Induction of Meiotic Initiation in Long-Term Mouse Spermatogonial Stem Cells Under Retinoid Acid and Nutrient Restriction Conditions.

Spermatogonial stem cells (SSCs) produce haploid sperm via mitosis and meiosis in vivo. Although the technique to culture mouse SSCs has been well established, induction of meiosis in vitro has remained a challenge. Retinoic acid (RA) is required for meiosis in vivo; however, RA alone is not sufficient to induce meiosis in vitro. Here, we describe a method in which nutrient restriction and RA synergistically induce meiotic initiation into meiotic prophase I in cultured mouse SSCs.

Gene regulation during meiosis.

Meiosis is essential for gamete production in all sexually reproducing organisms. It entails two successive cell divisions without DNA replication, producing haploid cells from diploid ones. This process involves complex morphological and molecular differentiation that varies across species and between sexes. Specialized genomic events like meiotic recombination and chromosome segregation are tightly regulated, including preparation for post-meiotic development. Research in model organisms, notably yeast, has shed light on the genetic and molecular aspects of meiosis and its regulation. Although mammalian meiosis research faces challenges, particularly in replicating gametogenesis in vitro, advances in genetic and genomic technologies are providing mechanistic insights. Here we review the genetics and molecular biology of meiotic gene expression control, focusing on mammals.

[Loss of RBFOX2 inhibits meiotic initiation in male mice].

Meiotic initiation is a critical step in gametogenesis. Recently, some genes required for meiotic initiation have been identified. However, meiosis-initiating factors and the underlying mechanisms are far from being fully understood. We have established a long-term culture system of spermatogonial stem cells (SSCs) and an in vitro model of meiotic initiation using mouse SSCs. Our previous study revealed that the RNA-binding protein RBFOX2 may regulate meiotic initiation, but the role and the mechanism need to be further elucidated. In this study, we constructed RBFOX2 knockdown SSC lines by using lentivirus-mediated gene delivery method, and found that the knockdown SSCs underwent normal self-renewal, mitosis and differentiation. However, they were unable to initiate meiosis when treated with retinoic acid, and they underwent apoptosis. These results indicate that RBFOX2 plays an essential role in meiotic initiation of spermatogonia. This work provides new clues for understanding the functions of RNA-binding proteins in meiotic initiation.

Transcriptional metabolic reprogramming implements meiotic fate decision in mouse testicular germ cells.

Nutrient starvation drives yeast meiosis, whereas retinoic acid (RA) is required for mammalian meiosis through its germline target Stra8. Here, by using single-cell transcriptomic analysis of wild-type and Stra8-deficient juvenile mouse germ cells, our data show that the expression of nutrient transporter genes, including Slc7a5, Slc38a2, and Slc2a1, is downregulated in germ cells during meiotic initiation, and this process requires Stra8, which binds to these genes and induces their H3K27 deacetylation. Consequently, Stra8-deficient germ cells sustain glutamine and glucose uptake in response to RA and exhibit hyperactive mTORC1/protein kinase A (PKA) activities. Importantly, expression of Slc38a2, a glutamine importer, is negatively correlated with meiotic genes in the GTEx dataset, and Slc38a2 knockdown downregulates mTORC1/PKA activities and induces meiotic gene expression. Thus, our study indicates that RA via Stra8, a chordate morphogen pathway, induces meiosis partially by generating a conserved nutrient restriction signal in mammalian germ cells by downregulating their nutrient transporter expression.

Mechanism of initiation of meiosis in mouse germ cells.

Meiosis is critical for germ cell development in multicellular organisms. Initiation of meiosis coincides with pre-meiotic S phase, which is followed by meiotic prophase, a prolonged G2 phase that ensures numerous meiosis-specific chromosome events. Meiotic prophase is accompanied by robust alterations of gene expression. In mouse germ cells, MEIOSIN and STRA8 direct cell cycle switch from mitosis to meiosis. MEIOSIN and STRA8 coordinate meiotic initiation with cell cycle, by activating the meiotic genes to have meiotic prophase program installed at S phase. This review mainly focuses on the mechanism of meiotic initiation in mouse germ cells from the viewpoint of the transcription of meiotic genes. Furthermore, signaling pathways that regulate meiotic initiation will be discussed in the context of germ cell development, pointing out the sexual differences in the mode of meiotic initiation.

