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Chen B, Zhu G, Yan A, He J, Liu Y, Li L, Yang X, Dong C, Kee K. IGSF11 is required for pericentric heterochromatin dissociation during meiotic diplotene. PLoS Genet 2021; 17:e1009778. [PMID: 34491997 PMCID: PMC8448346 DOI: 10.1371/journal.pgen.1009778] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2021] [Revised: 09/17/2021] [Accepted: 08/16/2021] [Indexed: 02/03/2023] Open
Abstract
Meiosis initiation and progression are regulated by both germ cells and gonadal somatic cells. However, little is known about what genes or proteins connecting somatic and germ cells are required for this regulation. Our results show that deficiency for adhesion molecule IGSF11, which is expressed in both Sertoli cells and germ cells, leads to male infertility in mice. Combining a new meiotic fluorescent reporter system with testicular cell transplantation, we demonstrated that IGSF11 is required in both somatic cells and spermatogenic cells for primary spermatocyte development. In the absence of IGSF11, spermatocytes proceed through pachytene, but the pericentric heterochromatin of nonhomologous chromosomes remains inappropriately clustered from late pachytene onward, resulting in undissolved interchromosomal interactions. Hi-C analysis reveals elevated levels of interchromosomal interactions occurring mostly at the chromosome ends. Collectively, our data elucidates that IGSF11 in somatic cells and germ cells is required for pericentric heterochromatin dissociation during diplotene in mouse primary spermatocytes. For sexually reproducing species, the number of chromosomes in a mature germ cell is half that of a typical somatic cell, and its chromosome sequence is not identical to that of parental cell, these changes result from a highly specialized cell division process named meiosis. In contrast to mitosis, germ cells undergo many meiotic-specific regulatory processes during prophase I of meiosis. In mammals, the development of male and female meiotic germ cells relies on completely different microenvironment provided by sexually specialized gonadal somatic cells, but what gene is required for germ cells and gonadal somatic cells to mediate meiosis progression is largely unclear. Here, we construct a fluorescent reporter to trace meiotic prophase in mice, and use it to examine the requirement of IGSF11 in mediating pericentric heterochromatin dissociation during meiosis.
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Affiliation(s)
- Bo Chen
- Center for Stem Cell Biology and Regenerative Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China
| | - Gengzhen Zhu
- Institute of Immunology and School of Medicine, Tsinghua University, Beijing, China
- Beijing Key Lab for Immunological Research on Chronic Diseases, Tsinghua University, Beijing, China
| | - An Yan
- Center for Stem Cell Biology and Regenerative Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China
| | - Jing He
- Center for Stem Cell Biology and Regenerative Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China
| | - Yang Liu
- MOE Key Laboratory of Bioinformatics, Center for Synthetic & Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Lin Li
- Central Laboratory, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China
| | - Xuerui Yang
- MOE Key Laboratory of Bioinformatics, Center for Synthetic & Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Chen Dong
- Institute of Immunology and School of Medicine, Tsinghua University, Beijing, China
- Beijing Key Lab for Immunological Research on Chronic Diseases, Tsinghua University, Beijing, China
| | - Kehkooi Kee
- Center for Stem Cell Biology and Regenerative Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China
- * E-mail:
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EIF2S3Y suppresses the pluripotency state and promotes the proliferation of mouse embryonic stem cells. Oncotarget 2017; 7:11321-31. [PMID: 26863630 PMCID: PMC4905476 DOI: 10.18632/oncotarget.7187] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2015] [Accepted: 01/23/2016] [Indexed: 12/15/2022] Open
Abstract
Eukaryotic translation initiation factor 2, subunit 3, and structural gene Y-linked (EIF2S3Y) is essential for spermatogenesis in mouse models. However, its effect on embryonic stem (ES) cells remains unknown. In our observation, differentiated ES cells showed higher levels of EIF2S3Y. To further elucidate its role in ES cells, we utilized ES-derived EIF2S3Y-overexpressing cells and found that EIF2S3Y down-regulated the pluripotency state of ES cells, which might be explained by decreased histone methylation levels because of reduced levels of ten-eleven translocation 1 (TET1). Moreover, EIF2S3Y-overexpressing cells showed an enhanced proliferation rate, which might be due to increased Cyclin A and Cyclin E levels. This study highlighted novel roles of EIF2S3Y in the pluripotency maintenance and proliferation control of ES cells, which would provide an efficient model to study germ cell generation as well as cancer development using ES cells, thus providing valuable target for clinical applications of ES cells.
