CYP26B1 promotes male germ cell differentiation by suppressing STRA8-dependent meiotic and STRA8-independent mitotic pathways

Germ cell sex is defined by factors derived from somatic cells. CYP26B1 is known to be a male sex-promoting factor that inactivates retinoic acid (RA) in somatic cells. In CYP26B1-null XY gonads, germ cells are exposed to a higher level of RA than in normal XY gonads and this activates Stra8 to induce meiosis while male-specific gene expression is suppressed. However, it is unknown whether meiotic entry by an elevated level of RA is responsible for the suppression of male-type gene expression. To address this question, we have generated Cyp26b1/Stra8 double knockout (dKO) embryos. We successfully suppressed the induction of meiosis in CYP26B1-null XY germ cells by removing the Stra8 gene. Concomitantly, we found that the male genetic program represented by the expression of NANOS2 and DNMT3L was totally rescued in about half of dKO germ cells, indicating that meiotic entry causes the suppression of male differentiation. However, half of the germ cells still failed to enter the appropriate male pathway in the dKO condition. Using microarray analyses together with immunohistochemistry, we found that KIT expression was accompanied by mitotic activation, but was canceled by inhibition of the RA signaling pathway. Taken together, we conclude that inhibition of RA is one of the essential factors to promote male germ cell differentiation, and that CYP26B1 suppresses two distinct genetic programs induced by RA: a Stra8-dependent meiotic pathway, and a Stra8-independent mitotic pathway.     

RALDH2, the enzyme for retinoic acid synthesis, mediates meiosis initiation in germ cells of the female embryonic chickens

Meiosis is a process unique to the differentiation of germ cells and exhibits sex-specific in timing. Previous studies showed that retinoic acid (RA) as the vitamin A metabolite is crucial for controlling Stra8 (Stimulated by retinoic acid gene 8) expression in the gonad and to initiate meiosis; however, the mechanism by which retinoid-signaling acts has remained unclear. In the present study, we investigated the role of the enzyme retinaldehyde dehydrogenase 2 (RALDH2) which catalyzes RA synthesizes by initiating meiosis in chicken ovarian germ cells. Meiotic germ cells were first detected at day 15.5 in chicken embryo ovary when the expression of synaptonemal complex protein 3 (Scp3) and disrupted meiotic cDNA 1 homologue (Dmc1) became elevated, while Stra8 expression was specifically up-regulated at day 12.5 before meiosis onset. It was observed from the increase in Raldh2 mRNA expression levels and decreases in Cyp26b1 (the enzyme for RA catabolism) expression levels during meiosis that requirement for RA accumulation is essential to sustain meiosis. This was also revealed by RA stimulation of the cultured ovaries with the initiation of meiosis response, and the knocking down of the Raldh2 expression during meiosis, leading to abolishment of RA-dependent action. Altogether, these studies indicate that RA synthesis by the enzyme RALDH2 and signaling through its receptor is crucial for meiosis initiation in chicken embryonic ovary.     

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes

During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63γ expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway.     

Notch pathway regulates female germ cell meiosis progression and early oogenesis events in fetal mouse

A critical process of early oogenesis is the entry of mitotic oogonia into meiosis, a cell cycle switch regulated by a complex gene regulatory network. Although Notch pathway is involved in numerous important aspects of oogenesis in invertebrate species, whether it plays roles in early oogenesis events in mammals is unknown. Therefore, the rationale of the present study was to investigate the roles of Notch signaling in crucial processes of early oogenesis, such as meiosis entry and early oocyte growth. Notch receptors and ligands were localized in mouse embryonic female gonads and 2 Notch inhibitors, namely DAPT and L-685,458, were used to attenuate its signaling in an in vitro culture system of ovarian tissues from 12.5 days post coitum (dpc) fetus. The results demonstrated that the expression of Stra8, a master gene for germ cell meiosis, and its stimulation by retinoic acid (RA) were reduced after suppression of Notch signaling, and the other meiotic genes, Dazl, Dmc1, and Rec8, were abolished or markedly decreased. Furthermore, RNAi of Notch1 also markedly inhibited the expression of Stra8 and SCP3 in cultured female germ cells. The increased methylation status of CpG islands within the Stra8 promoter of the oocytes was observed in the presence of DAPT, indicating that Notch signaling is probably necessary for maintaining the epigenetic state of this gene in a way suitable for RA stimulation. Furthermore, in the presence of Notch inhibitors, progression of oocytes through meiosis I was markedly delayed. At later culture periods, the rate of oocyte growth was decreased, which impaired subsequent primordial follicle assembly in cultured ovarian tissues. Taken together, these results suggested new roles of the Notch signaling pathway in female germ cell meiosis progression and early oogenesis events in mammals.     

