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.

Ectopically Expressed Meiosis-Specific Cancer Testis Antigen HORMAD1 Promotes Genomic Instability in Squamous Cell Carcinomas.

Genomic instability is a prominent hallmark of cancer, however the mechanisms that drive and sustain this process remain elusive. Research demonstrates that numerous cancers with increased levels of genomic instability ectopically express meiosis-specific genes and undergo meiomitosis, the clash of mitotic and meiotic processes. These meiotic genes may represent novel therapeutic targets for the treatment of cancer. We studied the relationship between the expression of the meiosis protein HORMAD1 and genomic instability in squamous cell carcinomas (SCCs). First, we assessed markers of DNA damage and genomic instability following knockdown and overexpression of HORMAD1 in different cell lines representing SCCs and epithelial cancers. shRNA-mediated depletion of HORMAD1 expression resulted in increased genomic instability, DNA damage, increased sensitivity to etoposide, and decreased expression of DNA damage response/repair genes. Conversely, overexpression of HORMAD1 exhibited protective effects leading to decreased DNA damage, enhanced survival and decreased sensitivity to etoposide. Furthermore, we identified a meiotic molecular pathway that regulates HORMAD1 expression by targeting the upstream meiosis transcription factor STRA8. Our results highlight a specific relationship between HORMAD1 and genomic instability in SCCs, suggesting that selectively inhibiting HORMAD1, possibly, through STRA8 signaling, may provide a new paradigm of treatment options for HORMAD1-expressing SCCs.

Generation of a novel Stra8-driven Cre recombinase strain for use in pre-meiotic germ cells in mice†.

The development of oocytes occurs over a broad time frame, starting at the earliest stages of embryogenesis and continuing into adulthood. Conditional knockout technologies such as the Cre/loxP recombination system are useful for analyzing oocyte development at specific stages, but not every time frame has appropriate Cre drivers, for instance, during oocyte meiotic initiation through early prophase I in the embryo. Here, we generated a novel knockin mouse line that produces a bicistronic transcript from the endogenous Stra8 locus that includes a "self-cleaving" 2A peptide upstream of cre. This allows for high efficiency cleavage and production of both proteins individually and results in expression of cre in both male and female gonads at the biologically relevant stage. Fluorescent reporter analysis confirms that this line recapitulates endogenous Stra8 expression in both sexes and does not affect fertility of heterozygous nor homozygous mice. This line, named Stra8P2Acre, adds to the repertoire of germ-cell specific cre driver lines and, importantly, allows for deletion of target genes during key embryonic oocyte developmental stages, including early events in meiosis. Summary Sentence Generation of a novel cre recombinase knockin to the Stra8 locus allows production of Stra8 and cre without affecting fertility.

Cell cycle regulation for meiosis in mammalian germ cells.

In mouse fetal gonads, germ cell development is accompanied by changes in cell cycle mode in response to external signals and intrinsic mechanisms of cells. During fetal development, male germ cells undergo G0/G1 arrest, while female germ cells exit the mitotic cell cycle and enter meiosis. In fetal testes, NANOS2 and CYP26B1 force germ cells to stay in G0/G1 arrest phase, preventing them from entering the meiotic cell cycle. In the fetal ovary, external signals, such as RA, BMP, and WNT, promote the competency of female germ cells to enter the meiotic cell cycle. MEIOSIN and STRA8 ensure the establishment of the meiotic cell cycle by activating meiotic genes, such that meiotic entry coincides with the S phase. This review discusses germ cell development from the viewpoint of cell cycle regulation and highlights the mechanism of the entry of germ cells into meiosis.

Jun-mediated lncRNA-IMS promotes the meiosis of chicken spermatogonial stem cells via gga-miR-31-5p/stra8.

Meiosis, a key step in spermatogenesis, is affected by many factors. Current studies have shown that long noncoding RNAs (lncRNAs) are potential factors regulating meiosis, and their regulatory mechanisms have received much attention. However, little research has been done on its regulatory mechanism in the spermatogenesis of roosters. Here, we found that lncRNA involved in meiosis and spermatogenesis (lncRNA-IMS) was involved in the regulation of Stra8 by gga-miR-31-5p and hindered the inhibition of Stra8 by gga-miR-31-5p. The acquisition and loss of function experiments demonstrated that lncRNA-IMS was involved in meiosis and spermatogenesis. In addition, we predicted and determined the core promoter region of lncRNA-IMS. Prediction of transcription factors, deletion/overexpression of binding sites, knockdown/overexpression of Jun, and dual-luciferase reporter analysis confirmed that Jun positively activated transcription of lncRNA-IMS. Our findings further enrich the TF-lncRNA-miRNA-mRNA regulatory network during male meiosis and provide new ideas for studying the molecular mechanism of meiosis and spermatogenesis in chicken spermatogonial stem cells.

