Stimulated By Retinoic Acid Gene 8 Research Articles

Female-specific mechanisms of meiotic initiation and progression in mammalian oocyte development.

Meiosis is regulated in sexually dimorphic manners in mammals. In females, the commitment to and entry into meiosis are coordinated with the developmental program of oocytes. Female germ cells initiate meiosis within a short time window during the fetal period and then undergo meiotic arrest until puberty. However, the genetic mechanisms underlying the orchestration of oocyte development and meiosis to maximize the reproductive lifespan of mammalian females remain largely elusive. While meiotic initiation is regulated by a sexually common mechanism, where meiosis initiator and Stimulated by Retinoic Acid Gene 8 (STRA8) activate the meiotic genes, the female-specific mode of meiotic initiation is mediated by the interaction between retinoblastoma (RB) and STRA8. This review highlights the female-specific mechanisms of meiotic initiation and meiotic prophase progression in the context of oocyte development. Furthermore, the downstream pathway of the RB-STRA8 interaction that may regulate meiotic arrest will be discussed in the context of oocyte development, highlighting a potential genetic link between the female-specific mode of meiotic entry and meiotic arrest.

Alternative splicing for germ cell-specific Mga transcript can be eliminated without compromising mouse viability or fertility.

The stimulated by retinoic acid gene 8 (STRA8)/MEIOSIN complex and polycomb repressive complex (PRC) 1.6, a PRC1 subtype, are believed to be positive and negative regulators of meiotic onset, respectively. During meiotic initiation, the transcription repressive activity of PRC1.6 must be attenuated so that meiosis-related genes can be effectively activated by the STRA8/MEIOSIN complex. However, the molecular mechanisms that control the impairment of PRC1.6 function remain unclear. We recently demonstrated that the Mga gene, which encodes a scaffolding component of PRC1.6, produces variant mRNA by alternative splicing specifically during meiosis. Furthermore, the anomalous MGA protein encoded by the variant mRNA bears an intrinsic ability to function as a dominant negative regulator against the construction of PRC1.6 and is therefore assumed to be, at least in part, involved in impairment of the complex. Therefore, to unequivocally evaluate the physiological significance of Mga variant mRNA production in gametogenesis, we examined the consequences of a genetic manipulation that renders mice unable to produce Mga variant mRNA. Our data revealed that mutant mice were equivalent to wild-type mice in terms of viability and fertility. Our detailed examination of spermatogenesis also revealed that this genetic alteration is not associated with any apparent abnormalities in testis size, spermatogenic cycle, timing of meiotic onset, or marker gene expression of spermatogonia and spermatocytes. Taken together, these data indicate that the production of germ cell-specific Mga variant mRNA is dispensable not only for viability but also for gametogenesis.

Retinoic acid induced meiosis initiation in female germline stem cells by remodelling three-dimensional chromatin structure.

ObjectivesThis study aimed to clarify the regulation and mechanism of meiotic initiation in FGSC development.Materials and MethodsFGSCs were induced to differentiate into meiosis in differentiation medium. RNA sequencing was performed to analysis the difference of transcription level. High‐through chromosome conformation capture sequencing (Hi‐C) was performed to analysis changes of three‐dimensional chromatin structure. Chromosome conformation capture further confirmed a spatial chromatin loop. ChIP‐qPCR and dual luciferase reporter were used to test the interaction between Stimulated by retinoic acid gene 8 (STRA8) protein and Trip13 promoter.ResultsCompared with FGSCs, the average diameter of STRA8‐positive germ cells increased from 13 μm to 16.8 μm. Furthermore, there were 4788 differentially expressed genes between the two cell stages; Meiosis and chromatin structure‐associated terms were significantly enriched. Additionally, Hi‐C results showed that FGSCs underwent A/B compartment switching (switch rate was 29.81%), the number of topologically associating domains (TADs) increasing, the average size of TADs decreasing, and chromatin loop changes at genome region of Trip13 from undifferentiated stage to meiosis‐initiation stage. Furthermore, we validated that Trip13 promoter contacted distal enhancer to form spatial chromatin loop and STRA8 could bind Trip13 promoter to promote gene expression.ConclusionFGSCs underwent chromatin structure remodelling from undifferentiated stage to meiosis‐initiation stage, which facilitated STRA8 binding to Trip13 promoter and promoting its expression.