Znhit1 controls meiotic initiation in male germ cells by coordinating with Stra8 to activate meiotic gene expression.

The switch from mitosis to meiosis ensures the successive formation of gametes. However, it remains unclear how meiotic initiation occurs within the context of chromatin. Recent studies have shown that zinc finger HIT-type containing 1 (Znhit1), a subunit of the SRCAP chromatin remodeling complex, plays essential roles in modulating the chromatin structure. Herein, we report that the germline-conditional deletion of Znhit1 in male mice specifically blocks meiotic initiation. We show that Znhit1 is required for meiotic prophase events, including synapsis, DNA double-strand break formation, and meiotic DNA replication. Mechanistically, Znhit1 controls the histone variant H2A.Z deposition, which facilitates the expression of meiotic genes, such as Meiosin, but not the expression of Stra8. Interestingly, Znhit1 deficiency disrupts the transcription bubbles of meiotic genes. Thus, our findings identify the essential role of Znhit1-dependent H2A.Z deposition in allowing activation of meiotic gene expression, thereby controlling the initiation of meiosis.

The microRNA miR-202 prevents precocious spermatogonial differentiation and meiotic initiation during mouse spermatogenesis.

Spermatogonial differentiation and meiotic initiation during spermatogenesis are tightly regulated by a number of genes, including those encoding 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 key regulators such as STRA8 and DMRT6. In miR-202 knockout (KO) mice, the undifferentiated spermatogonial pool is reduced, accompanied by age-dependent decline of fertility. In KO mice, SYCP3, STRA8 and DMRT6 are expressed earlier than in wild-type 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 partially rescued by the knockdown of Dmrt6. Therefore, we have not only shown that miR-202 is a regulator of meiotic initiation but also identified a previously unknown module in the underlying regulatory network.

Critical roles of mRNA m6A modification and YTHDC2 expression for meiotic initiation and progression in female germ cells.

Meiotic entry and progression require dynamic regulation of germline gene expression. m6A on mRNAs and recognition by YTHDC2 has been known as post-transcriptional regulatory complex, but the roles of this regulator remain unclear for meiotic initiation and progression in female germ cells (FGCs). This study showed that m6A modification occurred mainly in FGCs rather than ovarian somatic cells (SOMAs), and m6A levels in FGCs increased significantly with meiotic initiation. m6A inhibition suppressed expression of the meiotic markers and affected the percent of FGCs at zygotene, pachytene and diplotene stage respectively. YTHDC2 expression also increased in the same pattern with m6A. Ythdc2 knockdown decreased the percent of STRA8-positive FGCs and altered the percent of FGCs at zygotene and pachytene stage respectively. Taken together, these results suggest that mRNA m6A modification and YTHDC2 expression are essential for meiotic initiation and progression in FGCs.

Retinoic acid and androgen influence germ cells development and meiotic initiation in juvenile orange-spotted grouper, Epinephelus coioides.

Meiosis is essential for germ cells development for all sexually reproducing species. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in mammals, birds and tetrapods. Here, we investigated the effects of RA on meiotic initiation and sex determination in protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides). Expression profile investigations of meiotic marker genes during gonadal development indicated that germ cells undergone meiosis approximately at 180 days after hatching in the orange-spotted grouper. RA synthase inhibitor treatments on juvenile orange-spotted groupers resulted in impeded germ cells development and delayed meiotic initiation with simultaneous down-regulation of vasa, dazl, sycp3 and rec8, which was rescued by exogenous RA administration. Additionally, exogenous androgen treated fish showed a delayed meiotic initiation consistent with decreased sycp3 and rec8 expression and were directed to a spermiogenesis fate. Our results imply that meiotic initiation in the orange-spotted grouper is strongly influenced by RA and androgen, and the regulation of meiotic initiation may involve in the spermatogenesis induced by exogenous androgen.

Polycomb repressive complex 1 (PRC1) regulates meiotic initiation of ovarian germ cells in chick embryos.

