Regulation Of Germ Cell Development Research Articles

Characterization of Two Gonadal Genes, zar1 and wt1b, in Hermaphroditic Fish Asian Seabass (Lates calcarifer)

Zygote arrest-1 (Zar1) and Wilms’ tumor 1 (Wt1) play an important role in oogenesis, with the latter also involved in testicular development and gender differentiation. Here, Lczar1 and Lcwt1b were identified in Asian seabass (Lates calcarifer), a hermaphrodite fish, as the valuable model for studying sex differentiation. The cloned cDNA fragments of Lczar1 were 1192 bp, encoding 336 amino acids, and contained a zinc-binding domain, while those of Lcwt1b cDNA were 1521 bp, encoding a peptide of 423 amino acids with a Zn finger domain belonging to Wt1b family. RT-qPCR analysis showed that Lczar1 mRNA was exclusively expressed in the ovary, while Lcwt1b mRNA was majorly expressed in the gonads in a higher amount in the testis than in the ovary. In situ hybridization results showed that Lczar1 mRNA was mainly concentrated in oogonia and oocytes at early stages in the ovary, but were undetectable in the testis. Lcwt1b mRNA was localized not only in gonadal somatic cells (the testis and ovary), but also in female and male germ cells in the early developmental stages, such as those of previtellogenic oocytes, spermatogonia, spermatocytes and spermatids. These results indicated that Lczar1 and Lcwt1b possibly play roles in gonadal development. Therefore, the findings of this study will provide a basis for clarifying the mechanism of Lczar1 and Lcwt1b in regulating germ cell development and the sex reversal of Asian seabass and even other hermaphroditic species.

MosGILT controls innate immunity and germ cell development in Anopheles gambiae

Gene-edited mosquitoes lacking a gamma-interferon-inducible lysosomal thiol reductase-like protein, namely (mosGILTnull) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILTnull Anopheles gambiae was therefore compared to wild type (WT) mosquitoes by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILTnull A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg, an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILTnull mosquitoes. These results provide a crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.

Identification and Analysis of Genes Related to Testicular Size in 14-Day-Old Piglets.

The RNA-Seq technology was used to screen the key genes that affect the early development of the testes of Duroc × Landrace × Yorkshire piglets, to determine the regulatory pathway and provide reference for subsequent reproductive performance research, breeding, and other production practices. This study selected 14-day-old Duroc × Landrace × Yorkshire piglets as the trial animals. Testes from piglets with similar weights and no pathological changes were divided into small testis (ST) and large testis (LT) groups, and the RNA-Seq screening of differentially expressed genes (DEGs) was performed to find candidate genes and regulatory pathways related to early testicular development. The results show that 570 DEGs were found in the ST and LT groups, with 281 upregulated and 289 downregulated. The DEGs were mainly enriched on 47 gene ontology (GO) functional items. The Kyoto encyclopedia of genes and genotypes (KEGG) enrichment analysis found that there were 44 significantly enriched KEGG signal pathways, and the regulation of testicular development mainly focused on the arachidonic acid metabolism, Wnt signaling pathway and GnRH secretion pathways. The PTGES, SFRP1, SPP1, PLA2G4E, KCNJ5, PTGS2, and HCN1 genes were found to be as closely related to the testicular development of these Duroc × Landrace × Yorkshire piglets, and the differential gene expression was consistent with the real-time quantitative reverse transcription PCR (real-time qRT-PCR) validation results. This study was validated by high-throughput sequencing analysis and real-time qRT-PCR, and showed that the PTGES, SFRP1, SPP1, PLA2G4E, KCNJ5, PTGS2, and HCN1 genes may be involved in the regulation of germ cell development, spermatogenesis and semen traits. These should be further studied as candidate genes for early testicular development and reproductive trait regulation in boars.

