic changes in niche properties may underlie the fate plasticity of spermatogonia evident during testis regeneration. While formation of spermatogenic colonies in germ-cell-depleted testis upon transplantation is a standard assay for SSCs, differentiation-primed spermatogonial fractions have transplantation potential and this assay provides readout of regenerative rather than steady-state stem cell capacity. The characterisation of spermatogonial populations with regenerative capacity is essential for the development of clinical applications aimed at restoring fertility in individuals following germline depletion by genotoxic treatments. This review will discuss regulatory mechanisms of SSCs in homeostatic and regenerative testis and the conservation of these mechanisms between rodent models and man.
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Affiliation(s)
- Hue M La
- Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC, 3800, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne, VIC, 3800, Australia
| | - Robin M Hobbs
- Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC, 3800, Australia.
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne, VIC, 3800, Australia.
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42
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Oliver E, Stukenborg JB. Rebuilding the human testis in vitro. Andrology 2019; 8:825-834. [PMID: 31539453 PMCID: PMC7496374 DOI: 10.1111/andr.12710] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2019] [Revised: 08/21/2019] [Accepted: 09/16/2019] [Indexed: 12/22/2022]
Abstract
Increasing rates of male infertility have led to a greater need for relevant model systems to gain further insight into male fertility and its failings. Spermatogenesis and hormone production occur within distinct regions of the testis. Defined by specialized architecture and a diverse population of cell types, it is no surprise that disruption of this highly organized microenvironment can lead to infertility. To date, no robust in vitro system has facilitated full spermatogenesis resulting in the production of fertilization‐competent human spermatozoa. Here, we review a selection of current in vitro systems available for modelling the human testis microenvironment with focus on the progression of spermatogenesis and recapitulation of the testis microenvironment.
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Affiliation(s)
- E Oliver
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Solna, Sweden
| | - J-B Stukenborg
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Solna, Sweden
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Zhao H, Li T, Yang H, Mehmood MU, Lu Y, Liang X, Yang X, Xu H, Lu K, Lu S. The effects of growth factors on proliferation of spermatogonial stem cells from Guangxi Bama mini-pig. Reprod Domest Anim 2019; 54:1574-1582. [PMID: 31544277 DOI: 10.1111/rda.13566] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2019] [Revised: 09/10/2019] [Accepted: 09/16/2019] [Indexed: 12/25/2022]
Abstract
The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini-pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line-derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha-1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT-PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1-, DBA- and CDH1-positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.
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Affiliation(s)
- Huimin Zhao
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, College of Animal Science and Technology, Guangxi University, Nanning, China.,College of Life Science and Technology, Guangxi University, Nanning, China
| | - Tingting Li
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, College of Animal Science and Technology, Guangxi University, Nanning, China
| | - Huan Yang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, College of Animal Science and Technology, Guangxi University, Nanning, China
| | - Muhammad Usman Mehmood
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, College of Animal Science and Technology, Guangxi University, Nanning, China
| | - Yangqing Lu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, College of Animal Science and Technology, Guangxi University, Nanning, China
| | - Xingwei Liang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, College of Animal Science and Technology, Guangxi University, Nanning, China
| | - Xiaogan Yang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, College of Animal Science and Technology, Guangxi University, Nanning, China
| | - Huiyan Xu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, College of Animal Science and Technology, Guangxi University, Nanning, China
| | - Kehuan Lu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, College of Animal Science and Technology, Guangxi University, Nanning, China
| | - Shengsheng Lu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, College of Animal Science and Technology, Guangxi University, Nanning, China
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Yan M, Li L, Mao B, Li H, Li SYT, Mruk D, Silvestrini B, Lian Q, Ge R, Cheng CY. mTORC1/rpS6 signaling complex modifies BTB transport function: an in vivo study using the adjudin model. Am J Physiol Endocrinol Metab 2019; 317:E121-E138. [PMID: 31112404 PMCID: PMC6689739 DOI: 10.1152/ajpendo.00553.2018] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/19/2018] [Revised: 04/30/2019] [Accepted: 05/05/2019] [Indexed: 12/13/2022]
Abstract
Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII-IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky." This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.