Meiosis is essential for gametogenesis and exhibits sex-specific property during embryonic development. Retinoic acid (RA) signalling initiates germ cell meiosis by activating Stra8 (stimulated by RA gene 8). Although additional factors are involved in regulating the meiotic initiation of germ cells, their regulatory mechanisms are unclear. In this study, we found that Polycomb repressive complex 1 (PRC1) is largely expressed in chicken ovarian germ and somatic cells during early stages of meiosis. We demonstrated that PRC1 regulates Stra8, pluripotent factors and paracrine factors (Notch ligands) leading to a synergistic effect on the suppression of germ cell meiotic initiation. Finally, we observed that repression of PRC1 resulted in precocious meiotic initiation and apoptosis of ovarian cells in vivo. These results aid in understanding the regulation of meiotic initiation in germ cells by PRC1 and provide evidence to support the hypothesis that regulation of meiotic initiation is conserved in higher vertebrates.

Retinoic acid triggers meiosis initiation via stra8-dependent pathway in Southern catfish, Silurus meridionalis.

Existing studies demonstrated that retinoic acid (RA) regulates meiotic initiation via stra8-independent pathway in teleosts which lack stra8 in their genomes. However, stra8 was recently identified from several fish species including Southern catfish (Silurus meridionalis). To explore the existence of stra8-dependent pathway in RA mediated meiotic initiation in fishes, in the present study, the genes encoding RA synthase aldh1a2 and catabolic enzyme cyp26a1 and cyp26b1 were cloned from the Southern catfish. By immunohistochemistry, Aldh1a2 signal was observed in gonads of both sexes during the meiotic initiation period. By real-time PCR, differentially expressed gene was observed for cyp26a1, but not for cyp26b1, in gonads during the meiotic initiation. Administration of exogenous RA or inhibition of endogenous RA degradation by either KET (RA catabolic enzyme inhibitor) or cyp26a1 knockdown using CRISPR/Cas9 induced advanced meiotic initiation in the ovaries as demonstrated by increased Stra8/stra8 expression and appearance of oocytes. In contrast, treatment with RA synthase inhibitor DEAB resulted in delayed meiotic initiation and Stra8/stra8 expression in the ovaries, which was rescued by exogenous RA administration. These results indicated that (1) RA triggers the onset of meiosis via stra8-dependent pathway in stra8 existing teleosts, as it does in tetrapods; (2) exogenous RA can rescue the endogenous RA deficiency; (3) Cyp26a1, instead of Cyp26b1, is the key catabolic enzyme involved in meiosis initiation in teleosts. Taken together, RA might trigger meiotic initiation via stra8-dependent and -independent pathway in different teleosts.

Progesterone regulates chicken embryonic germ cell meiotic initiation independent of retinoic acid signaling.

The signaling molecule retinoic acid (RA) is known to trigger germ cells to enter meiosis. However, RA may not be the only secreted inducer of meiosis. Our previous data indicate that luteinizing hormone also promotes germ cell meiotic initiation by upregulating 3ßHSDII transcription. Here, using chicken embryos, we investigate the role of progesterone (P4) in regulating germ cell meiotic initiation. Progesterone treatment at embryonic Day 9.5 accelerated germ cell meiosis entry in the female chicken embryos. However, P4 treatment in vivo did no influence on testicular germ cells but triggered their meiotic initiation in the cultured testes. As treatment with an RA receptor (RAR) inhibitor did not block the stimulatory effect of P4 on germ cell meiotic initiation, this P4 stimulatory effect seems to be independent of RAR-mediated signaling. The abundance of RA metabolism-related enzymes and RAR (RARß) mRNAs did not differ significantly between P4-treated and control individuals. The RA concentration in the ovaries remained unchanged by P4 treatment in vivo. Because no inhibition by the P4 receptor (PR) nuclear receptor antagonist mifepristone on P4 effect was observed in either in vitro or in vivo experiments, the effect of P4 on germ cell meiotic initiation is probably mediated by membrane PRs (mPR). The mPRα, mPRß, and mPRγ mRNAs were all expressed in the embryonic ovaries. The expression of mPRα and mPRß was higher than that of mPRγ. Immunohistochemical results showed that mPRα-positive cells were mainly scattered in the ovarian cortex area where most germ cells were distributed. The mPRß-positive cells were widely distributed in the ovaries, and positive cells were clustered with a similar morphology to that of germ cell clusters. In conclusion, P4 may regulate embryonic germ cell meiotic initiation independent of RA signaling through the membrane PRs. This study provides a new insight into the mechanisms of germ cell meiotic initiation in the chicken.