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.

Comparative effects of retinoic acid, granulosa cells conditioned medium or forskolin in combination with granulosa cell co-culturing on mouse germ cell differentiation.

BACKGROUND: Devising of an appropriate in vitro culture method for germ cells differentiation in the presence of soluble factors has attracted considerable attention, which results will provide new insight into reproductive biology. In this study, we compared the effects of forskolin, retinoic acid (RA) or granulosa cell-conditioned medium in the presence or absence of granulosa cell co-culturing on germ cell differentiation from embryonic stem cells (ESCs). METHODS AND RESULTS: Embryonic stem cells were differentiated using embryoid bodies (EBs) for 5 days, and then EB-derived cells were co-cultured with or without adult mouse granulosa cells using monolayer protocol and treated with 50 µM forskolin, 1 µM RA and 50% granulosa cell-conditioned medium for 4 days. Granulosa cell-conditioned medium significantly increased the levels of Scp3, Rec8, Mvh and Gdf9 expression in the granulosa cell co-culture method compared to untreated cells. A significant elevation of Stra8, Rec8 and Mvh was observed after treatment with RA in the absence of granulosa cells and there was no significant increase in the levels of expression of germ cell-specific genes after treatment with forskolin compared to control. Furthermore, forskolin and RA significantly increased viability and proliferation of germ-like cells, compared with granulosa cell-conditioned medium. CONCLUSIONS: Our study revealed that granulosa cell-conditioned medium and RA effectively can induce germ cell differentiation from ESCs, however combined application of granulosa cell-conditioned medium and co-culturing with granulosa cells had synergic effect on germ cell development in vitro as optimized protocol.

Fertilization, but Not Post-Implantation Development, Can Occur in the Absence of Sperm and Oocyte Beta1 Integrin in Mice.

Fertilization is a complex process that requires successive stages and culminates in the adhesion/fusion of gamete membranes. If the question of the involvement of oocyte integrins has been swept away by deletion experiments, that of the involvement of sperm integrins remains to be further characterized. In the present study, we addressed the question of the feasibility of sperm-oocyte adhesion/fusion and early implantation in the absence of sperm ß1 integrin. Males and females with ß1 integrin-depleted sperm and oocytes were mated, and fertilization outcome was monitored by a gestational ultrasound analysis. Results suggest that although the sperm ß1 integrin participates in gamete adhesion/fusion, it is dispensable for fertilization in mice. However, sperm- and/or oocyte-originated integrin ß1 is essential for post-implantation development. Redundancy phenomena could be at the origin of a compensatory expression or alternative dimerization pattern.

The Beginning of Meiosis in Mammalian Female Germ Cells: A Never-Ending Story of Intrinsic and Extrinsic Factors.

Meiosis is the unique division of germ cells resulting in the recombination of the maternal and paternal genomes and the production of haploid gametes. In mammals, it begins during the fetal life in females and during puberty in males. In both cases, entering meiosis requires a timely switch from the mitotic to the meiotic cell cycle and the transition from a potential pluripotent status to meiotic differentiation. Revealing the molecular mechanisms underlying these interrelated processes represents the essence in understanding the beginning of meiosis. Meiosis facilitates diversity across individuals and acts as a fundamental driver of evolution. Major differences between sexes and among species complicate the understanding of how meiosis begins. Basic meiotic research is further hindered by a current lack of meiotic cell lines. This has been recently partly overcome with the use of primordial-germ-cell-like cells (PGCLCs) generated from pluripotent stem cells. Much of what we know about this process depends on data from model organisms, namely, the mouse; in mice, the process, however, appears to differ in many aspects from that in humans. Identifying the mechanisms and molecules controlling germ cells to enter meiosis has represented and still represents a major challenge for reproductive medicine. In fact, the proper execution of meiosis is essential for fertility, for maintaining the integrity of the genome, and for ensuring the normal development of the offspring. The main clinical consequences of meiotic defects are infertility and, probably, increased susceptibility to some types of germ-cell tumors. In the present work, we report and discuss data mainly concerning the beginning of meiosis in mammalian female germ cells, referring to such process in males only when pertinent. After a brief account of this process in mice and humans and an historical chronicle of the major hypotheses and progress in this topic, the most recent results are reviewed and discussed.

[Construction of a testis Elovl4 gene knockout mouse model based on Cre/loxP system].