Male differentiation of germ cells induced by embryonic age-specific Sertoli cells in mice

Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells.     

Effect of BMP4 preceded by retinoic acid and co‐culturing ovarian somatic cells on differentiation of mouse embryonic stem cells into oocyte‐like cells

Bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) signaling are the key regulators for germ cell and meiosis induction, respectively. Gonadal tissue also provides an appropriate microenvironment for oocyte differentiation in vivo. The current study aimed to determine whether mimicking in vivo niche is more efficient for oocyte differentiation from embryonic stem (ES) cells. Here, differentiation of mouse ES cells toward oocyte‐like cells using embryoid body (EB) and monolayer protocols was induced in the presence (+BMP4) or absence (‐BMP4) of BMP4. On day 5, each group was co‐cultured with ovarian somatic cells in the presence or absence of RA (+RA or –RA) for an additional 14 days. Our results showed a significant increase in expression of meiotic markers in the +BMP4 condition in EB differentiation protocol. Further differentiation with ovarian somatic cells led to a subpopulation of oocyte‐like cell formation. Compared to the controls, the +RA condition resulted in a significant elevation of the meiotic gene expression in contrast to Oct4 that significantly decreased in both protocols. In the cells pre‐treated with BMP4 and then exposed to RA in the monolayer differentiation protocol, the gene expression levels of germ cell, Mvh, and maturation markers, Cx37, Zp2, and Gdf9, were also upregulated significantly. Therefore, it can be concluded that +BMP4 and +RA along with ovarian somatic cell co‐culture improved the rate of in vitro oocyte differentiation.     

Epidermal growth factor and three‐dimensional scaffolds provide conducive environment for differentiation of mouse embryonic stem cells into oocyte‐like cells

Three‐dimensional (3D) culture provides a biomimicry of the naive microenvironment that can support cell proliferation, differentiation, and regeneration. Some growth factors, such as epidermal growth factor (EGF), facilitate normal meiosis during oocyte maturation in vivo. In this study, a scaffold‐based 3D coculture system using purified alginate was applied to induce oocyte differentiation from mouse embryonic stem cells (mESCs). mESCs were induced to differentiate into oocyte‐like cells using embryoid body protocol in the two‐dimensional or 3D microenvironment in vitro. To increase the efficiency of the oocyte‐like cell differentiation from mESCs, we employed a coculture system using ovarian granulosa cells in the presence or absence of epidermal growth factor (+EGF or ?EGF) for 14 days and then the cells were assessed for germ cell differentiation, meiotic progression, and oocyte maturation markers. The cultures exposed to EGF in the alginate‐based 3D microenvironment showed the highest level of premeiotic (Oct4 and Mvh), meiotic (Scp1, Scp3, Stra8, and Rec8), and oocyte maturation (Gdf9, Cx37, and Zp2) marker genes (p < .05) in comparison to other groups. According to the gene‐expression patterns, we can conclude that alginate‐based 3D coculture system provided a highly efficient protocol for oocyte‐like cell differentiation from mESCs. The data showed that this culture system along with EGF improved the rate of in vitro oocyte‐like cell differentiation.     

