scholarly journals Genomic stability of mouse spermatogonial stem cells in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shinichiro Chuma ◽  
Mito Kanatsu-Shinohara ◽  
Ami Katanaya ◽  
Mihoko Hosokawa ◽  
Takashi Shinohara
Keyword(s):  
Stem Cells ◽  
Mutation Rate ◽  
Chromosomal Abnormalities ◽  
Embryonic Stem ◽  
Germline Mutations ◽  
Spermatogonial Stem Cells ◽  
Population Doubling ◽  
Seminiferous Tubules ◽  
Selective Growth Advantage

AbstractGermline mutations underlie genetic diversity and species evolution. Previous studies have assessed the theoretical mutation rates and spectra in germ cells mostly by analyzing genetic markers and reporter genes in populations and pedigrees. This study reported the direct measurement of germline mutations by whole-genome sequencing of cultured spermatogonial stem cells in mice, namely germline stem (GS) cells, together with multipotent GS (mGS) cells that spontaneously dedifferentiated from GS cells. GS cells produce functional sperm that can generate offspring by transplantation into seminiferous tubules, whereas mGS cells contribute to germline chimeras by microinjection into blastocysts in a manner similar to embryonic stem cells. The estimated mutation rate of GS and mGS cells was approximately 0.22 × 10−9 and 1.0 × 10−9 per base per cell population doubling, respectively, indicating that GS cells have a lower mutation rate compared to mGS cells. GS and mGS cells also showed distinct mutation patterns, with C-to-T transition as the most frequent in GS cells and C-to-A transversion as the most predominant in mGS cells. By karyotype analysis, GS cells showed recurrent trisomy of chromosomes 15 and 16, whereas mGS cells frequently exhibited chromosomes 1, 6, 8, and 11 amplifications, suggesting that distinct chromosomal abnormalities confer a selective growth advantage for each cell type in vitro. These data provide the basis for studying germline mutations and a foundation for the future utilization of GS cells for reproductive technology and clinical applications.

scholarly journals Propagation of bovine spermatogonial stem cells in vitro

Reproduction ◽  
2008 ◽  
Vol 136 (5) ◽  
pp. 543-557 ◽  
Author(s):  
Pedro M Aponte ◽  
Takeshi Soda ◽  
Katja J Teerds ◽  
S Canan Mizrak ◽  
Henk J G van de Kant ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Cultured Cells ◽  
Somatic Cells ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Type A Spermatogonia ◽  
Stimulation Of

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study thein vitrobehavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

scholarly journals Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2265
Author(s):  
Whitney F. Alpaugh ◽  
Anna L. Voigt ◽  
Rkia Dardari ◽  
Lin Su ◽  
Iman Al Khatib ◽  
...  
Keyword(s):  
Stem Cells ◽  
Metabolic Regulation ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Testis Weight ◽  
Stem And Progenitor Cells ◽  
Cell Transcriptome ◽  
Carboxy Terminal

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1−/− mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1−/− mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1−/− compared to wild-type mice. Furthermore, cultured Uch-l1−/− SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

scholarly journals Cryopreserved fragments of testicular seminiferous tubules of rats as a source of spermatogonial stem cells

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
N. Volkova ◽  
◽  
M. Yukhta ◽  
L. Sokil ◽  
L. Chernyschenko ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Morphological Characteristics ◽  
Relative Number ◽  
Spermatogonial Stem Cells ◽  
Blue Staining ◽  
Seminiferous Tubules ◽  
Proliferative Potential ◽  
Tissue Samples

The use of modern technologies of cryopreservation of testicular tissue samples in prepubertal patients is one of the ways to maintain their fertility in the future. The purpose of the study was to investigate the proliferative potential, morphological characteristics and expression of specific markers of cell culture obtained from cryopreserved and vitrified fragments of seminiferous tubules (FSTs) of rats' testis. Materials and methods. The isolation of cells from native, cryopreserved and vitrified FSTs of immature rats was performed by incubation in a solution of collagenase type IV (1 mg/mL) + DNase (500 μg/mL). Cell viability was determined by Trypan blue staining. Monoclonal antibodies CD9-FITC, CD24-PE, CD45-FITC, CD90-FITC were used for immunophenotype analysis. Morphological characteristics, proliferative activity (MTT assay), relative number of cells positive for MAGE-B1 and vimentin were assessed in the obtained cultures. Results. The analysis of phenotypic characteristics showed that cells from native, cryopreserved and vitrified FSTs were characterized by high expression level of CD9 (≥ 40 %), CD24 (≥ 70 %), CD90 (≥ 70 %) and low expression of the CD45 (≤ 1 %). In cell culture in vitro, the studied cells from cryopreserved and vitrified rat's FSTs had the ability to adhere and proliferate while maintaining a cells population positive for MAGE-B1 and vimentin. Conclusions. The results can be the basis for the development of effective protocols for the cultivation and cryopreservation of testicular spermatogonial stem cells in order to restore fertility in men.