Very long chain polyunsaturated fatty acids (VLC-PUFAs) are unique fatty acids in tissues of mammals such as retina and testis, and the key enzyme of its biosynthesis is very long chain fatty acid elongase 4 (Elovl4). Development of an animal model of tissue-specific knockout of Elovl4 gene is conducive to the in-depth study of the biological function of VLC-PUFAs. Therefore, we constructed Stra8-Cre mice and Elovl4 floxed mice based on Cre/loxP system, and obtained the (Elovl4[flox/+], Stra8-Cre) heterozygous knockout mice by hybridization. Subsequently, female mice were selected to cross with male mice with homozygous Elovl4[flox/flox] to gain homozygous mice (Elovl4[flox/flox], Stra8-Cre) through genotype identification and screening. RT-PCR, qRT-PCR, Western blotting, immunohistochemistry and immunofluorescence techniques were used to detect the knock-out efficiency of Elovl4 in testis. The expression of Elovl4 in testis of both heterozygous and homozygous knockout mice were significantly down-regulated at mRNA and protein levels, but were not affected in other tissues. In summary, we constructed a mouse model with specific knockout of Elovl4 gene in testis, which provides a reliable animal model for studying the effect of VLC-PUFAs on the reproductive function of male mice and the underpinning molecular mechanisms.

Retinoic Acid Receptor Alpha Is Essential in Postnatal Sertoli Cells but Not in Germ Cells.

Retinoic acid signaling is indispensable for the completion of spermatogenesis. It is known that loss of retinoic acid nuclear receptor alpha (RARA) induces male sterility due to seminiferous epithelium degeneration. Initial genetic studies established that RARA acts in Sertoli cells, but a recent paper proposed that RARA is also instrumental in germ cells. In the present study, we have re-assessed the function of RARA in germ cells by genetically ablating the Rara gene in spermatogonia and their progenies using a cell-specific conditional mutagenesis approach. We show that loss of Rara in postnatal male germ cells does not alter the histology of the seminiferous epithelium. Furthermore, RARA-deficient germ cells differentiate normally and give rise to normal, living pups. This establishes that RARA plays no crucial role in germ cells. We also tested whether RARA is required in Sertoli cells during the fetal period or after birth. For this purpose, we deleted the Rara gene in Sertoli cells at postnatal day 15 (PN15), i.e., after the onset of the first spermatogenic wave. To do so, we used temporally controlled cell-specific mutagenesis. By comparing the testis phenotypes generated when Rara is lost either at PN15 or at embryonic day 13, we show that RARA exerts all of its functions in Sertoli cells not at the fetal stage but from puberty.

Function of Retinoic Acid in Development of Male and Female Gametes.

Retinoic acid, an active metabolite of vitamin A, is necessary for many developmental processes in mammals. Much of the field of reproduction has looked toward retinoic acid as a key transcriptional regulator and catalyst of differentiation events. This review focuses on the effects of retinoic acid on male and female gamete formation and regulation. Within spermatogenesis, it has been well established that retinoic acid is necessary for the proper formation of the blood-testis barrier, spermatogonial differentiation, spermiation, and assisting in meiotic completion. While many of the roles of retinoic acid in male spermatogenesis are known, investigations into female oogenesis have provided differing results.

Instructing Mouse Germ Cells to Adopt a Female Fate.

BACKGROUND: Germ cells are critical for the survival of our species. They are the only cells that undergo meiosis - the reductive form of cell division that is necessary for genetic reassortment of chromosomes and production of the haploid gametes, the sperm and eggs. Remarkably, the initial female/male fate decision in fetal germ cells does not depend on whether they are chromosomally XX or XY; rather, initial sexual fate is imposed by influences from the surrounding tissue. In mammals, the female germline is particularly precious: despite recent suggestions that germline stem cells exist in the ovary, it is still generally accepted that the ovarian reserve is finite, and its size is dependant on germ cells of the fetal ovary initiating meiosis in a timely manner. SUMMARY: Prior to 2006, evidence suggested that gonadal germ cells initiate meiotic prophase I by default, but more recent data support a key role for the signalling molecule retinoic acid (RA) in instructing female germ cell fate. Newer findings also support a key meiosis-inducing role for another signalling molecule, bone morphogenic protein (BMP). Nonetheless, many questions remain. KEY MESSAGES: Here, we review knowledge thus far regarding extrinsic and intrinsic determinants of a female germ cell fate, focusing on the mouse model.

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.

Roles of Stra8 and Tcerg1l in retinoic acid induced spermatogonial differentiation in mouse†.