A Gene Regulatory Program for Meiotic Prophase in the Fetal Ovary

The chromosomal program of meiotic prophase, comprising events such as laying down of meiotic cohesins, synapsis between homologs, and homologous recombination, must be preceded and enabled by the regulated induction of meiotic prophase genes. This gene regulatory program is poorly understood, particularly in organisms with a segregated germline. We characterized the gene regulatory program of meiotic prophase as it occurs in the mouse fetal ovary. By profiling gene expression in the mouse fetal ovary in mutants with whole tissue and single-cell techniques, we identified 104 genes expressed specifically in pre-meiotic to pachytene germ cells. We characterized the regulation of these genes by 1) retinoic acid (RA), which induces meiosis, 2) Dazl, which is required for germ cell competence to respond to RA, and 3) Stra8, a downstream target of RA required for the chromosomal program of meiotic prophase. Initial induction of practically all identified meiotic prophase genes requires Dazl. In the presence of Dazl, RA induces at least two pathways: one Stra8-independent, and one Stra8-dependent. Genes vary in their induction by Stra8, spanning fully Stra8-independent, partially Stra8-independent, and fully Stra8-dependent. Thus, Stra8 regulates the entirety of the chromosomal program but plays a more nuanced role in governing the gene expression program. We propose that Stra8-independent gene expression enables the stockpiling of selected meiotic structural proteins prior to the commencement of the chromosomal program. Unexpectedly, we discovered that Stra8 is required for prompt down-regulation of itself and Rec8. Germ cells that have expressed and down-regulated Stra8 are refractory to further Stra8 expression. Negative feedback of Stra8, and subsequent resistance to further Stra8 expression, may ensure a single, restricted pulse of Stra8 expression. Collectively, our findings reveal a gene regulatory logic by which germ cells prepare for the chromosomal program of meiotic prophase, and ensure that it is induced only once.     

Expression of miR‐34c in response to overexpression of Boule and Stra8 in dairy goat male germ line stem cells (mGSCs)

Reproduction is required for the survival of all mammalian animals. Spermatogenesis is an essential and complex developmental process that ultimately results in production of haploid spermatozoa. Recent studies demonstrated that Boule and stimulated by retinoic acid 8 (Stra8) played important roles in initiation meiosis in male germ cells. miR‐34c is indispensable in the late steps of spermatogenesis; remarkably, the main function of miR‐34c is to reduce cell proliferation potentiality and promote cellular apoptosis. The objectives of this study were to investigate the expression patterns of Boule, Stra8, P53 and miR‐34c in dairy goat testis and their relationship in male germ line stem cells (mGSCs). The results first revealed the expression patterns of Boule, Stra8, P53 and miR‐34c in 30 dpp, 90 dpp and adult testes of dairy goats. The expression levels of Boule, Stra8, P53 and miR‐34c in adult dairy goat testes were significantly higher than that of 30 dpp. Overexpression of Boule and Stra8 promoted the expression of miR‐34c in dairy goat mGSCs. In our previous study, we showed that miR‐34c was P53 dependent in mGSCs. These results have shown that the up‐regulation of miR‐34c was not due to P53 protein activation but which might be caused by the up‐regulation of Boule and Stra8 promoting the advance of meiosis. In addition, we found retinoic acid would decrease the expression of P53 and miR‐34c, however, did not change the expression of c‐Myc greatly. It suggested that the function of driving differentiation of dairy goat mGSCs by retinoic acid might not be caused by P53. Copyright © 2013 John Wiley & Sons, Ltd.     

Cluster characterization of mouse embryonic stem cell-derived pluripotent embryoid bodies in four distinct developmental stages

The formation of embryoid bodies (EBs) is the principal step in the differentiation of embryonic stem (ES) cells. In this study, the morphological characteristics and gene expression patterns of EBs related to the sequential stages of embryonic development were well defined in four distinct developmental groups over 112 days of culture: early-stage EBs groups (1–7 days of differentiation), mid-stage EBs groups (9–15 days of differentiation), maturing EBs groups (17–45 days of differentiation) and matured EBs groups (50 days of differentiation). We first determined definite histological location of apoptosis within EBs and the sequential expression of molecular markers representing stem cells (Oct4, SSEA-1, Sox-2 and AKP), germ cells (Fragilis, Dazl, c-kit, StellaR, Mvh and Stra8), ectoderm (Neurod, Nestin and Neurofilament), mesoderm (Gata-1, Flk-1 and Hbb) and endoderm (AFP and Transthyretin). Our results revealed that developing EBs possess either pluripotent stem cell or germ cell states and that three-dimensional aggregates of EBs initiate mES cell differentiation during prolonged culture in vitro. Therefore, we suggest that this EB system to some extent recapitulates the early developmental processes occurring in vivo.     