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Costa GMJ, Avelar GF, Lacerda SMSN, Figueiredo AFA, Tavares AO, Rezende-Neto JV, Martins FGP, França LR. Horse spermatogonial stem cell cryopreservation: feasible protocols and potential biotechnological applications. Cell Tissue Res 2017; 370:489-500. [PMID: 28831567 DOI: 10.1007/s00441-017-2673-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2017] [Accepted: 07/09/2017] [Indexed: 01/04/2023]
Abstract
The establishment of proper conditions for spermatogonial stem cells (SSCs) cryopreservation and storage represents an important biotechnological approach for the preservation of the genetic stock of valuable animals. This study demonstrates the effects of different cryopreservation protocols on the survival rates and phenotypic expression of SSCs in horses. The cells were enzymatically isolated from testes of eight adult horses. After enrichment and characterization of germ cells in the suspension, the feasibility of several cryopreservation protocols were evaluated. Three different cryomedia compositions, associated with three different methods of freezing (vitrification, slow-freezing and fast-freezing) were evaluated. Based on the rates of viable SSCs found before and after thawing, as well as the number of recovered cells after cryopreservation, the best results were obtained utilizing the DMSO-based cryomedia associated with the slow-freezing method. In addition, when isolated cells were cultured in vitro, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and immunofluorescence analysis indicated that the cryopreserved cells were as metabolically active as the fresh cells and were also expressing typical SSCs proteins (VASA, NANOS2 and GFRA1). Therefore, our results indicate that equine SSCs can be cryopreserved without impairment of structure, function, or colony-forming abilities.
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Affiliation(s)
- Guilherme M J Costa
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil.
| | - Gleide F Avelar
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil
| | - Samyra M S N Lacerda
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil
| | - André F A Figueiredo
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil
| | - Amanda O Tavares
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil
| | - José V Rezende-Neto
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil
| | - Felipe G P Martins
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil
| | - Luiz R França
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil.
- National Institute for Amazonian Research (INPA), Manaus, AM, Brazil.
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Ma F, Zhou Z, Li N, Zheng L, Wu C, Niu B, Tang F, He X, Li G, Hua J. Lin28a promotes self-renewal and proliferation of dairy goat spermatogonial stem cells (SSCs) through regulation of mTOR and PI3K/AKT. Sci Rep 2016; 6:38805. [PMID: 27941834 PMCID: PMC5150521 DOI: 10.1038/srep38805] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2016] [Accepted: 11/15/2016] [Indexed: 12/14/2022] Open
Abstract
Lin28a is a conserved RNA-binding protein that plays an important role in development, pluripotency, stemness maintenance, proliferation and self-renewal. Early studies showed that Lin28a serves as a marker of spermatogonial stem cells (SSCs) and promotes the proliferation capacity of mouse SSCs. However, there is little information about Lin28a in livestock SSCs. In this study, we cloned Capra hircus Lin28a CDS and found that it is evolutionarily conserved. Lin28a is widely expressed in different tissues of Capra hircus, but is expressed at a high level in the testis. Lin28a is specifically located in the cytoplasm of Capra hircus spermatogonial stem cells and may also be a marker of dairy goat spermatogonial stem cells. Lin28a promoted proliferation and maintained the self-renewal of GmGSCs-I-SB in vivo and in vitro. Lin28a-overexpressing GmGSCs-I-SB showed an enhanced proliferation rate, which might be due to increased PCNA expression. Moreover, Lin28a maintained the self-renewal of GmGSCs-I-SB by up-regulating the expression of OCT4, SOX2, GFRA1, PLZF and ETV5. Furthermore, we found that Lin28a may activate the AKT, ERK, and mTOR signaling pathways to promote the proliferation and maintain the self-renewal of GmGSCs-I-SB.
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Affiliation(s)
- Fanglin Ma
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering &Technology, Northwest A&F University, Yangling, Shaanxi, 712100 China
| | - Zhe Zhou
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering &Technology, Northwest A&F University, Yangling, Shaanxi, 712100 China
| | - Na Li
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering &Technology, Northwest A&F University, Yangling, Shaanxi, 712100 China
| | - Liming Zheng
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering &Technology, Northwest A&F University, Yangling, Shaanxi, 712100 China
| | - Chongyang Wu
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering &Technology, Northwest A&F University, Yangling, Shaanxi, 712100 China
| | - Bowen Niu
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering &Technology, Northwest A&F University, Yangling, Shaanxi, 712100 China
| | - Furong Tang
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering &Technology, Northwest A&F University, Yangling, Shaanxi, 712100 China
| | - Xin He
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering &Technology, Northwest A&F University, Yangling, Shaanxi, 712100 China
| | - Guangpeng Li
- Key Laboratory for Mammalian Reproductive Biology and Biotechnology, Ministry of Education, Inner Mongolia University, Hohhot, 010021, China
| | - Jinlian Hua
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering &Technology, Northwest A&F University, Yangling, Shaanxi, 712100 China
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Aponte PM. Spermatogonial stem cells: Current biotechnological advances in reproduction and regenerative medicine. World J Stem Cells 2015; 7:669-680. [PMID: 26029339 PMCID: PMC4444608 DOI: 10.4252/wjsc.v7.i4.669] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/28/2015] [Revised: 03/13/2015] [Accepted: 04/14/2015] [Indexed: 02/06/2023] Open
Abstract
Spermatogonial stem cells (SSCs) are the germ stem cells of the seminiferous epithelium in the testis. Through the process of spermatogenesis, they produce sperm while concomitantly keeping their cellular pool constant through self-renewal. SSC biology offers important applications for animal reproduction and overcoming human disease through regenerative therapies. To this end, several techniques involving SSCs have been developed and will be covered in this article. SSCs convey genetic information to the next generation, a property that can be exploited for gene targeting. Additionally, SSCs can be induced to become embryonic stem cell-like pluripotent cells in vitro. Updates on SSC transplantation techniques with related applications, such as fertility restoration and preservation of endangered species, are also covered on this article. SSC suspensions can be transplanted to the testis of an animal and this has given the basis for SSC functional assays. This procedure has proven technically demanding in large animals and men. In parallel, testis tissue xenografting, another transplantation technique, was developed and resulted in sperm production in testis explants grafted into ectopical locations in foreign species. Since SSC culture holds a pivotal role in SSC biotechnologies, current advances are overviewed. Finally, spermatogenesis in vitro, already demonstrated in mice, offers great promises to cope with reproductive issues in the farm animal industry and human clinical applications.