Cell-Cell Interactions in the Testis that Support Male Fertility

The testis has two important functions: the production of sperm for fertility and the synthesis and release of androgens, such as testosterone, that maintain sperm production, sexual function, health, and wellbeing. Sperm production takes place within the seminiferous tubules, supported by the somatic Sertoli cells, and is stimulated by gonadotrophins produced by the pituitary and androgens produced by the Leydig cells in the interstitium. Spermatogenesis is a highly complex and co-ordinated process regulated via cell-cell interactions between multiple cell types. Sertoli cells produce many factors, in a cyclic manner, that are required for germ cell development and, in turn, they receive signals from developing germ cells to maintain their function. Regulation of the spermatogenic cycle involves different cell types co-operating to produce a “pulse” of retinoic acid that stimulates immature germ cells to become committed to differentiation. Research in the past decade has shown that Sertoli cells do much more than regulate germ cell development; they maintain the development and function of peritubular myoid cells, the testicular vasculature and the Leydig cells. Sertoli cells send signals to interstitial immune cells to maintain an immunosuppressive environment and deposit thousands of proteins into the interstitium, including spermatid-specific proteins that likely help to promote peripheral immune tolerance. Leydig cells produce testosterone that acts on receptors in Sertoli cells to initiate and maintain spermatogenesis, and in peritubular myoid cells to support germ cell development. Leydig cells exist in a niche with specialised macrophages that can regulate Leydig cell function and steroidogenesis, highlighting that cross-talk between testis cells and the immune system is important for testis function. Defining the specific signals that operate between the different cell types that regulate key testis functions will help to uncover new therapies to support male fertility and testicular testosterone production over the life course.

Single-cell RNA sequencing identifies eggplant as a regulator of germ cell development in Drosophila.

Drosophila ovarian germline stem cells (GSCs) are a powerful model for stem cell research. In this study, we use single-cell RNA sequencing (scRNA-seq), an RNAi screen and bioinformatic analysis, to identify genes involved in germ cell differentiation, including 34 genes with upregulated expression during early germ cell development and 19 genes that may regulate germ cell differentiation. Among these, a gene we have named eggplant (eggpl) is highly expressed in GSCs and downregulated in early daughter cells. RNAi knockdown of eggpl causes germ cell proliferation and differentiation defects. In flies fed a rich yeast diet, the expression of eggpl is significantly lower and knockdown or knockout of eggpl phenocopies a rich diet. In addition, eggpl knockdown suppresses the reduction in germ cell proliferation caused by inhibition of the insulin effector PI3K. These findings suggest that downregulation of eggpl links nutritional status to germ cell proliferation and differentiation. Collectively, this study provides new insights into the signaling networks that regulate early germ cell development and identifies eggpl as a key player in this process.

Review: Embryonic stem cells as tools for in vitro gamete production in livestock.

The goal of in vitro gametogenesis is to reproduce the events of sperm and oocyte development in the laboratory. Significant advances have been made in the mouse in the last decade, but evolutionary divergence from the murine developmental program has prevented the replication of these advances in large mammals. In recent years, intensive work has been done in humans, non-human primates and livestock to elucidate species-specific differences that regulate germ cell development, due to the number of potential applications. One of the most promising applications is the use of in vitro gametes to optimize the spread of elite genetics in cattle. In this context, embryonic stem cells have been posed as excellent candidates for germ cell platforms. Here, we present the most relevant advances in in vitro gametogenesis of interest to livestock science, including new types of pluripotent stem cells with potential for germline derivation, characterization of the signaling environment in the gonadal niche, and experimental systems used to reproduce different stages of germ cell development in the laboratory.

The Divergent and Conserved Expression Profile of Turtle Nanog Gene Comparing with Fish and Mammals.