288 THE EXPRESSION PATTERN OF ACETYLATED ALPHA-TUBULIN IS CONSERVED IN PORCINE AND MURINE SPERMATOGONIAL STEM CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 223
Author(s):  
J. Luo ◽  
S. Megee ◽  
I. Dobrinski
Keyword(s):  
Stem Cells ◽  
Basement Membrane ◽  
Expression Pattern ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Histone Deacetylase 6 ◽  
Pgp 9.5

During mammalian spermatogenesis, spermatogonial stem cells (SSCs) reside in the stem cell niche on the basement membrane where they undergo self-renewing divisions. Differentiating daughter cells are located progressively more toward the tubular lumen where they ultimately form spermatozoa. The mechanisms responsible for maintenance of SSCs at the basement membrane are unclear. Microtubules consisting of α/β-tubulin heterodimers are associated with many cellular functions. Reversible acetylation of α-tubulin at Lys40 has been implicated in regulating microtubule stability and function. Acetylation of α-tubulin is abundant in stable microtubules but absent from dynamic cellular structures. Deacetylation of α-tubulin is controlled by histone deacetylase 6 which is predominantly expressed in mouse testis. Here, we tested the hypothesis that differential acetylation of α-tubulin might be involved in maintenance of SSCs. Immunohistochemistry for acetylated α-tubulin (Ac-α-Tu) and the spermatogonia specific proteins PGP 9.5, DAZL, and PLZF were used to characterize the expression pattern of Ac-α-Tu in porcine and murine germ cells at different stages of testis development. In immature boar testes, Ac-α-Tu was present exclusively in gonocytes but not in other testicular cells at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating to the basement membrane of the seminiferous tubules, and Ac-α-Tu appeared to be polarized toward the basement membrane. In immature mouse testes, Ac-α-Tu was present in germ cells and Sertoli cells at 6 days of age, whereas at 2 weeks of age, Ac-α-Tu expression was stronger in spermatogonia co-expressing PGP 9.5 and in spermatocytes than in Sertoli cells or PGP 9.5-negative spermatogonia. In adult boar and mouse testes, Ac-α-Tu was detected in a few single or paired spermatogonia expressing PGP 9.5 localized on the basement membrane as well as in spermatocytes, spermatids, and spermatozoa. Spermatogonia with high levels of Ac-α-Tu expressed PLZF but did not express DAZL, suggesting that only undifferentiated spermatogonia maintain a high level of Ac-α-Tu. When seminiferous tubules from 1-week-old and adult boar testes were maintained in vitro for 1–2 days, high levels of Ac-α-Tu were detected in single or paired round spermatogonia with a large nucleus, compared to low levels in elongated paired and aligned spermatogonia. The unique expression pattern of Ac-α-Tu in undifferentiated germ cells during postnatal development appears to be conserved in mammalian testes. Since Ac-α-Tu is a component of long-lived stable microtubules and reducing acetylation of α-tubulin enhances cell motility, these results suggest that stabilization of microtubules might contribute to the maintenance of spermatogonial stem cells. This work was supported by 1R01 RR 17359-05.

scholarly journals Compromised Chondrocyte Differentiation Capacity in TERC Knockout Mouse Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

2019 ◽  
Vol 20 (5) ◽  
pp. 1236 ◽  
Author(s):  
Wei-Fang Chang ◽  
Yun-Hsin Wu ◽  
Jie Xu ◽  
Li-Ying Sung
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Population Doubling ◽  
Chondrocyte Differentiation ◽  
Wild Type

Mammalian telomere lengths are primarily regulated by telomerase, consisting of a reverse transcriptase protein (TERT) and an RNA subunit (TERC). We previously reported the generation of mouse Terc+/− and Terc−/− embryonic stem cells (ntESCs) by somatic cell nuclear transfer. In the present work, we investigated the germ layer development competence of Terc−/−, Terc+/− and wild-type (Terc+/+) ntESCs. The telomere lengths are longest in wild-type but shortest in Terc−/− ntESCs, and correlate reversely with the population doubling time. Interestingly, while in vitro embryoid body (EB) differentiation assay reveals EB size difference among ntESCs of different genotypes, the more stringent in vivo teratoma assay demonstrates that Terc−/− ntESCs are severely defective in differentiating into the mesodermal lineage cartilage. Consistently, in a directed in vitro chondrocyte differentiation assay, the Terc−/− cells failed in forming Collagen II expressing cells. These findings underscore the significance in maintaining proper telomere lengths in stem cells and their derivatives for regenerative medicine.