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Choi NY, Park YS, Ryu JS, Lee HJ, Araúzo-Bravo MJ, Ko K, Han DW, Schöler HR, Ko K. A novel feeder-free culture system for expansion of mouse spermatogonial stem cells. Mol Cells 2014; 37:473-9. [PMID: 24854861 PMCID: PMC4086341 DOI: 10.14348/molcells.2014.0080] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2014] [Revised: 04/26/2014] [Accepted: 04/28/2014] [Indexed: 12/24/2022] Open
Abstract
Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.
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Affiliation(s)
- Na Young Choi
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
| | - Yo Seph Park
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
| | - Jae-Sung Ryu
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
| | - Hye Jeong Lee
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
| | - Marcos J. Araúzo-Bravo
- Group of Computational Biology and Bioinformatics, Biodonostia Health Research Institute, San Sebastián,
Spain
- IKERBASQUE, Basque Foundation for Science, Bilbao,
Spain
| | - Kisung Ko
- Department of Medicine, Medical Research Institute, College of Medicine, Chung-Ang University, Seoul 156-756,
Korea
| | - Dong Wook Han
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
| | - Hans R. Schöler
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster 48149,
Germany
- University of Münster, Medical Faculty, 48149 Münster,
Germany
| | - Kinarm Ko
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
- Research Institute of Medical Science, Konkuk University, Seoul 143-701,
Korea
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Santos Nassif Lacerda SM, Costa GMJ, da Silva MDA, Campos-Junior PHA, Segatelli TM, Peixoto MTD, Resende RR, de França LR. Phenotypic characterization and in vitro propagation and transplantation of the Nile tilapia (Oreochromis niloticus) spermatogonial stem cells. Gen Comp Endocrinol 2013; 192:95-106. [PMID: 23792279 DOI: 10.1016/j.ygcen.2013.06.013] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/18/2013] [Revised: 06/05/2013] [Accepted: 06/11/2013] [Indexed: 12/23/2022]
Abstract
In association with in vitro culture and transplantation, isolation of spermatogonial stem cells (SSCs) is an excellent approach for investigating spermatogonial physiology in vertebrates. However, in fish, the lack of SSC molecular markers represents a great limitation to identify/purify these cells, rendering it difficult to apply several valuable biotechnologies in fish-farming. Herein, we describe potential molecular markers, which served to phenotypically characterize, cultivate and transplant Nile tilapia SSCs. Immunolocalization revealed that Gfra1 is expressed exclusively in single type A undifferentiated spermatogonia (Aund, presumptive SSCs). Likewise, the expression of Nanos2 protein was observed in Aund cells. However, Nanos2-positive spermatogonia have also been identified in cysts with two to eight germ cells that encompass type A differentiated spermatogonia (Adiff). Moreover, we also established effective primary culture conditions that allowed the Nile tilapia spermatogonia to expand their population for at least one month while conserving their original undifferentiated (stemness) characteristics. The maintenance of Aund spermatogonial phenotype was demonstrated by the expression of early germ cell specific markers and, more convincingly, by their ability to colonize and develop in the busulfan-treated adult Nile tilapia recipient testes after germ cell transplantation. In addition to advancing our knowledge on the identity and physiology of fish SSCs, these findings provide the first step in establishing a system that will allow fish SSCs expansion in vitro, representing an important progress towards the development of new biotechnologies in aquaculture, including the possibility of producing transgenic fish.
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Affiliation(s)
- Samyra Maria Santos Nassif Lacerda
- Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil
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Martin LA, Seandel M. Serial enrichment of spermatogonial stem and progenitor cells (SSCs) in culture for derivation of long-term adult mouse SSC lines. J Vis Exp 2013:e50017. [PMID: 23462452 DOI: 10.3791/50017] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells(1-3). Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines(4). However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state. Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors(5,6). Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies(7). Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types(8). Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC(2,3). The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)(3). Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.
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Affiliation(s)
- Laura A Martin
- Department of Surgery, Weill Cornell Medical College, NY, USA
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