Simple SummaryChinese soft-shell turtle (Pelidiscus sinensis) is a widely farmed aquatic reptile in China. It has of high nutritional, pharmaceutical, and economic value, but wild Chinese soft-shell turtle has been faced with an extreme decline of populations. Thus, it is of great significance to study the germ cells biology and develop the techniques for turtle breeding and genetic resource preserving. Nanog is a homeodomain-containing transcription factor, and it plays a vital role in maintaining the pluripotency of embryonic stem cells; there is a lack of reporting on the Nanog gene in reptile. In this study, the cDNA sequence and promoter of a Nanog gene was identified in P. sinensis, designed as PsNanog. PsNanog gene products were mainly expressed in gonads and were especially rich in ovary. Particularly, PsNanog mRNA and protein was exclusively expressed in germ cells in ovary and testis. Collectively, this is the first report on the gene structure and expression of Nanog in turtle, which may lay a foundation for further functional analysis of the Nanog in turtle, even in the reptiles. Further, PsNanog probably has the conserved and divergent function in regulating cell pluripotency and germ cell development in turtle, compared with other species, including teleosts and mammals.Nanog is a homeodomain-containing transcription factor, and it plays a vital role in maintaining the pluripotency of embryonic stem cells. Nanog’s function has been well studied in many species. However, there is lack of reporting on the Nanog gene in reptile. Here, we identified a 1032 bp cDNA sequence of a Nanog gene in Pelidiscus sinensis, known as PsNanog. PsNanog has a highly conserved HD domain and shares a high identity with that of Chelonia mydas and the lowest identity with Oryzias latipes. Similarly, PsNanog presented a tight cluster with C. mydas Nanog, but was far from those of teleosts. Additionally, we cloned a length of 1870 bp PsNanog promoter. Dual luciferase assay showed that the DNA fragment of −1560 to +1 exhibited a high promoter activity. The RT-PCR and RT-qPCR results showed that PsNanog was predominantly expressed in ovary, and then in testis. The in situ hybridization and immunohistochemical analysis showed that PsNanog was expressed in the early primary oocytes and the cytoplasm of the cortical region of stage VIII oocytes in ovary, and distributed in most stages of germ cells in testis. Collectively, the results imply that PsNanog probably has the conserved function in regulating germ cell development across phyla and is also a pluripotent cell gene and expressed in germ cells, which is similar to that in teleosts and mammals.

Fancb deficiency causes premature ovarian insufficiency in mice

Fanconi anemia complementation group B (FANCB) protein is a major component of the Fanconi anemia (FA) core complex and plays an important role in hematopoiesis and germ cell development. Deletion of Fancb gene causes the defect of primordial germ cell (PGC) development and infertility in male mice. However, it remains unknown whether Fancb is required for female germ cell development. In this study, we found that the fertility of Fancb knockout male mice in C57/ICR mixed backgrounds was not affected. Female Fancb-/- mice were obtained by crossing Fancb+/- females with Fancb-/Y males. The number of PGCs was dramatically decreased in Fancb-/- females. Very few oocytes were observed after birth and the primordial follicle pool was completely depleted at 6weeks of age in Fancb-/- females. However, the remained oocytes from Fancb-/- mice were normal in fertilization and embryonic development from 2-cell to the blastocyst stage. We also found that Fancb and Fancl double-knockout males were also fertile and the number of sperm in epididymis was not reduced as compared to that of Fancb-/- and Fancl-/- single-knockout mice. Taken together, these results showed that Fancb is also essential for female germ cell development. Inactivation of Fancb causes massive germ cell loss and infertility in adult females. We also found that Fancb and Fancl do not act synergistically in regulating germ cell development.

Somatic regulation of female germ cell regeneration and development in planarians.