scholarly journals miRNA Signature in Mouse Spermatogonial Stem Cells Revealed by High-Throughput Sequencing

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Tao Tan ◽  
Yanfeng Zhang ◽  
Weizhi Ji ◽  
Ping Zheng
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Spermatogonial Stem Cells ◽  
Mouse Embryonic Stem Cells ◽  
Elevated Expression

Spermatogonial stem cells (SSCs) play fundamental roles in spermatogenesis. Although a handful of genes have been discovered as key regulators of SSC self-renewal and differentiation, the regulatory network responsible for SSC function remains unclear. In particular, small RNA signatures during mouse spermatogenesis are not yet systematically investigated. Here, using next generation sequencing, we compared small RNA signatures of in vitro expanded SSCs, testis-derived somatic cells (Sertoli cells), developing germ cells, mouse embryonic stem cells (ESCs), and mouse mesenchymal stem cells among mouse embryonic stem cells (ESCs) to address small RNA transition during mouse spermatogenesis. The results manifest that small RNA transition during mouse spermatogenesis displays overall declined expression profiles of miRNAs and endo-siRNAs, in parallel with elevated expression profiles of piRNAs, resulting in the normal biogenesis of sperms. Meanwhile, several novel miRNAs were preferentially expressed in mouse SSCs, and further investigation of their functional annotation will allow insights into the mechanisms involved in the regulation of SSC activities. We also demonstrated the similarity of miRNA signatures between SSCs and ESCs, thereby providing a new clue to understanding the molecular basis underlying the easy conversion of SSCs to ESCs.

Embryonic stem cell derived germ cells induce spermatogenesis after transplantation into the testes of an adult mouse azoospermia model

2017 ◽  
Vol 131 (18) ◽  
pp. 2381-2395 ◽  
Author(s):  
Zohreh Makoolati ◽  
Mansoureh Movahedin ◽  
Mehdi Forouzandeh-Moghadam ◽  
Majid Naghdi ◽  
Morteza Koruji
Keyword(s):  
Stem Cells ◽  
Sertoli Cells ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Leukaemia Inhibitory Factor ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Morphogenetic Protein ◽  
Cell Gene Expression

The present study aimed to: (i) identify the exogenous factors that allow in vitro differentiation of mouse spermatogonial stem cells (SSCs) from embryonic stem cells (ESCs); (ii) evaluate the effects of Sertoli cells in SSC enrichment; and (iii) assess the success of transplantation using in vitro differentiated SSCs in a mouse busulfan-treated azoospermia model. A 1-day-old embryoid body (EB) received 5 ng/ml of bone morphogenetic protein 4 (BMP4) for 4 days, 3 µM retinoic acid (RA) in a SIM mouse embryo-derived thioguanine and ouabain resistant (STO) co-culture system for 7 days, and was subsequently co-cultured for 2 days with Sertoli cells in the presence or absence of a leukaemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and RA composition, and in the presence of these factors in simple culture medium. Higher viability, proliferation and germ cell gene expression were seen in the presence of the LIF, bFGF and RA composition, on top of Sertoli cells. Immunocytochemistry results showed higher CDH1 expression in this group. Sertoli co-culture had no effects on SSC proliferation. Eight weeks after transplantation, injected cells were observed at the base of the seminiferous tubules and in the recipient testes. The number of spermatogonia and the mass of the testes were higher in transplanted testes relative to the control group. It seems that transplantation of these cells can be useful in infertility treatment.

scholarly journals Preservation of fertility in nature and ART

Reproduction ◽  
2002 ◽  
pp. 3-11 ◽  
Author(s):  
R Gosden ◽  
M Nagano
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Reproductive Technologies ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Natural Fertility ◽  
Fertilization In Vitro ◽  
Gonadal Failure