The essential roles of Mps1 in spermatogenesis and fertility in mice

Monopolar spindle 1 (MPS1), which plays a critical role in somatic mitosis, has also been revealed to be essential for meiosis I in oocytes. Spermatogenesis is an important process involving successive mitosis and meiosis, but the function of MPS1 in spermatogenesis remains unclear. Here, we generated Mps1 conditional knockout mice and found that Ddx4-cre-driven loss of Mps1 in male mice resulted in depletion of undifferentiated spermatogonial cells and subsequently of differentiated spermatogonia and spermatocytes. In addition, Stra8-cre-driven ablation of Mps1 in male mice led to germ cell loss and fertility reduction. Spermatocytes lacking Mps1 have blocked at the zygotene-to-pachytene transition in the prophase of meiosis I, which may be due to decreased H2B ubiquitination level mediated by MDM2. And the expression of many meiotic genes was decreased, while that of apoptotic genes was increased. Moreover, we also detected increased apoptosis in spermatocytes with Mps1 knockout, which may have been the reason why germ cells were lost. Taken together, our findings indicate that MPS1 is required for mitosis of gonocytes and spermatogonia, differentiation of undifferentiated spermatogonia, and progression of meiosis I in spermatocytes.Subject terms: Cell division, Spermatogenesis     

Expression pattern of Boule in dairy goat testis and its function in promoting the meiosis in male germline stem cells (mGSCs)

Boule is a conserved gene in meiosis, which encodes RNA binding protein required for spermatocyte meiosis. Deletion of Boule was found to block meiosis in spermatogenesis, which contributes to infertility. Up to date, the expression and function of Boule in the goat testis are not known. The objectives of this study were to investigate the expression pattern of Boule in dairy goat testis and their function in male germline stem cells (mGSCs). The results first revealed that the expression level of Boule in adult testes was significantly higher than younger and immature goats, and azoospermia and male intersex testis. Over‐expression of Boule promoted the expression of meiosis‐related genes in dairy goat mGSCs. The expression of Stra8 was up‐regulated by over‐expression of Boule analyzed by Western blotting and Luciferase reporter assay. While, Cdc25a, the downstream regulator of Boule, was found not to affect the expression of Stra8, and our data illustrated that Cdc25a did not regulate meiosis via Stra8. The expression of Stra8 and Boule was up‐regulated by RA induction. Taken together, results suggest the Boule plays an important role in dairy goat spermatogenesis and that over‐expression of Boule may promote spermatogenesis and meiosis in dairy goat. J. Cell. Biochem. 114: 294–302, 2013. © 2012 Wiley Periodicals, Inc.     

NANOS2 promotes male germ cell development independent of meiosis suppression

NANOS2 is an RNA-binding protein essential for fetal male germ cell development. While we have shown that the function of NANOS2 is vital for suppressing meiosis in embryonic XY germ cells, it is still unknown whether NANOS2 plays other roles in the sexual differentiation of male germ cells. In this study, we addressed the issue by generating Nanos2/Stra8 double knockout (dKO) mice, whereby meiosis was prohibited in the double-mutant male germ cells. We found that the expression of male-specific genes, which was decreased in the Nanos2 mutant, was hardly recovered in the dKO embryo, suggesting that NANOS2 plays a role in male gene expression other than suppression of meiosis. To investigate the molecular events that may be controlled by NANOS2, we conducted a series of microarray analyses to search putative targets of NANOS2 that fulfilled 2 criteria: (1) increased expression in the Nanos2 mutant and (2) the mRNA associated with NANOS2. Interestingly, the genes predominantly expressed in undifferentiated primordial germ cells (PGCs) were significantly selected, implying the involvement of NANOS2 in the termination of the characteristics of PGCs. Furthermore, we showed that NANOS2 is required for the maintenance of mitotic quiescence, but not for the initiation of the quiescence in fetal male germ cells. These results suggest that NANOS2 is not merely a suppressor of meiosis, but instead plays pivotal roles in the sexual differentiation of male germ cells.     

Derivation of male germ cells from induced pluripotent stem cells by inducers: A review

Induced pluripotent stem cells (iPSCs) refer to stem cells that are artificially produced using a new technology known as cellular reprogramming, which can use gene transduction in somatic cells. There are numerous potential applications for iPSCs in the field of stem cell biology becauase they are able to give rise to several different cell features of lineages such as three-germ layers. Primordial germ cells, generated via in vitro differentiation of iPSCs, have been demonstrated to produce functional gametes. Therefore, in this review we discussed past and recent advances in the in vitro differentiation of germ cells using pluripotent stem cells with an emphasis on iPSCs. Although this domain of research is still in its infancy, exploring development mechanisms of germ cells is promising, especially in humans, to promote future reproductive and developmental engineering technologies. While few studies have evaluated the ability and efficiency of iPSCs to differentiate toward male germ cells in vitro by different inducers, the given effect was investigated in this review.