SUMMARYFemale germ cells develop into oocytes, with the capacity for totipotency. In most animals, these remarkable cells are specified during development and cannot be regenerated. By contrast, planarians, known for their regenerative prowess, can regenerate germ cells. To uncover mechanisms required for female germ cell development and regeneration, we generated gonad-specific transcriptomes and identified genes whose expression defines progressive stages of female germ cell development. Strikingly, early female germ cells share molecular signatures with the pluripotent stem cells driving planarian regeneration. We observe spatial heterogeneity within somatic ovarian cells and find that a regionally enriched foxL homolog is required for oocyte differentiation, but not specification, suggestive of functionally distinct somatic compartments. Unexpectedly, a neurotransmitter-biosynthetic enzyme, aromatic L-amino acid decarboxylase (AADC), is also expressed in somatic gonadal cells, and plays opposing roles in female and male germ cell development. Thus, somatic gonadal cells deploy conserved factors to regulate germ cell development and regeneration in planarians.

Pou5f1 and Nanog Are Reliable Germ Cell-Specific Genes in Gonad of a Protogynous Hermaphroditic Fish, Orange-Spotted Grouper (Epinephelus coioides).

Pluripotency markers Pou5f1 and Nanog are core transcription factors regulating early embryonic development and maintaining the pluripotency and self-renewal of stem cells. Pou5f1 and Nanog also play important roles in germ cell development and gametogenesis. In this study, Pou5f1 (EcPou5f1) and Nanog (EcNanog) were cloned from orange-spotted grouper, Epinephelus coioides. The full-length cDNAs of EcPou5f1 and EcNanog were 2790 and 1820 bp, and encoded 475 and 432 amino acids, respectively. EcPou5f1 exhibited a specific expression in gonads, whereas EcNanog was expressed highly in gonads and weakly in some somatic tissues. In situ hybridization analyses showed that the mRNA signals of EcNanog and EcPou5f1 were exclusively restricted to germ cells in gonads. Likewise, immunohistofluorescence staining revealed that EcNanog protein was limited to germ cells. Moreover, both EcPou5f1 and EcNanog mRNAs were discovered to be co-localized with Vasa mRNA, a well-known germ cell maker, in male and female germ cells. These results implied that EcPou5f1 and EcNanog could be also regarded as reliable germ cell marker genes. Therefore, the findings of this study would pave the way for elucidating the mechanism whereby EcPou5f1 and EcNanog regulate germ cell development and gametogenesis in grouper fish, and even in other protogynous hermaphroditic species.

Different expression of LHR, PRLR, GH and IGF1 during testicular development of yak

AbstractLuteinizing hormone receptor (LHR), prolactin receptor (PRLR), growth hormone (GH) and insulin‐like growth factor 1 (IGF1) have been shown to be key regulators of germ cell development. However, the role of LHR, PRLR, GH and IGF1 in the development of yak testis remains unclear. In this study, we aimed to describe and compare gene expression and protein localization of LHR, PRLR, GH and IGF1 in the development of yak testes. Testes were collected from 6, 24, 36 and 72 months yak, and the kidney, liver, testicular, lung, skeletal muscle, heart and spleen tissues were collected from 36 months yak. The quantitative real‐time PCR (qRT‐PCR) results showed that the expression of these four genes was widely expressed in kidney, liver, testicular, lung, skeletal muscle, heart and spleen, while the LHR and PRLR were highly expressed in the kidney, skeletal muscle and testis, and higher levels of GH and IGF were expressed in spleen and testis. Moreover, the mRNA expression of these genes in adults was higher than in pre‐pubertal yak. In the testis, the LHR‐, PRLR‐, GH‐ and IGF1‐positive signals were detected in the Leydig cells of the 6 months, while the intense positive signals were discovered in Leydig cells, spermatogonia and spermatocytes of the 36 and 72 months. Thus, LHR, PRLR, GH and IGF1 may be involved in the development of spermatids and spermatocytes, and in the regulation of spermatogonia proliferation and Leydig cell function.

Impacts of Immunometabolism on Male Reproduction.