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Affiliation(s)
- Ming Yan
- Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council , New York, New York
| | - Linxi Li
- Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council , New York, New York
- Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University , Wenzhou, Zhejiang , China
| | - Baiping Mao
- Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council , New York, New York
- Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University , Wenzhou, Zhejiang , China
| | - Huitao Li
- Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council , New York, New York
- Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University , Wenzhou, Zhejiang , China
| | - Stephen Y T Li
- Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council , New York, New York
| | - Dolores Mruk
- Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council , New York, New York
| | | | - Qingquan Lian
- Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University , Wenzhou, Zhejiang , China
| | - Renshan Ge
- Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University , Wenzhou, Zhejiang , China
| | - C Yan Cheng
- Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council , New York, New York
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GDNF family receptor alpha 1 is a reliable marker of undifferentiated germ cells in bulls. Theriogenology 2019; 132:172-181. [DOI: 10.1016/j.theriogenology.2019.04.016] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2019] [Revised: 04/11/2019] [Accepted: 04/14/2019] [Indexed: 12/27/2022]
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Mohammadzadeh E, Mirzapour T, Nowroozi MR, Nazarian H, Piryaei A, Alipour F, Modarres Mousavi SM, Ghaffari Novin M. Differentiation of spermatogonial stem cells by soft agar three-dimensional culture system. ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2019; 47:1772-1781. [DOI: 10.1080/21691401.2019.1575230] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Affiliation(s)
- Elham Mohammadzadeh
- Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
- Shefa Neuroscience Research Center, Khatam Alanbia Hospital, Tehran, Iran
| | - Tooba Mirzapour
- Department of Biology, Faculty of Science, University of Guilan, Guilan, Iran
| | | | - Hamid Nazarian
- Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Abbas Piryaei
- Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
- Urogenital Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Fatemeh Alipour
- Shefa Neuroscience Research Center, Khatam Alanbia Hospital, Tehran, Iran
| | | | - Marefat Ghaffari Novin
- Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Sharma S, Wistuba J, Pock T, Schlatt S, Neuhaus N. Spermatogonial stem cells: updates from specification to clinical relevance. Hum Reprod Update 2019; 25:275-297. [DOI: 10.1093/humupd/dmz006] [Citation(s) in RCA: 55] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2018] [Revised: 11/23/2018] [Accepted: 02/22/2019] [Indexed: 12/20/2022] Open
Affiliation(s)
- Swati Sharma
- Centre of Reproductive Medicine and Andrology, Institute of Reproductive and Regenerative Biology, Albert-Schweitzer Campus 1, Building D11, Münster, Germany
| | - Joachim Wistuba
- Centre of Reproductive Medicine and Andrology, Institute of Reproductive and Regenerative Biology, Albert-Schweitzer Campus 1, Building D11, Münster, Germany
| | - Tim Pock
- Centre of Reproductive Medicine and Andrology, Institute of Reproductive and Regenerative Biology, Albert-Schweitzer Campus 1, Building D11, Münster, Germany
| | - Stefan Schlatt
- Centre of Reproductive Medicine and Andrology, Institute of Reproductive and Regenerative Biology, Albert-Schweitzer Campus 1, Building D11, Münster, Germany
| | - Nina Neuhaus
- Centre of Reproductive Medicine and Andrology, Institute of Reproductive and Regenerative Biology, Albert-Schweitzer Campus 1, Building D11, Münster, Germany
