A Gene Regulatory Program for Meiotic Prophase in the Fetal Ovary

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

The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation

The signal for somatic sex determination in mammals, Caenorhabditis elegans and Drosophila melanogaster is chromosomal, but the overall mechanisms do not appear to be conserved between the phyla. However it has been found quite recently that the C. elegans sex-determining gene Mab-3 contains a domain highly homologous to the Drosophila sex-determining gene doublesex (dsx) and shares a similar role. These data suggest that at least some aspects of the regulation of sex determination might be conserved. In humans, a doublesex-related gene (DMRT1) was identified at less than 30 kb from the critical region for sex reversal on chromosome 9p24 (TD9). In order to get insights into the role of DMRT1 in sex determination/differentiation, we have isolated DMRT1 mouse homologue (Dmrt1) and analysed its expression pattern. The gene is expressed in the genital ridges of both sexes during the sex-determining switch and it shows male/female dimorphism at late stages of sex differentiation.     

PLZF suppresses differentiation of mouse spermatogonial progenitor cells via binding of differentiation associated genes

Promyelocytic leukaemia zinc finger (PLZF) is a key factor in inhibiting differentiation of spermatogonial progenitor cells (SPCs), but the underlying mechanisms are still largely unknown. In this study, the regulation of PLZF on Kit, Stra8, Sohlh2, and Dmrt1 (SPCs differentiation related genes) was investigated. We found some PLZF potential binding sites existed in the promoters of Kit, Stra8, Sohlh2, and Dmrt1. Additionally, the expressions of KIT, STRA8, SOHLH2, and DMRT1 were upregulated when PLZF was knockdown in SPCs. Furthermore, chromatin immunoprecipitation quantitative polymerase chain reaction revealed PLZF directly bound to the promoters of Kit, Stra8, Sohlh2, and Dmrt1. Besides, dual luciferase assay verified PLZF repressed those gene expressions. Collectively, our finding indicate that PLZF binds to the promoter regions of Kit, Stra8, Sohlh2, and Dmrt1 to regulate SPCs differentiation, which facilitate us to further understand the regulatory mechanism of PLZF in SPCs fates.     

Medaka dead end encodes a cytoplasmic protein and identifies embryonic and adult germ cells

dead end (dnd) was identified in zebrafish as a gene encoding an RNA-binding protein essential for primordial germ cell (PGC) development and gametogenesis in vertebrates. The adult dnd RNA expression has been restricted to the ovary in Xenopus or to the testis in mouse. Its protein product is nuclear in chicken germ cells but both cytosolic and nuclear in mouse cell cultures. Here we report the cloning and expression pattern of Odnd, the medakafish (Oryzias latipes) dnd gene. Sequence comparison, gene structure, linkage analysis and expression demonstrate that Odnd encodes the medaka Dnd orthologue. A systematic comparison of Dnd proteins from five fishes and tetrapod representatives led to the identification of five previously unidentified conserved regions besides the RNA recognition motif. The Odnd RNA is maternally supplied and preferentially segregated with PGCs. Its adult expression occurs in both sexes and is restricted to germ cells. In the testis, Odnd is abundant in spermatogonia and meiotic cells but absent in sperm. In the ovary, Odnd RNA persists throughout oogenesis. Furthermore, we developed a dual color fluorescent in situ hybridization procedure allowing for precise comparisons of expression and distribution patterns between two genes in medaka embryos and adult tissues. Importantly, this procedure co-localized Odnd and Ovasa in testicular germ cells and PGCs. Surprisingly, by cell transfection and embryo RNA injection we show that ODnd is cytoplasmic in cell cultures, cleavage embryos and PGCs. Therefore, medaka dnd encodes a cytoplasmic protein and identifies embryonic and adult germ cells of both sexes.     