The physiological process of male reproduction relies on the orchestration of neuroendocrine, immune, and energy metabolism. Spermatogenesis is controlled by the hypothalamic-pituitary-testicular (HPT) axis, which modulates the production of gonadal steroid hormones in the testes. The immune cells and cytokines in testes provide a protective microenvironment for the development and maturation of germ cells. The metabolic cellular responses and processes in testes provide energy production and biosynthetic precursors to regulate germ cell development and control testicular immunity and inflammation. The metabolism of immune cells is crucial for both inflammatory and anti-inflammatory responses, which supposes to affect the spermatogenesis in testes. In this review, the role of immunometabolism in male reproduction will be highlighted. Obesity, metabolic dysfunction, such as type 2 diabetes mellitus, are well documented to impact male fertility; thus, their impacts on the immune cells distributed in testes will also be discussed. Finally, the potential significance of the medicine targeting the specific metabolic intermediates or immune metabolism checkpoints to improve male reproduction will also be reassessed.

Novel circGFRα1 Promotes Self-Renewal of Female Germline Stem Cells Mediated by m6A Writer METTL14

Circular RNAs (circRNAs) play important roles in the self-renewal of stem cells. However, their significance and regulatory mechanisms in female germline stem cells (FGSCs) are largely unknown. Here, we identified an N6-methyladenosine (m6A)-modified circRNA, circGFRα1, which is highly abundant in mouse ovary and stage-specifically expressed in mouse FGSC development. Knockdown of circGFRα1 in FGSCs significantly reduced their self-renewal. In contrast, overexpression of circGFRα1 enhanced FGSC self-renewal. Mechanistically, circGFRα1 promotes FGSC self-renewal by acting as a competing endogenous RNA (ceRNA) that sponges miR-449, leading to enhanced GFRα1 expression and activation of the glial cell derived neurotrophic factor (GDNF) signaling pathway. Furthermore, circGFRα1 acts as a ceRNA based on METTL14-mediated cytoplasmic export through the GGACU motif. Our study should help to understand the mechanisms regulating germ cell development, add new evidence on the mechanism of action of circRNA, and deepen our understanding of the development of FGSCs.

The RNA polymerase II subunit RPB-9 recruits the integrator complex to terminate Caenorhabditis elegans piRNA transcription.

PIWI‐interacting RNAs (piRNAs) are genome‐encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA polymerase II (RNA Pol II) core subunit RPB‐9 as required for piRNA‐mediated silencing in the nematode Caenorhabditis elegans. We show that rpb‐9 initiates heritable piRNA‐mediated gene silencing at two DNA transposon families and at a subset of somatic genes in the germline. We provide genetic and biochemical evidence that RPB‐9 is required for piRNA biogenesis by recruiting the Integrator complex at piRNA genes, hence promoting transcriptional termination. We conclude that, as a part of its rapid evolution, the piRNA pathway has co‐opted an ancient machinery for high‐fidelity transcription.

Rbm46, a novel germ cell-specific factor, modulates meiotic progression and spermatogenesis.

It has been suggested that many novel RNA-binding proteins (RBPs) are required for gametogenesis, but the necessity of few of these proteins has been functionally verified. Here, we identified one RBP, Rbm46, and investigated its expression pattern and role in zebrafish reproduction. We found that rbm46 is maternally provided and specifically expressed in the germ cells of gonadal tissues using in situ hybridization, reverse transcription-PCR, and quantitative real-time polymerase chain reaction (qRT-PCR). Two independent rbm46 mutant zebrafish lines were generated via the transcription activator-like effector nuclease technique. Specific disruption of rbm46 resulted in masculinization and infertility in the mutants. Although the spermatogonia appeared grossly normal in the mutants, spermatogenesis was impaired, and meiosis events were not observed. The introduction of a tp53M214K mutation could not rescue the female-to-male sex-reversal phenotype, indicating that rbm46 acts independently of the p53-dependent apoptotic pathway. RNA sequencing and qRT-PCR subsequently indicated that Rbm46 might be involved in the posttranscriptional regulation of functional genes essential for germ cell development, such as nanos3, dazl, and sycp3, during gametogenesis. Together, our results reveal for the first time the crucial role of rbm46 in regulating germ cell development in vivo through promotion of germ cell progression through meiosis prophase I.

