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During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We have previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, Tsga8, plays an essential role in spermatid morphogenesis. Loss of Tsga8 in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. Tsga8 depletion leads to dysregulation of thousands of genes, including the X-chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, Tsga8 is a KMT2B target that is vitally necessary for spermiogenesis and fertility., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Mouse trophoblast stem cells (TSCs) form colonies of different sizes and morphologies, which might reflect their degrees of differentiation. Therefore, each colony type can have a characteristic gene expression profile; however, the expression levels of internal reference genes may also change, causing fluctuations in their estimated gene expression levels. In this study, we validated seven housekeeping genes by using a geometric averaging method and identified Gapdh as the most stable gene across different colony types. Indeed, when Gapdh was used as the reference, expression levels of Elf5, a TSC marker gene, stringently classified TSC colonies into two groups: a high expression groups consisting of type 1 and 2 colonies, and a lower expression group consisting of type 3 and 4 colonies. This clustering was consistent with our putative classification of undifferentiated/differentiated colonies based on their time-dependent colony transitions. By contrast, use of an unstable reference gene (Rn18s) allowed no such clear classification. Cdx2, another TSC marker, did not show any significant colony type-specific expression pattern irrespective of the reference gene. Selection of stable reference genes for quantitative gene expression analysis might be critical, especially when cell lines consisting of heterogeneous cell populations are used.

Mouse trophoblast stem cells (TSCs) proliferate indefinitely in vitro, despite their highly heterogeneous nature. In this study, we sought to characterize TSC colony types by using methods based on cell biology and biochemistry for a better understanding of how TSCs are maintained over multiple passages. Colonies of TSCs could be classified into four major types: type 1 is compact and dome-shaped, type 4 is flattened but with a large multilayered cell cluster, and types 2 and 3 are their intermediates. A time-lapse analysis indicated that type 1 colonies predominantly appeared after passaging, and a single type 1 colony gave rise to all other types. These colony transitions were irreversible, but at least some type 1 colonies persisted throughout culture. The typical cells comprising type 1 colonies were small and highly motile, and they aggregated together to form primary colonies. A hierarchical clustering based on global gene expression profiles suggested that a TSC line containing more type 1 colony cells was similar to in vivo extraembryonic tissues. Among the known TSC genes examined, Elf5 showed a differential expression pattern according to colony type, indicating that this gene might be a reliable marker of undifferentiated TSCs. When aggregated with fertilized embryos, cells from types 1 and 2, but not from type 4, distributed to the polar trophectoderm in blastocysts. These findings indicate that cells typically found in type 1 colonies can persist indefinitely as stem cells and are responsible for the maintenance of TSC lines. They may provide key information for future improvements in the quality of TSC lines., (© 2016 by the Society for the Study of Reproduction, Inc.)

Spermatogonial stem cells (SSCs) are required for spermatogenesis. Earlier studies showed that glial cell line-derived neurotrophic factor (GDNF) was indispensable for SSC self-renewal by binding to the GFRA1/RET receptor. Mice with mutations in these molecules showed impaired spermatogenesis, which was attributed to SSC depletion. Here we show that SSCs undergo GDNF-independent self-renewal. A small number of spermatogonia formed colonies when testis fragments from a Ret mutant mouse strain were transplanted into heterologous recipients. Moreover, fibroblast growth factor 2 (FGF2) supplementation enabled in vitro SSC expansion without GDNF. Although GDNF-mediated self-renewal signaling required both AKT and MAP2K1/2, the latter was dispensable in FGF2-mediated self-renewal. FGF2-depleted testes exhibited increased levels of GDNF and were enriched for SSCs, suggesting that the balance between FGF2 and GDNF levels influences SSC self-renewal in vivo. Our results show that SSCs exhibit at least two modes of self-renewal and suggest complexity of SSC regulation in vivo., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Spermatogonial stem cells (SSCs) are the only stem cells in the body that transmit genetic information to the next generation. The long-term propagation of rodent SSCs is now possible in vitro, and their genetic modification is feasible. However, their differentiation into sperm is possible only under in vivo conditions. Here we show a new in vitro system that can induce full spermatogenesis from SSC lines or any isolated SSCs. The method depends on an organ culture system onto which SSCs are transplanted. The settled SSCs form colonies and differentiate up into sperm. The resultant haploid cells are fertile, and give rise to healthy offspring through micro-insemination. In addition, the system can induce spermatogenesis from SSCs that show spermatogenic failure due to a micro-environmental defect in their original testes. Thus, an in vitro system is established that can be used to correct or manipulate the micro-environmental conditions required for proper spermatogenesis from murine SSC lines.