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Medrano JV, Vilanova-Pérez T, Fornés-Ferrer V, Navarro-Gomezlechon A, Martínez-Triguero ML, García S, Gómez-Chacón J, Povo I, Pellicer A, Andrés MM, Novella-Maestre E. Influence of temperature, serum, and gonadotropin supplementation in short- and long-term organotypic culture of human immature testicular tissue. Fertil Steril 2019; 110:1045-1057.e3. [PMID: 30396549 DOI: 10.1016/j.fertnstert.2018.07.018] [Citation(s) in RCA: 49] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2018] [Revised: 07/06/2018] [Accepted: 07/18/2018] [Indexed: 12/11/2022]
Abstract
OBJECTIVE To study how temperature, serum, and gonadotropin supplementation affect the organotypic culture of human immature testicular tissue (ITT) in vitro. DESIGN Experimental basic science study. SETTING Reproductive biology laboratory. PATIENT(S) ITT from 4 boys with cancer that had testicular tissue cryopreserved as part of their fertility preservation treatment. INTERVENTION(S) In vitro organotypic culture of ITT, exposed to different temperatures (37°C vs. 34°C), serum (fetal bovine serum [FBS] vs. Knockout Serum Replacement [KOS]), and gonadotropin supplementation (with and without FSH and LH). MAIN OUTCOME MEASURE(S) Characterization of the tissue was performed at days 0, 14, and 70 with the use of reverse-transcription quantitative polymerase chain reaction, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, histologic analysis by means of hematoxylin-eosin staining, and immunohistochemical staining. Hormonal secretion was determined at days 3, 14, 28, and 70 by means of immunofluorescent assay. RESULT(S) The 37°C conditions showed an accelerated loss of tubular morphology and higher intratubular apoptosis. KOS supplementation triggered the up-regulation of STAR, SOX9, DAZL, DDX4, PLZF, and UTF1, the percentage of SOX9+/androgen receptor (AR)-positive mature Sertoli cells at day 14, and testosterone secretion. Gonadotropin supplementation increased the numbers of both undifferentiated UTF1+ spermatogonia and premeiotic VASA+/SYCP3+ spermatogonia at day 14, and the number of SOX9+ Sertoli cells at day 70. The low SOX9+/AR+ colocalization, the disorganized pattern of ZO-1, and the progressive decrease of antimüllerian hormone secretion indicated inefficient Sertoli cell maturation in vitro. CONCLUSION(S) The 34°C condition in KOS showed the best results for the survival of both spermatogonia and Sertoli cells. FSH/LH supplementation also improved long-term survival of Sertoli cells and the maturation of spermatogonia up to meiotic initiation in short-term culture.
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Affiliation(s)
- Jose V Medrano
- Instituto de Investigación Sanitaria La Fe, Valencia, Spain.
| | | | | | | | | | - Sofía García
- Instituto de Investigación Sanitaria La Fe, Valencia, Spain; Hospital Universitario y Politécnico La Fe, Valencia, Spain
| | | | - Ivan Povo
- Hospital Universitario y Politécnico La Fe, Valencia, Spain
| | - Antonio Pellicer
- Instituto de Investigación Sanitaria La Fe, Valencia, Spain; Fundación IVI, Valencia, Spain
| | - María M Andrés
- Instituto de Investigación Sanitaria La Fe, Valencia, Spain; Hospital Universitario y Politécnico La Fe, Valencia, Spain
| | - Edurne Novella-Maestre
- Instituto de Investigación Sanitaria La Fe, Valencia, Spain; Hospital Universitario y Politécnico La Fe, Valencia, Spain
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de Siqueira-Silva DH, da Silva Rodrigues M, Nóbrega RH. Testis structure, spermatogonial niche and Sertoli cell efficiency in Neotropical fish. Gen Comp Endocrinol 2019; 273:218-226. [PMID: 30195025 DOI: 10.1016/j.ygcen.2018.09.004] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/19/2018] [Revised: 07/23/2018] [Accepted: 09/04/2018] [Indexed: 11/23/2022]
Abstract
Neotropical icthyofauna represents one of the most diverse and extreme ecosystems in the world. Likewise, reproduction showed enormous diversity with different reproductive systems, modes and behavior. On the other hand, information on Neotropical fish species, in particular on male reproductive physiology is restricted to few species. This mini-review aimed to compile the existing information on spermatogenesis of Neotropical teleosts focusing on testis structure, spermatogonial niche and Sertoli cell efficiency. The first topic covers the histological analysis of the testicular structure, showing a conserved testicular pattern in relation to