Mitochondrial RBP Complex AMG-1/SLRP-1 is Required for Germline Development and Reproductive Success in <i>Caenorhabditis elegans</i>

Mitochondria with their own genome (mtDNA) are the powerhouse for many biological processes. The post-transcriptional regulation of mtDNA is conducted by nuclear-encoded mitochondrial RNA-binding proteins (mtRBPs). However, only a few mtRBPs have been characterized. Here, we identified an mtRBP AMG-1 (Abnormal Mitochondria in Germline) which is crucial for the reproductive development especially spermatogenesis in C. elegans. amg-1 mutation caused mitochondrial dysfunction associated with structure defects and disturbed mtDNA transcription in germ cells. Furthermore, AMG-1 deficiency triggers sperm apoptosis and gonadal mitochondrial unfolded protein response non-cell autonomously. SLRP-1 with RNA recognition motif interacts with AMG-1 genetically to regulate germ cell development. AMG-1 is an ortholog of mammalian LRPPRC, whose mutations cause the French-Canadian type of Leigh syndrome (FCLS) characterized by neurodegeneration. Our findings that mtRBP is involved in spermatogenesis by post-transcriptionally regulating mtDNA might provide implications for understanding of the pathogenic mechanisms of FCLS and for the development of mitochondrial medicine.

Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system

The application of genome engineering techniques to understand the mechanisms that regulate germ cell development opens promising new avenues to develop methods to control sexual maturation and mitigate associated detrimental effects in fish. In this study, the functional role of piwil2 in primordial germ cells (PGCs) was investigated in Nile tilapia using CRISPR/Cas9 and the resultant genotypes were further explored. piwil2 is a gonad-specific and maternally deposited gene in Nile tilapia eggs which is known to play a role in repression of transposon elements and is therefore thought to be important for maintaining germline cell fate. A functional domain of piwil2, PIWI domain, was targeted by injecting Cas9 mRNA and sgRNAs into Nile tilapia embryos at 1 cell stage. Results showed 54% of injected mutant larvae had no or less putative PGCs compared to control fish, suggesting an essential role of piwil2 in survival of PGCs. The genotypic features of the different phenotypic groups were explored by next generation sequencing (NGS) and other mutant screening methods including T7 endonuclease 1 (T7E1), CRISPR/Cas-derived RNA-guided engineered nuclease (RGEN), high resolution melt curve analysis (HRMA) and fragment analysis. Linking phenotypes to genotypes in F0 was hindered by the complex mosacism and wide indel spectrum revealed by NGS and fragment analysis. This study strongly suggests the functional importance of piwil2 in PGCs survival. Further studies should focus on reducing mosaicism when using CRISPR/Cas9 system to facilitate direct functional analysis in F0.

Dynamic and regulated TAF gene expression during mouse embryonic germ cell development.

Germ cells undergo many developmental transitions before ultimately becoming either eggs or sperm, and during embryonic development these transitions include epigenetic reprogramming, quiescence, and meiosis. To begin understanding the transcriptional regulation underlying these complex processes, we examined the spatial and temporal expression of TAF4b, a variant TFIID subunit required for fertility, during embryonic germ cell development. By analyzing published datasets and using our own experimental system to validate these expression studies, we determined that both Taf4b mRNA and protein are highly germ cell-enriched and that Taf4b mRNA levels dramatically increase from embryonic day 12.5–18.5. Surprisingly, additional mRNAs encoding other TFIID subunits are coordinately upregulated through this time course, including Taf7l and Taf9b. The expression of several of these germ cell-enriched TFIID genes is dependent upon Dazl and/or Stra8, known regulators of germ cell development and meiosis. Together, these data suggest that germ cells employ a highly specialized and dynamic form of TFIID to drive the transcriptional programs that underlie mammalian germ cell development.