Spermatogonial stem cells (SSCs) undergo self-renewal divisions to support spermatogenesis. Although several in vitro SSC culture systems have been developed, these systems include serum or fibroblast feeders, which complicate SSC self-renewal analyses. Here, we developed a serum- and feeder-free culture system for long-term propagation of SSCs. In addition to the SSC self-renewal factors, including glial cell line-derived neurotrophic factor, supplementation with fetuin and lipid-associated molecules was required to drive SSC proliferation in vitro. Cultured cells proliferated for at least 6 mo at a rate comparable to that of serum-supplemented cultured cells. However, germline potential was reduced under serum- and feeder-free conditions, as indicated by a lower SSC frequency after germ cell transplantation. Nevertheless, the cultured cells completed spermatogenesis and produced offspring following spermatogonial transplantation into seminiferous tubules of infertile mice. This culture system provides a basic platform for understanding the regulation of SSC fate commitment in vitro and for improving SSC culture medium.

Spermatogonial stem cells (SSCs) are slowly dividing cells that undergo self-renewal division to support spermatogenesis. Although the effects of genetic background in stem cell self-renewal have been well studied in hematopoietic stem cells, little is known about its effect on stem cells in other self-renewing tissues, including SSCs. To examine whether genetic factors are involved in regulation of SSC self-renewal, we first studied spermatogenesis in different inbred mouse strains (C57BL/6, DBA/2, AKR, BALB/C and C3H) after chemical damage caused by busulfan. Spermatogenesis in the DBA/2 and AKR strains was relatively resistant to busulfan treatment, whereas spermatogenesis was diminished in C57BL/6 mice and nearly ablated in C3H and BALB/C mice. Serial germ cell transplantation experiments provided functional evidence that SSCs with the DBA/2 background expanded more rapidly than those with the B6 background. Finally, we also employed the Germline Stem (GS) cell culture technique to examine the self-renewal activity in vitro. Although genetic manipulation of GS cells has been limited to those from the DBA/2 background, we produced transgenic offspring of the C3H background by electroporation of GS cells with a plasmid vector. Our results underscore the importance of genetic factors in SSC self-renewal. Furthermore, application of genetic modification techniques to GS cells with non-DBA/2 backgrounds extends the potential of a SSC-based approach in male germline modification.

Although pluripotent stem cells were recently discovered in postnatal testis, attempts to analyze their developmental potential have led to conflicting claims that spermatogonial stem cells are pluripotent or that they lose spermatogenic potential after conversion into pluripotent stem cells. To examine this issue, we analyzed the developmental fate of a single spermatogonial stem cell that appeared during transfection experiments. After transfection of a neomycin-resistance gene into germline stem cells, we obtained an embryonic stem-like, multipotent germline stem cell line. Southern blot analysis revealed that the germline stem and multipotent germline stem clones have the same transgene integration pattern, demonstrating their identical origin. The two lines, however, have different DNA methylation patterns. The multipotent germline stem cells formed chimeras after blastocyst injection but did not produce sperm after germ cell transplantation, whereas the germline stem cells could produce only spermatozoa and did not differentiate into somatic cells. Interestingly, the germline stem cells expressed several transcription factors (Pou5f1, Sox2, Myc, and Klf4) required for reprogramming fibroblasts into a pluripotent state, suggesting that they are potentially pluripotent. Thus, our study provides evidence that a single spermatogonial stem cell can acquire pluripotentiality but that conversion into a pluripotent cell type is accompanied by loss of spermatogenic potential.