the phylogenetic position: basal species present anastomosing tubular testis (e.g. Astyanax altiparanae, Conorhynchos conirostris, Pimelodus maculatus, Lophiosilurus alexandri, Rhinelepis aspera, among others), while derived teleosts showed lobular testis (e.g. Cichlasoma dimerus, Cichla kelberi, Odontesthes bonariensis, Synbranchus marmoratus and others). Next to testicular structure, existing data showed that type A undifferentiated spermatogonia (Aund) is differentially distributed among the Neotropical species. Aund can be restricted at the blind-end of the germinal compartment (O. bonariensis), or spread along the germinal epithelium (A. altiparanae), or even distributed along the germinal epithelium but concentrated at the blind-end (C. kelberi and C. intermedia). Moreover, recent studies in A. altiparanae have demonstrated that within the germinal compartment, Aund have a preferential distribution in areas neighboring the interstitial compartment - the spermatogonial niche. The proximity with the interstitium suggests that interstitial cells, such as Leydig cells, are important for Aund maintenance in the testis. Finally, this mini-review highlighted Sertoli cell efficiency, showing that a single Sertoli cell can support a higher number of germ cells (80-140 spermatids) in Neotropical species evaluated at the moment (e.g. A. altiparanae, Hoplias malabaricus, Poecilia reticulata, Serrasalmus spilopleura, C. intermedia). Overall, this review provided basic and functional information on spermatogenesis of Neotropical species. More studies in this field are necessary since Neotropical region is considered one of the hotspot regions to discovery new species providing, therefore, new opportunities to investigate spermatogenesis in fish.
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Affiliation(s)
- Diógenes Henrique de Siqueira-Silva
- Group of Studies on the Reproduction of Amazon fish (GERPA/LANEC), PPG in Biodiversity and Biotechnology (BIONORTE), University of South and Southern of Pará (Unifesspa), Marabá, Pará, Brazil.
| | - Maira da Silva Rodrigues
- Reproductive and Molecular Biology Group, Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP) - Botucatu Campus, Botucatu, Brazil; Aquaculture Center of São Paulo State University (CAUNESP), São Paulo State University (UNESP) - Jaboticabal Campus, Jaboticabal, Brazil
| | - Rafael Henrique Nóbrega
- Reproductive and Molecular Biology Group, Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP) - Botucatu Campus, Botucatu, Brazil.
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Medrano JV, Luján S, Andrés MDM, Pellicer A. [Current state regarding fertility cryopreservation in pre-pubertal boys]. Rev Int Androl 2018; 18:27-34. [PMID: 30477959 DOI: 10.1016/j.androl.2018.07.006] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2018] [Revised: 05/08/2018] [Accepted: 07/25/2018] [Indexed: 01/15/2023]
Abstract
Some treatments for any cancer therapy and hematological diseases may have gonadotoxic side effects that can result in infertility, and thus sperm cryopreservation is routinely offered to patients as the strategy to preserve their fertility. However, there are many cases where sperm banking cannot be applied, as is the case of pre-pubertal cancer patients and others unable to produce mature gametes at the moment of diagnosis. Regarding this, recent breakthroughs have gained public attention to the fertility preservation options that Regenerative Medicine can offer to these patients. In this review, we tried to compile and discuss the latest updates about all these strategies from a critical point of view.
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Affiliation(s)
- Jose V Medrano
- Unidad de Medicina Reproductiva, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, España.
| | - Saturnino Luján
- Unidad de Medicina Reproductiva, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, España
| | - María Del Mar Andrés
- Unidad de Oncología pediátrica, Hospital Universitario y Politécnico La Fe, Valencia, Spain
| | - Antonio Pellicer
- Unidad de Medicina Reproductiva, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, España
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