Npat-dependent programmed Sertoli cell proliferation is indispensable for testis cord development and germ cell mitotic arrest.

As the major somatic cell type, Sertoli cells undergo active proliferation and play essential roles to establish testis cord at fetal stage. They also function to maintain germ cell development throughout the life of testicular development. However, the significance of Sertoli cell number for testis cord development and gonocyte fate is still unclear. Nuclear protein ataxia-telangiectasia (NPAT, also known as p220), a substrate of cyclin E/cyclin-dependent kinase 2, is well known as a regulator of cell proliferation through regulating histone expression. To study the role of NPAT during Sertoli cell development, we generated a mouse strain carrying conditional floxed Npat alleles, when crossing with anti-Müllerian hormone-cre, leading to the specific deletion of Npat in Sertoli cells. Npat disruption in Sertoli cells inhibited the programmed proliferation of fetal Sertoli cells resulting in disruption of developing testis cords, and subsequent postnatal mutant testes were severely hypoplastic. Germ cells, which are presumed to be in quiescent status during perinatal stage, exited G0 phase arrest and re-enter mitotic cell cycle prematurely. Of particular note, some germ cells possessed the meiotic signal in Npat-deficient testes. Our data thus indicates that the function of Npat-dependent Sertoli cells is essential at multiple steps in testis development, and this study also identifies Sertoli cells as a major regulator of germ cell development, which are required to maintain a local growth niche to repress premature mitosis and meiosis of gonocytes.-Jiang, X., Yin, S., Fan, S., Bao, J., Jiao, Y., Ali, A., Iqbal, F., Xu, J., Zhang, Y., Shi, Q. Npat-dependent programmed Sertoli cell proliferation is indispensable for testis cord development and germ cell mitotic arrest.

Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions.

MicroRNAs (miRNAs) play a critical role in multiple aspects of biology. Dicer, an RNase III endonuclease, is essential for the biogenesis of miRNAs, and the germ cell-specific Dicer1 knockout mouse shows severe defects in gametogenesis. How miRNAs regulate germ cell development is still not fully understood. In this study, we identified germ cell-specific miRNAs (miR-741-3p, miR-871-3p, miR-880-3p) by analyzing published RNA-seq data of mouse. These miRNA genes are contiguously located on the X chromosome near other miRNA genes. We named them X chromosome-linked miRNAs (XmiRs). To elucidate the functions of XmiRs, we generated knockout mice of these miRNA genes using the CRISPR/Cas9-mediated genome editing system. Although no histological abnormalities were observed in testes of F0 mice in which each miRNA gene was disrupted, a deletion covering miR-871 and miR-880 or covering all XmiRs (ΔXmiRs) resulted in arrested spermatogenesis in meiosis in a few seminiferous tubules, indicating their redundant functions in spermatogenesis. Among candidate targets of XmiRs, we found increased expression of a gene encoding a WNT receptor, FZD4, in ΔXmiRs testis compared with that in wildtype testis. miR-871-3p and miR-880-3p repressed the expression of Fzd4 via the 3′-untranslated region of its mRNA. In addition, downstream genes of the WNT/β-catenin pathway were upregulated in ΔXmiRs testis. We also found that miR-871, miR-880, and Fzd4 were expressed in spermatogonia, spermatocytes and spermatids, and overexpression of miR-871 and miR-880 in germ stem cells in culture repressed their increase in number and Fzd4 expression. Previous studies indicated that the WNT/β-catenin pathway enhances and represses proliferation and differentiation of spermatogonia, respectively, and our results consistently showed that stable β-catenin enhanced GSC number. In addition, stable β-catenin partially rescued reduced GSC number by overexpression of miR-871 and miR-880. The results together suggest that miR-871 and miR-880 cooperatively regulate the WNT/β-catenin pathway during testicular germ cell development.