Offspring production of haploid spermatid-like cells derived from mouse female germline stem cells with chromatin condensation

BackgroundDuring male meiosis, the Y chromosome can form perfect pairing with the X chromosome. However, it is unclear whether mammalian Female germline stem cells (FGSCs) without a Y chromosome can transdifferentiate into functional haploid spermatid-like cells (SLCs).ResultsWe found that spermatogenesis was restarted by transplanting FGSCs into Kitw/wv mutant testes. Complete meiosis and formation of SLCs was induced in vitro by testicular cells of Kitw/wv mutant mice, cytokines and retinoic acid. Healthy offspring were produced by sperm and SLCs derived from the in vivo and in vitro transdifferentiation of FGSCs, respectively. Furthermore, high-throughput chromosome conformation capture sequencing(Hi-C-seq) and “bivalent” (H3K4me3-H3K27me3) micro chromatin immunoprecipitation sequencing (μChIP-seq) experiments showed that stimulated by retinoic acid gene 8 (STRA8)/protamine 1 (PRM1)-positive transdifferentiated germ cells (tGCs) and male germ cells (mGCs) display similar chromatin dynamics and chromatin condensation during in vitro spermatogenesis.ConclusionThis study demonstrates that sperm can be produced from FGSCs without a Y chromosome. This suggests a strategy for dairy cattle breeding to produce only female offspring with a high-quality genetic background.

The role of retinoic acid in the commitment to meiosis

Male meiosis is a complex process whereby spermatocytes undergo cell division to form haploid cells. This review focuses on the role of retinoic acid (RA) in meiosis, as well as several processes regulated by RA before cell entry into meiosis that are critical for proper meiotic entry and completion. Here, we discuss RA metabolism in the testis as well as the roles of stimulated by retinoic acid gene 8 (STRA8) and MEIOSIN, which are responsive to RA and are critical for meiosis. We assert that transcriptional regulation in the spermatogonia is critical for successful meiosis.

Stimulated by retinoic acid gene 8 (STRA8) interacts with the germ cell specific bHLH factor SOHLH1 and represses c‐KIT expression in vitro

STRA8 (Stimulated by Retinoic Acid Gene 8) controls the crucial decision of germ cells to engage meiotic division up and down‐regulating genes involved in the meiotic programme. It has been proven as an amplifier of genes involved in cell cycle control and chromosome events, however, how STRA8 functions as negative regulator are not well understood. In this study, we demonstrate that STRA8 can interact with itself and with other basic Helix‐Loop‐Helix (bHLH) transcription factors through its HLH domain and that this domain is important for its ability to negatively interfere with the Ebox‐mediated transcriptional activity of bHLH transcription factors. Significantly, we show that STRA8 interacts with TCF3/E47, a class I bHLH transcription factors, and with SOHLH1, a gonadal‐specific bHLH, in male germ cells obtained from prepuberal mouse testis. We demonstrated that STRA8, indirectly, is able to exert a negative control on the SOHLH1‐dependent stimulation of c‐KIT expression in late differentiating spermatogonia and preleptotene spermatocytes. Although part of this results were obtained only ‘in vitro’, they support the notion that STRA8 interacting with different transcription factors, besides its established role as ‘amplifier’ of meiotic programme, is able to finely modulate the balance between spermatogonia proliferation, differentiation and acquisition of meiotic competence.

Meiotic gatekeeper STRA8 regulates cell cycle by interacting with SETD8 during spermatogenesis.

STRA8 (Stimulated By Retinoic Acid Gene 8) is a retinoic acid (RA) induced gene that plays vital roles in spermatogonial proliferation, differentiation and meiosis. The SETD8 and STRA8 protein interaction was discovered using the yeast two‐hybrid technique using a mouse spermatogonial stem cell (SSC) cDNA library. The interaction of these two proteins was confirmed using co‐immunoprecipitation and identification of key domains governing the protein: protein complex. STRA8 and SETD8 showed a mutual transcriptional regulation pattern that provided evidence that SETD8 negatively regulated transcriptional activity of the STRA8 promoter. The SETD8 protein directly bound to the proximal promoter of the STRA8 gene. STRA8 increased the transcriptional activity of SETD8 promoter in a dose‐dependent manner. For the first time, we have discovered that STRA8 and SETD8 display a cell cycle‐dependent expression pattern in germline cells. Expression levels of SETD8 and H4K20me1 in S phase of STRA8 overexpression GC1 cells were different from that previously observed in tumour cell lines. In wild‐type mice testis, SETD8, H4K20me1 and PCNA co‐localized with STRA8 in spermatogonia. Further, our studies quantitated abnormal expression levels of cell cycle and ubiquitination‐related factors in STRA8 dynamic models. STRA8 and SETD8 may regulate spermatogenesis via Cdl4‐Clu4A‐Ddb1 ubiquitinated degradation axis in a PCNA‐dependent manner.