Retinoic acid (RA) induces spermatogonial differentiation, but the mechanism by which it operates remains largely unknown. We developed a germ cell culture assay system to study genes involved in spermatogonial differentiation triggered by RA. Stimulated by RA 8 (Stra8), a RA-inducible gene, is indispensable for meiosis initiation, and its deletion results in a complete block of spermatogenesis at the pre-leptotene/zygotene stage. To interrogate the role of Stra8 in RA mediated differentiation of spermatogonia, we derived germ cell cultures from the neonatal testis of both wild type and Stra8 knock-out mice. We provide the first evidence that Stra8 plays a crucial role in modulating the responsiveness of undifferentiated spermatogonia to RA and facilitates transition to a differentiated state. Stra8-mediated differentiation is achieved through the downregulation of a large portfolio of genes and pathways, most notably including genes involved in the spermatogonial stem cell self-renewal process. We also report here for the first time the role of transcription elongation regulator-1 like (Tcerg1l) as a downstream effector of RA-induced spermatogonial differentiation.

Identification of regulatory elements required for Stra8 expression in fetal ovarian germ cells of the mouse.

In mice, the entry of germ cells into meiosis crucially depends on the expression of stimulated by retinoic acid gene 8 (Stra8). Stra8 is expressed specifically in pre-meiotic germ cells of females and males, at fetal and postnatal stages, respectively, but the mechanistic details of its spatiotemporal regulation are yet to be defined. In particular, there has been considerable debate regarding whether retinoic acid is required, in vivo, to initiate Stra8 expression in the mouse fetal ovary. We show that the distinctive anterior-to-posterior pattern of Stra8 initiation, characteristic of germ cells in the fetal ovary, is faithfully recapitulated when 2.9 kb of the Stra8 promoter is used to drive eGFP expression. Using in vitro transfection assays of cutdown and mutant constructs, we identified two functional retinoic acid responsive elements (RAREs) within this 2.9 kb regulatory element. We also show that the transcription factor DMRT1 enhances Stra8 expression, but only in the presence of RA and the most proximal RARE. Finally, we used CRISPR/Cas9-mediated targeted mutation studies to demonstrate that both RAREs are required for optimal Stra8 expression levels in vivo.

Differential expression patterns of the two paralogous Rec8 from Nile tilapia and their responsiveness to retinoic acid signaling.

REC8 (meiotic recombination protein 8) is an essential component of meiotic cohesion complexes. Interestingly, two paralogous rec8 genes happen to exist in the stra8 (stimulated by retinoic acid gene 8)-absent fishes but not in stra8-existing fishes. Stra8 is usually considered as the prerequirement during RA (retinoic acid)-mediated meiosis initiation in mammals. However, how RA triggers meiosis in the stra8-absent fishes just like Nile tilapia (Oreochromis niloticus) remains elusive. Here we characterized the two paralogous rec8 genes in Nile tilapia (Onrec8a and Onrec8b), and investigated their expression patterns and responsiveness to RA signaling by treatment of ex vivo testicular culture and promoter luciferase reporter assay. OnRec8a and OnRec8b share 36% identity to each other and are true orthologs of REC8. Their expression was predominantly restricted to meiotic germline cells with differential spatiotemporal patterns. During spermatogenesis, OnRec8b predominantly exhibited nuclear expression in spermatocytes from 60 dah (days after hatching), while OnRec8a exhibited cytoplasmic expression from 90 dah. During oogenesis, OnRec8a was expressed from 30 dah, while OnRec8b from 90 dah. Further study shows that RA signaling could upregulate the expression of both Onrec8a and Onrec8b. Collectively, our data implies that OnRec8a and OnRec8b might have differential function during meiosis and be involved in RA-mediated meiosis program.

STRA8 induces transcriptional changes in germ cells during spermatogonial development.

Spermatogonial development is a key process during spermatogenesis to prepare germ cells to enter meiosis. While the initial point of spermatogonial differentiation is well-characterized, the development of spermatogonia from the onset of differentiation to the point of meiotic entry has not been well defined. Further, STRA8 is highly induced at the onset of spermatogonial development but its function in spermatogonia has not been defined. To better understand how STRA8 impacts spermatogonia, we performed RNA-sequencing in both wild-type and STRA8 knockout mice at multiple timepoints during retinoic acid (RA)-stimulated spermatogonial development. As expected, in spermatogonia from wild-type mice we found that steady-state levels of many transcripts that define undifferentiated progenitor cells were decreased while transcripts that define the differentiating spermatogonia were increased as a result of the actions of RA. However, the spermatogonia from STRA8 knockout mice displayed a muted RA response such that there were more transcripts typical of undifferentiated cells and fewer transcripts typical of differentiating cells following RA action. While spermatogonia from STRA8 knockout mice can ultimately form spermatocytes that fail to complete meiosis, it appears that the defect likely begins as a result of altered messenger RNA levels during spermatogonial differentiation.

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.

Localized Induction of Gene Expression in Embryonic Stem Cell Aggregates Using Holographic Optical Tweezers to Create Biochemical Gradients.

Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses. Graphical abstract.