Individuals may regard reproduction as optional but sufficient number of them must be productive to perpetuate the species. The reproductive system is surprisingly vulnerable and depends, among other things, on a limited endowment of oocytes, controlled proliferation of spermatogonial stem cells and the genetic integrity of both. The developmental competence of oocytes and spermatogonial stem cells is maintained by evolved mechanisms for cellular detoxification and genomic stability, and excess or damaged cells are eliminated by apoptosis. Gonadal failure as a result of germ cell depletion can occur at any age, and from the effects of chemical cytotoxicity, disease and infection as well as genetic predisposition. Among extrinsic factors, alkylating agents and ionizing radiation are important causes of iatrogenic gonadal failure in young women and men. In animal models, there is evidence that hormonal manipulation, deletion of genes involved in apoptotic pathways and dietary manipulation can protect against natural and induced germ cell loss, but evidence in humans is absent or unclear. Assisted reproductive technologies (ARTs) provide an ensemble of strategies for preserving fertility in patients and commercially valuable or endangered species. Semen cryopreservation was the first technology for preserving male fertility, but this cannot serve prepubertal boys, for whom banking of testicular biopsies may provide a future option. In sterilized rodents, cryopreserved spermatogonial stem cells can recolonize seminiferous tubules and reinitiate spermatogenesis, and subcutaneous implantation of intact tubules can generate spermatozoa for fertilization in vitro by intracytoplasmic sperm injection. Transplantation of frozen-banked ovarian tissue is well-established for restoring cyclicity and fertility and is currently undergoing clinical evaluation for cancer patients. When restoration of natural fertility is unnecessary or reimplantation is unsafe, it is desirable to culture the germ cells from thawed tissue in vitro until they reach the stage at which they can be fertilized. Low temperature banking of immature germ cells is potentially very versatile, but storage of embryos and, to a lesser extent, mature oocytes is already practised in a number of species, including humans, and is likely to remain a mainstay for fertility preservation.

scholarly journals Mouse thy1-positive spermatogonia suppress the proliferation of spermatogonial stem cells by Extracellular vesicles in vitro

2020 ◽  
Author(s):  
Yu Lin ◽  
Qian Fang ◽  
Yue He ◽  
Xiaowen Gong ◽  
Yinjuan Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Vesicles ◽  
Magnetic Beads ◽  
Spermatogonial Stem Cells ◽  
The Self ◽  
Seminiferous Tubules ◽  
Self Renewal

ABSTRACTThe self-renewal of mammalian spermatogonial stem cells (SSCs) supports spermatogenesis to produce spermatozoa, and this is precisely controlled in a stem niche microenvironment in the seminiferous tubules. Although studies have revealed the role of the surrounding factors in SSCs, little is known about whether the division of SSCs is controlled by extracellular vesicles. Here, extracellular vesicles were found in the basal compartment of seminiferous tubules in mouse, rat, rabbit and human testes. In the mice, the testicular extracellular vesicles are secreted by spermatogonia and are taken up by SSCs. Further, the extracellular vesicles from thy1-positive spermatogonia were purified by anti-Thy1-coupled magnetic beads, and which suppress their proliferation of SSCs but not lead to the apoptosis in vitro.

scholarly journals MicroRNA-202 prevents precocious spermatogonial differentiation and meiotic initiation during mouse spermatogenesis

2021 ◽  
Author(s):  
Jian Chen ◽  
Chenxu Gao ◽  
Xiwen Lin ◽  
Yan Ning ◽  
Wei He ◽  
...  
Keyword(s):  
Stem Cells ◽  
Regulatory Network ◽  
Spermatogonial Stem Cells ◽  
Mirna Biogenesis ◽  
Seminiferous Tubules ◽  
Wild Type ◽  
Number Of Genes ◽  
Direct Target ◽  
Meiotic Initiation

Spermatogonial differentiation and meiotic initiation during spermatogenesis are tightly regulated by a number of genes including those coding enzymes for miRNA biogenesis. However, whether and how single miRNAs regulate these processes remain unclear. Here, we report that miR-202, a member of the let-7 family, prevents precocious spermatogonial differentiation and meiotic initiation in spermatogenesis by regulating the timely expression of many genes including those for other key regulators. In miR-202 knockout (KO) mice, the undifferentiated spermatogonial pool is reduced, ultimately causing agametic seminiferous tubules. SYCP3, STRA8 and DMRT6 are expressed earlier in KO mice than in wild-type (WT) littermates, and Dmrt6 mRNA is a direct target of miR-202-5p. Moreover, the precocious spermatogonial differentiation and meiotic initiation were also observed in KO spermatogonial stem cells when cultured and induced in vitro, and could be rescued by the knockdown of Dmrt6. Therefore, we have not only shown that miR-202 is a novel regulator of meiotic initiation but also added a new module to the underlying regulatory network.

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