Expression and localization of meiosis-associated protein in gonads of female rats at different stages

It was well known that a critical process of oogenesis in the female mammalian was the entry of mitotic oogonia into meiosis. Early studies from model animal mice suggested that the retinoic acid (RA) response signal protein STRA8 (stimulated by retinoic acid gene 8) and the meiosis-specific chromosomal behavior marker protein SCP3 (Synaptonemal Complex Protein 3) were two crucial molecular markers during meiosis. The expression of STRA8 and SCP3 at different stages in rat ovaries was investigated by immunohistochemistry, qRT-PCR and Western Blot. Immunohistochemistry results showed that STRA8 and SCP3 were mainly expressed in embryonic stage. And STRA8 was expressed in the cytoplasm and nucleus of the ovaries after birth. qRT-PCR and Western Blot results showed that the mRNA and protein levels of STRA8 and SCP3 were expressed in embryonic stage. The expression of STRA8 and SCP3 indicated germ cells enter meiosis in rats embryo, and STRA8 and SCP3 could serve as molecular markers for the meiosis in rats. The localization of STRA8 in the nucleus increased the possibility that STRA8 might act as transcription factor or activate transcription to function after birth.

Effects of body temperature on the expression and localization of meiosis-related proteins STRA8 and SCP3 in boar testes

Body temperature could lead to interruption of spermatogenesis, but the molecular mechanism was still unclear. Cryptorchidism was defined as the failure of testes to enter the scrotum, which exposed the testes to body temperature. Meiosis was a unique feature of germ cell development. Whether cryptorchidism damage the initiation of meiosis in boars had not been reported. The aim of this study was to determine whether spermatogonia in the cryptorchid testes entered into meiosis by detecting meiosis-related markers stimulated by retinoic acid gene 8 (STRA8) and synaptonemal complex protein 3 (SCP3). Three boars with spontaneous unilateral abdominal cryptorchidism were used. The testis located in the abdomen was cryptorchidism group, the scrotal testis of the same animal was used as control. HE results showed that only Sertoli cells, and a few spermatogonia remained in the seminiferous tubules, and no spermatids were seen compared with the control. Immunohistochemistry results showed that in both control and cryptorchidism group, STRA8 was mainly expressed in the nucleus of spermatogonia and spermatocytes. In control group, SCP3 was expressed in the nucleus of spermatocytes. In cryptorchidism group, SCP3 immunopositive cells were also observed. qRT-PCR and Western Blot results showed that the mRNA and protein levels of STRA8 and SCP3 were significantly decreased in cryptorchid boars. The expression of STRA8 and SCP3 in cryptorchidism suggested that spermatogonia could still enter meiosis in cryptorchid boars.

Immunolocalization of proteins in the spermatogenesis process of canine.

Spermatogenesis is a process in which differentiated cells are produced and the adult stem cell population-known as spermatogonial stem cells (SSCs)-is continuously replenished. However, the molecular mechanisms underlying these processes are not fully understood in the canine species. We addressed this in this study by analysing the expression of specific markers in spermatogonia of seminiferous tubules of canine testes. SSCs at different stages of reproductive development (prepubertal and adult) were examined by immunohistochemistry and flow cytometry. Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRA1), deleted in azoospermia-like (DAZL) and promyelocytic leukaemia zinc finger (PLZF) were expressed in SSCs, while stimulated by retinoic acid gene 8 (STRA8) was detected only in undifferentiated spermatogonia in prepubertal testis and differentiated spermatogonia and spermatocytes in adult canine. Octamer-binding transcription factor 4 (OCT4) showed an expression pattern, and the levels did not differ between the groups examined. However, C-kit expression varied as a function of reproductive developmental stage. Our results demonstrate that these proteins play critical roles in the self-renewal and differentiation of SSCs and can serve as markers to identify canine spermatogonia at specific stages of development.

Exogenous Retinoic Acid and Cytochrome P450 26B1 Inhibitor Modulate Meiosis‐Associated Genes Expression in Canine Testis, an In Vitro Model