基于Cre/loxP系统的睾丸组织特异性敲除Elovl4基因小鼠模型的构建及敲除效率检测

极长链多不饱和脂肪酸(very long chain polyunsaturated fatty acids,VLC-PUFAs)是哺乳动物视网膜、睾丸等极少数组织中特有的脂肪酸,其生物合成的关键酶为极长链脂肪酸延长酶4(very long chain fatty acid elongase 4,Elovl4)。建立组织特异性敲除Elovl4基因的动物模型有利于深入研究VLC-PUFAs的生物学功能,因此,本研究基于Cre/loxP系统,先分别构建了Stra8-Cre小鼠和Elovl4 floxed小鼠,通过杂交获得(Elovl4[flox/+],Stra8-Cre)杂合子基因敲除小鼠,再选择雌鼠与Elovl4 floxed纯合子雄鼠即Elovl4 [flox/flox]雄鼠杂交,通过基因型鉴定筛选获得(Elovl4[flox/flox], Stra8-Cre)纯合子小鼠。利用RT-PCR、qRT-PCR、Western blotting、免疫组化和免疫荧光检测Elovl4在睾丸组织中的敲除效率,结果表明,无论是杂合子还是纯合子基因敲除小鼠,其睾丸组织中Elovl4的表达在mRNA及蛋白水平显著下调,但其他组织未受影响。本研究成功构建了睾丸组织特异性敲除Elovl4基因小鼠,为后续研究VLC-PUFAs对雄性小鼠生殖功能的影响及相关分子机制提供可靠的动物模型。     

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

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

Expression characterization of testicular DMRT1 in both Sertoli cells and spermatogenic cells of polyploid gibel carp

Dmrt1 has been suggested to play significant roles in sex determination and differentiation, but various expression patterns and cell types have been observed in the testis of vertebrates. Polyploid gibel carp, because of the multiple modes of unisexual gynogenesis and sexual reproduction, has become a unique case to explore the evolution of sex determination and differentiation. However, the sex-determination related genes in gibel carp have remained unknown. In this study, we identified and characterized 4 cDNAs of Dmrt1 genes. Subsequently, a polyclonal antibody specific to CagDMRT1 was prepared to examine its expression and distribution patterns at protein level. Significantly, both relative real-time PCR and Western blot detection confirmed predominant expression of CagDmrt1 in the adult testis of gibel carp. Moreover, the intensive expression of CagDMRT1 around spermatogenic cysts was revealed during spermatogenesis. And, following immunofluorescence co-localization of CagDMRT1 and CagVASA, a prominent CagDMRT1 expression in Sertoli cells and a mild CagDMRT1 expression in spermatogenic cells including spermatogonia and primary spermatocytes were clearly characterized. The CagDMRT1 signal in Sertoli cells is extensively distributed in both nuclei and cytoplasm, while the CagDMRT1 in spermatogonia and primary spermatocytes is mainly expressed in nuclei, and there is only the remained CagDMRT1 signal in the cytoplasm of secondary spermatocytes. These findings suggest that DMRT1 should be related to testis differentiation and spermatogenesis in gibel carp.     

Normal fertility in male mice with deletion of β‐catenin gene in germ cells

As a dual function protein, β‐catenin affects both cell adhesion and mediates canonical Wnt/β‐catenin cell signaling. β‐Catenin is prominently expressed in somatic Sertoli cells in the testis and postmeiotic germ cells, suggesting an additional role in spermatogenesis. It was reported previously that Cre/loxP‐mediated conditional inactivation of the β‐catenin gene (Ctnnb1) in male gonads using a protamine promoter‐driven Cre transgene (Prm‐cre) resulted in partial infertility, reduced sperm count, and abnormal spermatogenesis. In this report, we demonstrated that the conditional deletion of Ctnnb1 using a germ cell specific Cre transgene (Stra8‐icre) had no effect on male fertility. We have shown that the Stra8‐icre transgene was highly efficient in generating deletion in early pre‐meiotic and post‐meiotic cells. No differences in anatomical or histological presentation were found in the mutant testis, the production of viable sperm was similar, and no abnormalities in DNA sperm content were detected. We concluded that β‐catenin is fully dispensable in germ cells for spermatogenesis. The conflicting results from the earlier study may have been due to off‐target expression of Prm‐cre in testicular somatic cells. In future studies, the analysis of conditional mutants using several Cre‐transgenes should be encouraged to reduce potential errors. genesis 52:328–332, 2014. © 2014 Wiley Periodicals, Inc.     