Although ovarian theca cells play an indispensable role in folliculogenesis by providing follicular structural integrity and steroid substrates for estrogen production, little information is available about their recruitment, growth, and differentiation because their immature forms have not been identified. We have isolated putative thecal stem cells with the ability to self-renew and differentiate in vivo and in vitro. They are similar to fibroblasts in morphology and proliferate in vitro as round colonies with a homogenous cell population. They were induced to differentiate into early precursors and steroidogenic cells in a stepwise manner after treatment with serum, luteinizing hormone, and paracrine factors from granulosa cells. At each differentiation step, these cells displayed appropriate gene expression and morphological markers and later secreted androstenedione. The fully mature morphology was achieved by coculture with isolated granulosa cells. When transplanted into the ovaries, the putative thecal stem cells colonized exclusively in the ovarian interstitium and the thecal layer of follicles as differentiated cells. Thus, thecal stem cells appear to be present in neonatal ovaries and can be isolated, purified, and induced to differentiate in vitro. Thecal stem cells could provide an invaluable in vitro experimental system to study interactions among the oocytes, granulosa cells, and theca cells during normal folliculogenesis and to study ovarian pathology caused by theca cell dysfunction.

Spermatogonial stem cells represent a self-renewing population of spermatogonia, and continuous division of these cells supports spermatogenesis throughout the life of adult male animals. Previous attempts to introduce adenovirus vectors into spermatogenic cells, including spermatogonial stem cells, have failed to yield evidence of infection, suggesting that male germ cells may be resistant to adenovirus infection. In this study we show the feasibility of transducing spermatogonial stem cells by adenovirus vectors. When testis cells from ROSA26 Cre reporter mice were incubated in vitro with a Cre-expressing adenovirus vector, Cre-mediated recombination occurred at an efficiency of 49-76%, and the infected spermatogonial stem cells could reinitiate spermatogenesis after transplantation into seminiferous tubules of infertile recipient testes. No evidence of germ-line integration of adenovirus vector could be found in offspring from infected stem cells that underwent Cre-mediated recombination, which suggests that the adenovirus vector infected the cells but did not stably integrate into the germ line. Nevertheless, these results suggest that adenovirus may inadvertently integrate into the patient's germ line and indicate that there is no barrier to adenovirus infection in spermatogonial stem cells.

Testicular germ cell transplantation into the seminiferous tubules is at present the only way to induce spermatogenesis from a given source of spermatogonial stem cells. Here we show an alternative method that harnesses the self-organizing ability of testicular somatic cells. The testicular cells of embryonic or neonatal mice or rats and of newborn pigs were dissociated into single cells. Each of them reorganized into a tubular structure following implantation into the subcutis of immunodeficient mice. When mouse germline stem (GS) cells derived from spermatogonial stem cells and expanded in culture were intermingled with testicular cells of rodents, they were integrated in the reconstituted tubules and differentiated beyond meiosis into spermatids. Normal offspring were produced by the microinjection of those spermatids into oocytes. This method could be applicable to various mammalian species and useful for producing functional gametes from GS cells in a xenoectopic environment.

Spermatogonial stem cells continuously divide in the testis to support spermatogenesis throughout the life of adult male animals. Although very few spermatogonial stem cells are present in vivo, we recently succeeded in expanding these cells in vitro. Germ cells from postnatal testes were able to proliferate in the presence of several types of cytokines, and they formed uniquely shaped colonies of spermatogonia (germline stem or GS cells). These cells reinitiated normal spermatogenesis when transplanted into seminiferous tubules. However, much remains unknown about the contributions of cytokines to successful stem cell culture. In the present study, we examined the role of leukemia inhibitory factor (LIF) in GS cell culture. We found that the addition of LIF to newborn testis cell culture enhances the formation of germ cell colonies. Ciliary neurotrophic factor, but not oncostatin M, had the same effect, although they both bind to the IL-6ST (gp130) receptor. On the other hand, GS cells could be established from pup or adult testes in the absence of LIF. No phenotypic or functional difference was found between GS cells established from different stages, and normal offspring were born from pup-derived GS cells that had been maintained in the absence of LIF, indicating that LIF per se is not involved in the self-renewal of GS cells. These results demonstrate that LIF is useful in the initiation of GS cell culture and suggest that LIF or a related cytokine is involved in the maturation of gonocytes into spermatogonia.