Pharmacological approaches to control spermatogenesis are required to resolve overpopulation in dogs. The objective of the study was to investigate the regulation of meiosis-associated and male germ cell-related genes, stimulated by retinoic acid gene 8 (STRA8), synaptonemal complex protein 3 (SYCP3), dosage suppressor of mck1 (DMC1), doublesex and mab-3 related transcription factor 1 (DMRT1) and deleted in azoospermia-like (DAZL) following exogenous administration of retinoic acid (RA) and after the modulation of endogenous RA by a cytochrome P450, family 26, subfamily B, polypeptide 1 inhibitor (CYP26B1-I; R115866) in an in vitro testis model. Testicles of five healthy, medium-sized and mixed-breed dogs were used for the organotypic cultures. All-trans-RA at 2 μM, CYP26B1-I at 1 μM and the control dimethyl sulphoxide (DMSO) were administered to the testes cultures, and the cultures were maintained for 24 h. Genes STRA8, DAZL and DMRT1 were significantly up-regulated as a result of the direct and indirect increase in the RA levels in the testis, subsequent to the exogenous administration of all-trans-RA and CYP26B1 inhibitor. Up-regulation of STRA8 was very prominent compared to DAZL and DMRT, and the drastic up-regulation of STRA8 was also observed with CY26B1-I than with all-trans-RA. No significant differences were found with the early meiotic markers, SYCP3 and DMC1 with RA, CY26B1-I and vehicle treatments. Because DAZL encodes a germ cell-specific RNA-binding protein, required for the induction of STRA8 and initiation of meiosis, we might see the expression differences temporally with the stage of spermatogenesis. DMRT1 is a unique gonad- and stage-specific transcription factor, directly activates STRA8 and has the temporal influence on its expression. Protein expression of DAZL and STRA8 was greater in RA- and CYP26B1-I-treated testis culture, whereas DMRT1 showed greater protein expression for RA treatment, but not for CYP26B1-I treatment compared to control. Relative protein expression of STRA8 was greatest for the CYP26B1-I treatment compared to DMSO and RA treatments. In conclusion, pharmacological intervention of spermatogenesis pertinent to RA signalling is plausible, and the effect of modulation differs upon the types of molecules and the key stages of signalling being targeted.

Expression of CYP26b1 and Related Retinoic Acid Signalling Molecules in Young, Peripubertal and Adult Dog Testis

The objective of the study was to elucidate mRNA expression of CYP26b1 (cytochrome P450, family 26, subfamily B, polypeptide 1) and signalling molecules ALDH1 (aldehyde dehydrogenase 1), CRABPII (cellular retinoic acid-binding protein II), RARα (retinoic acid receptor alpha) and STRA8 (stimulated by retinoic acid gene 8) in dog testis from different post-natal developmental ages. Testicular tissue samples were collected from medium-sized mixed breed dogs at different ages such as young (<4 months; N = 4), peripubertal (4-8 months; N = 3) and adult (>8 months; N = 4) were used to evaluate relative mRNA expression. Genes of RA-degrading enzyme CYP26b1, ALDH1 involved in RA synthesis and genes of carrier protein CRABPII involved in RA metabolism were turned on during the post-natal testicular development in dogs. Their expression pattern differs at different developmental ages (p < 0.05), and the levels of mRNA expression were compensated towards a normal developmental response for the sexual maturity and continuous spermatogenesis. The mRNA expression of RARα, one of the RA receptors participates in RA signalling in connection to spermatogenesis, was recorded in young and adult stages at varying degree. STRA8 is one of the responsive genes with regard to meiosis, and this functional gene product was expressed in all ages with the changing level (p < 0.01). In summary, the expression pattern of RA signalling molecules differed from young to adult ages, and it is expected that these changes are to compensate towards a normal developmental response for the sexual maturity and continuous spermatogenesis.

Early Meiotic‐Specific Protein Expression in Post‐natal Rat Ovaries

Recent studies in mice challenged the basic doctrine that most mammalian females lose neo-oogenesis in post-natal ovaries. In order to provide more information in other species, we examined post-natal rat ovaries by histological sections and detected the germline cell marker protein RVLG (rat vasa-like gene), BrdU (5-bromodeoxyuridine) incorporation in RVLG-expressing cells, for identification of germline cells undergoing mitosis and meiosis in the ovarian surface epithelium (OSE). We also detected the expression of early meiotic-specific proteins disruption of meiotic control 1 (DMC1), stimulated by retinoic acid gene 8 (STRA8) and synaptonemal complex protein 3 (SCP3) by immunohistochemical analysis and Western blotting, and the transcript of SCP1, SCP3 and Sporulation-specific protein 11 (SPO11) by RT-PCR in the post-natal ovarian cortex. However we failed in detecting large ovoid cells in the OSE, which may represent the putative germline stem cells (GSCs) that are supposed to sustain neo-oogenesis, and the transcription of the meiotic-specific genes SCP1, SCP3 and SPO11 by RT-PCR as well as the translation of DMC1, STRA8 and SCP3 by Western blotting. Our data support the postulation that there is no neo-oogenesis occurring in the OSE of rat post-natal ovary through meiosis of GSCs.