Expression pattern of Boule in dairy goat testis and its function in promoting the meiosis in male germline stem cells (mGSCs)

Boule is a conserved gene in meiosis, which encodes RNA binding protein required for spermatocyte meiosis. Deletion of Boule was found to block meiosis in spermatogenesis, which contributes to infertility. Up to date, the expression and function of Boule in the goat testis are not known. The objectives of this study were to investigate the expression pattern of Boule in dairy goat testis and their function in male germline stem cells (mGSCs). The results first revealed that the expression level of Boule in adult testes was significantly higher than younger and immature goats, and azoospermia and male intersex testis. Over‐expression of Boule promoted the expression of meiosis‐related genes in dairy goat mGSCs. The expression of Stra8 was up‐regulated by over‐expression of Boule analyzed by Western blotting and Luciferase reporter assay. While, Cdc25a, the downstream regulator of Boule, was found not to affect the expression of Stra8, and our data illustrated that Cdc25a did not regulate meiosis via Stra8. The expression of Stra8 and Boule was up‐regulated by RA induction. Taken together, results suggest the Boule plays an important role in dairy goat spermatogenesis and that over‐expression of Boule may promote spermatogenesis and meiosis in dairy goat. J. Cell. Biochem. 114: 294–302, 2013. © 2012 Wiley Periodicals, Inc.     

Conservation and function of Dazl in promoting the meiosis of goat male germline stem cells

Dazl (deleted in azoospermia-like) is a conserved gene in mammalian meiosis, which encodes RNA binding protein required for spermatocyte meiosis. Up to date, the expression and function of Dazl in the goat testis are unknown. The objectives of this study were to investigate the expression pattern of Dazl in dairy goat testis and their function in male germline stem cells (mGSCs). The results first revealed that the expression level of Dazl in adult testes was significantly higher than younger and immature goats, and azoospermia and male intersex testis. The dairy goat Dazl is highly conserved analysed by several online and bioinformatics software, respectively. Over-expression of Dazl promoted the expression of meiosis-related genes in dairy goat mGSCs. The expression of Stra8 was up-regulated by over-expression of Dazl analysed by Luciferase reporter assay. Taken together, results suggest the Dazl plays an important role in dairy goat spermatogenesis and that over-expression of Dazl may promote Stra8 expression in dairy goat mGSCs.     

Figla基因过表达促进小鼠胚胎干细胞向雌性生殖细胞分化

生殖系a因子(Figla）是最早表达的生殖细胞特异性转录因子之一,对卵泡的发育、Zp基因的表达和透明带的形成具有调节作用. Figla基因异常会引起卵巢早衰的发生. 本研究通过 PCR自小鼠基因组中扩增出Figla基因,将其克隆到真核报告载体pDsRed1 N1,构建了携带609 bp 的Figla重组载体pDsRed1 N1 Figla. 用该载体转染小鼠胚胎干细胞（mESCs）系J1、小鼠成纤维细胞系NIH 3T3、小鼠畸胎瘤细胞P19和小鼠精原细胞系GC1,在荧光显微镜下观察红色荧光蛋白(RFP)在细胞中的表达,同时检测转染细胞中Figla基因及其它生殖细胞特异性基因的表达. 结果显示,转染2 d,mESCs内Figla总表达量明显增加,且内源性表达量亦有所提高,即转入的外源性Figla基因可以促进内源性Figla的启动和表达. 免疫荧光染色显示,表达RFP 的细胞同时表达生殖特异性基因Vasa,减数分裂特异性基因Stra8、Scp3及卵母细胞标志基因Zp3. 通过QRT PCR检测发现,在转染3 d的细胞中,Vasa、Scp3和Zp1的表达较对照组均有明显上调,而Oct4和Stra8的表达量下降. 研究表明,Figla基因对生殖特异性基因的表达具有调控作用,可以激活雌性生殖基因表达,为更清楚地了解Figla基因在生殖细胞生长发育过程中的调控机制,以及发现该基因在生殖细胞中的新功能奠定了基础.     

Expression of miR‐34c in response to overexpression of Boule and Stra8 in dairy goat male germ line stem cells (mGSCs)

Reproduction is required for the survival of all mammalian animals. Spermatogenesis is an essential and complex developmental process that ultimately results in production of haploid spermatozoa. Recent studies demonstrated that Boule and stimulated by retinoic acid 8 (Stra8) played important roles in initiation meiosis in male germ cells. miR‐34c is indispensable in the late steps of spermatogenesis; remarkably, the main function of miR‐34c is to reduce cell proliferation potentiality and promote cellular apoptosis. The objectives of this study were to investigate the expression patterns of Boule, Stra8, P53 and miR‐34c in dairy goat testis and their relationship in male germ line stem cells (mGSCs). The results first revealed the expression patterns of Boule, Stra8, P53 and miR‐34c in 30 dpp, 90 dpp and adult testes of dairy goats. The expression levels of Boule, Stra8, P53 and miR‐34c in adult dairy goat testes were significantly higher than that of 30 dpp. Overexpression of Boule and Stra8 promoted the expression of miR‐34c in dairy goat mGSCs. In our previous study, we showed that miR‐34c was P53 dependent in mGSCs. These results have shown that the up‐regulation of miR‐34c was not due to P53 protein activation but which might be caused by the up‐regulation of Boule and Stra8 promoting the advance of meiosis. In addition, we found retinoic acid would decrease the expression of P53 and miR‐34c, however, did not change the expression of c‐Myc greatly. It suggested that the function of driving differentiation of dairy goat mGSCs by retinoic acid might not be caused by P53. Copyright © 2013 John Wiley & Sons, Ltd.     