Spermatogenesis originates from a small number of spermatogonial stem cells that can reinitiate spermatogenesis and produce germ cell colonies following transplantation into infertile recipient testes. Although several previous studies have suggested a single-cell origin of germ cell colonies, only indirect evidence has been presented. In this investigation, we tested the clonal origin hypothesis using a retrovirus, which could specifically mark an individual spermatogonial stem cell. Spermatogonial stem cells were infected in vitro with an enhanced green fluorescence protein-expressing retrovirus and subsequently transplanted into infertile recipient mice. Live haploid germ cells were recovered from individual colonies and were microinjected into eggs to create offspring. In total, 45 offspring were produced from five colonies, and 23 (51%) of the offspring were transgenic. Southern blot analysis indicated that the transgenic offspring from the single colony carried a common integration site, and the integration site was different among the transgenic offspring from different colonies. These results provide evidence that germ cell colonies develop from single spermatogonial stem cells.

Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successful gene trapping and homologous recombination in spermatogonial stem cells. Cultured spermatogonial stem cells were transfected with gene trap or gene targeting vectors. Mutagenized stem cells were expanded clonally by drug selection. These cells underwent spermatogenesis and produced heterozygous offspring after transplantation into the seminiferous tubules of infertile mouse testes. Heterozygous mutant mice were intercrossed to produce homozygous gene knockouts. Using this strategy, the efficiency of homologous recombination for the occludin gene locus was 1.7% using a nonisogenic DNA construct. These results demonstrate the feasibility of altering genes in tissue-specific stem cells in a manner similar to embryonic stem cells and have important implications for gene therapy and animal transgenesis.

Spermatogenesis originates from a small number of spermatogonial stem cells that reside on the basement membrane and undergo self-renewal division to support spermatogenesis throughout the life of adult animals. Although the recent development of a technique to culture spermatogonial stem cells allowed reproduction of self-renewal division in vitro, much remains unknown about how spermatogonial stem cells are regulated. In this study, we found that spermatogonial stem cells could be cultured in an anchorage-independent manner, which is characteristic of stem cells from other types of self-renewing tissues. Although the cultured cells grew slowly (doubling time, approximately 4.7 days), they expressed markers of spermatogonia, and grew exponentially for at least 5 months to achieve 1.5 x 10(10) -fold expansion. The cultured cells underwent spermatogenesis following transplantation into the seminiferous tubules of infertile animals and fertile offspring were obtained by microinsemination of germ cells that had developed within the testes of recipients of the cultured cells. These results indicate that spermatogonial stem cells can undergo anchorage-independent, self-renewal division, and suggest that stem cells have the common property to survive and proliferate in the absence of exogenous substrata.

Although stem cells are believed to divide infinitely by self-renewal division, there is little evidence that demonstrates their infinite replicative potential. Spermatogonial stem cells are the founder cell population for spermatogenesis. Recently, in vitro culture of spermatogonial stem cells was described. Spermatogonial stem cells can be expanded in vitro in the presence of glial cell line-derived neurotrophic factor (GDNF), maintaining the capacity to produce spermatogenesis after transplantation into testis. Here, we examined the stability and proliferative capacity of spermatogonial stem cells using cultured cells. Spermatogonial stem cells were cultured over 2 years and achieved approximately 10(85)-fold expansion. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, spermatogonial stem cells retained the euploid karyotype and androgenetic imprint during the 2-year experimental period, and produced normal spermatogenesis and fertile offspring. However, the telomeres in spermatogonial stem cells gradually shortened during culture, suggesting that they are not immortal. Nevertheless, the remarkable stability and proliferative potential of spermatogonial stem cells suggest that they have a unique machinery to prevent transmission of genetic and epigenetic damages to the offspring, and these characteristics make them an attractive target for germline modification.

Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to the next generation. These cells can be cultured for extended periods in the presence of serum and feeder cells. However, little is known about factors that regulate self-renewal division of spermatogonial stem cells. In this investigation we examined the possibility of establishing culture systems for spermatogonial stem cells that lack serum or a feeder cell layer. Spermatogonial stem cells could expand in serum-free conditions on mouse embryonic fibroblasts (MEFs), or were successfully cultivated without feeder cells on a laminin-coated plate. However, they could not expand when both serum and feeder cells were absent. Although the cells cultured on laminin differed phenotypically from those on feeder cells, they grew exponentially for at least 6 mo, and produced normal, fertile progeny following transplantation into infertile mouse testis. This culture system will provide a new opportunity for understanding the regulatory mechanism that governs spermatogonial stem cells.

Spermatogenesis is a complex process that originates in a small population of spermatogonial stem cells. Here we report the in vitro culture of spermatogonial stem cells that proliferate for long periods of time. In the presence of glial cell line-derived neurotrophic factor, epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor, gonocytes isolated from neonatal mouse testis proliferated over a 5-month period (>10(14)-fold) and restored fertility to congenitally infertile recipient mice following transplantation into seminiferous tubules. Long-term spermatogonial stem cell culture will be useful for studying spermatogenesis mechanism and has important implications for developing new technology in transgenesis or medicine.

ESCs are most commonly derived from embryos originating from oocytes that reached metaphase II. We describe here a novel approach where ESCs with all pluripotency parameters were established from oocytes in which metaphase I was converted, from the cell cycle perspective, directly into metaphase II-like stage without the intervening anaphase to telophase I transition. The resulting embryos initiate development and reach the blastocyst stage from which the ESC lines are then established. Thus, our approach could represent an ethically acceptable method that can exploit oocytes that are typically discarded in in vitro fertilization clinics. Moreover, our results also indicate that the meiotic cell cycle can be converted into mitosis by modulating chromosomal contacts that are typical for meiosis with subsequent licensing of chromatin for DNA replication. S TEM C ELLS 2011;29:517-527 [ABSTRACT FROM AUTHOR]

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%–5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency. [ABSTRACT FROM AUTHOR]

Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility¹. Here we show that Cnot7-/- males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor², is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7-/- mice. Transplantation of spermatogonial stem cells from male Cnot7-/- mice to seminiferous tubules of Kit mutant mice (KitW/W-v) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7-/- condition. The testicular phenotypes of Cnot7-/- mice are similar to those of mice deficient in retinoid X receptor beta (Rut)3. We further show that Cnot7-/- binds the AF-1 domain of Rxrh and that Rxrh malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, we assessed the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells. [ABSTRACT FROM AUTHOR]

Abstract: Spermatogonial stem cells can convert into embryonic stem (ES) cell-like multipotent germline stem (mGS) cells in vitro and produce germline chimeras by blastocyst microinjection. Although homologous recombination was previously demonstrated in mGS cells, spermatogenesis was not found in chimeras, suggesting that they are not competent for germline modification. Here we conducted detailed analysis of chimeric animals to determine whether mGS cells retain germline potential after genetic manipulation. Spermatozoa that were deficient in the occludin gene could be recovered from animals that were chimeric with mGS cells that underwent homologous recombination. The phenotypes of the occludin knockout (KO) mice were similar to those reported for KO mice produced using ES cells, and the animals showed growth retardation, gastritis and male infertility. Furthermore, we found that heterozygous mGS cells acquire two copies of the G418-resistant genes and become homozygous for the targeted allele by culturing at high concentrations of G418. Cytogenetic analysis showed that the aneuploid mGS cells observed during genetic manipulation were trisomic for chromosome 8 or 11, which is a common chromosomal abnormality in ES cells. Thus, mGS cells can be used to produce KO animals, and this novel method of germline manipulation may prove useful in diverse mammalian species. [Copyright &y& Elsevier]