Bisphenol A exposure modifies DNA methylation of imprint genes in mouse fetal germ cells

Bisphenol A (BPA) is an estrogenic environmental toxin widely used for the production of plastics. Human frequent exposure to this chemical has been proposed to be a potential public health risk. The objective of this study was to assess the effects of BPA on DNA methylation of imprinting genes in fetal mouse germ cell. Pregnant mice were treated with BPA at doses of 0, 40, 80 and 160 μg BPA/kg body weight/day from 0.5 day post coitum. DNA methylation of imprinting genes, Igf2r, Peg3 and H19, was decreased with the increase of BPA concentration in fetal mouse germ cells (p < 0.01).The relative mRNA levels of Nobox were lower in BPA-treated group compared to control (BPA free) in female fetal germ cells, but in male fetal germ cells, a significant higher in Nobox expression was observed in BPA-treated group compared to control. Decreased mRNA expression of specific meiotic genes including Stimulated by Stra8 and Dazl were obtained in the female fetal germ cells. In conclusion, BPA exposure can affect the DNA methylation of imprinting genes in fetal mouse germ cells.     

Gene structure, multiple alternative splicing, and expression in gonads of zebrafish Dmrt1

Many basic cellular processes are shared across vast phylogenetic distances, whereas sex-determining mechanisms are highly variable between phyla although the existence of two sexes is nearly universal in the animal kingdom. The only molecular similarity in sex determination found so far between phyla is among the fly doublesex, worm mab-3, and vertebrate Dmrt1/DMY, which contain a zinc-finger-like DNA-binding motif, DM domain. Here we report that three isoforms of the zebrafish Dmrt1 were generated in gonads by multiple alternative splicing, which encoded predicted proteins with 267, 246, and 132 amino acids, respectively. By cDNA cloning and genomic structure analysis, we found that there were seven exons of Dmrt1, which were alternatively spliced to generate the Dmrt1 isoforms. Northern blotting analysis revealed that expression of zebrafish Dmrt1 was higher in testis than ovary. Real time fluorescent quantitative RT-PCR indicated that expression of isoform a of Dmrt1 was dominantly higher than those of Dmrt1 b and c. Furthermore, in situ hybridization to gonads sections showed that Dmrt1 was expressed in developing germ cells of both testis and ovary, suggesting that the Dmrt1 gene is not only associated with testis development, but also, may be important in ovary differentiation of zebrafish.     

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

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

Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice

We have generated a transgenic mouse line,Tg(Stra8-cre)1Reb (Stra8-cre), which expresses improved Cre recombinase under the control of a 1.4 Kb promoter region of the germ cell-specific stimulated by retinoic acid gene 8 (Stra8). cre is expressed only in males beginning at postnatal day (P)3 in early-stage spermatogonia and is detected through preleptotene-stage spermatocytes. To further define when cre becomes active, we crossed Stra8-cre males with Tg(ACTB-Bgeo/GFP)21Lbe (Z/EG) reporter females and compared the expression of enhanced green fluorescent protein (EGFP) with the protein encoded by the zinc finger and BTB domain containing 16 (Zbtb16) gene, PLZF-a marker for undifferentiated spermatogonia. Co-expression of EGFP is observed in the majority of PLZF+ cells. We also tested recombination efficiency by mating Stra8-cre;Z/EG males and females with wild-type mice and examining EGFP expression in the offspring. Recombination is detected in >95% of Z/EG+ pups born to Stra8-cre;Z/EG fathers but in none of the offspring born to transgenic mothers, a verification that cre is not functional in females. The postnatal, premeiotic, male germ cell-specific activity of Stra8-cre makes this mouse line a unique resource to study testicular germ cell development.