绒山羊Scp3基因的克隆及睾丸中第一轮减数分裂的发生

联会复合体蛋白3（synaptonemal complex protein 3, Scp3）是雄性生殖细胞减数分裂中联会复合体形成必需的组成部分,在哺乳动物生殖细胞减数分裂中具有重要功能。通过RT-PCR扩增和分子克隆的方法首次克隆到了绒山羊Scp3基因的编码区片段。测序结果表明,绒山羊与牛的Scp3基因编码序列同源率达到98％。根据测得的DNA序列,设计引物,用RT-PCR方法分析测定了青春前期绒山羊不同个体中睾丸组织的Scp3的转录表达。结果显示,雄性绒山羊青春前期睾丸发育存在个体差异,已分析的样品第一轮减数分裂发生的时间普遍为73天之后。这一结果为绒山羊的睾丸发育和精子发生过程的相关研究打下了基础。     

MiR-34c在奶山羊雄性生殖干细胞增殖分化过程中的作用

microRNA (miRNA)在奶山羊雄性生殖细胞和精子发生过程有重要的调控功能。为研究miR-34c对雄性生殖干细胞增殖与分化中的作用,本文利用视黄酸效应基因8（Stra8）在雄性生殖细胞中随年龄增长,以其表达量上调的表达特征为指针,使用实时定量PCR技术筛选分析miRNAs。结果发现,miR-34c与Stra8的表达规律基本一致。在无精症奶山羊的睾丸组织中,发现miR-34c在无精症奶山羊睾丸组织中表达缺失。利用miR-34c模拟物及抑制剂转染奶山羊雄性生殖干细胞,体外转染miR-34c模拟物及其抑制剂,发现miR-34c能够下调Rarg、Stra8与c-Myc基因的表达,减缓奶山羊雄性生殖干细胞的增殖。结果提示,miR-34c可能具有调控奶山羊雄性生殖干细胞的减数分裂的作用,同时抑制其增殖。     

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.     

Epidermal growth factor and three‐dimensional scaffolds provide conducive environment for differentiation of mouse embryonic stem cells into oocyte‐like cells

Three‐dimensional (3D) culture provides a biomimicry of the naive microenvironment that can support cell proliferation, differentiation, and regeneration. Some growth factors, such as epidermal growth factor (EGF), facilitate normal meiosis during oocyte maturation in vivo. In this study, a scaffold‐based 3D coculture system using purified alginate was applied to induce oocyte differentiation from mouse embryonic stem cells (mESCs). mESCs were induced to differentiate into oocyte‐like cells using embryoid body protocol in the two‐dimensional or 3D microenvironment in vitro. To increase the efficiency of the oocyte‐like cell differentiation from mESCs, we employed a coculture system using ovarian granulosa cells in the presence or absence of epidermal growth factor (+EGF or ?EGF) for 14 days and then the cells were assessed for germ cell differentiation, meiotic progression, and oocyte maturation markers. The cultures exposed to EGF in the alginate‐based 3D microenvironment showed the highest level of premeiotic (Oct4 and Mvh), meiotic (Scp1, Scp3, Stra8, and Rec8), and oocyte maturation (Gdf9, Cx37, and Zp2) marker genes (p < .05) in comparison to other groups. According to the gene‐expression patterns, we can conclude that alginate‐based 3D coculture system provided a highly efficient protocol for oocyte‐like cell differentiation from mESCs. The data showed that this culture system along with EGF improved the rate of in vitro oocyte‐like cell differentiation.     

Stra 8基因的激活与精原干细胞的特异性分化研究

视黄酸对维持正常的雄性睾丸结构和功能起着重要的作用。近来的研究发现,在雄性生殖腺发育过程中有一组基因,它们可以被视黄酸特异性的诱导活化,称为Stra(Stimulated by Retinoic Acid)基因。从鼠源分离得到的Stra8基因编码一种细胞质蛋白,该基因只特异性的在成熟雄性生殖细胞中表达,其功能被认为与精子形成有关。为研究Stra8基因的表达特性,我们从小鼠的基因组中克隆了Stra8基因的启动子序列(1.4kb)。将Stra8基因的1.4kb启动子序列克隆到pEGFP-1载体的EGFP基因之前,构建成由Stra8基因1.4kb启动子序列调控表达绿色荧光蛋白的pStra8-EGFP载体。将其分别转化到不同类型的细胞中,如小鼠ES-129细胞、人胎儿胰腺干细胞、小鼠骨髓间充质干细胞和小鼠精原干细胞等,通过荧光显微镜观察发现,绿色荧光蛋白只在小鼠精原干细胞中表达,表明Stra8基因是组织特异性表达的基因。将pStra8-EGFP转化小鼠骨髓间充质干细胞,经G418筛选2周后,用视黄酸诱导,12h培养后,有一部分转化pStra8-EGFP载体的细胞表达绿色荧光蛋白。RT-PCR证明这些细胞中有精原干细胞特异表达基因Stra8的转录,还有生殖细胞特异表达基因CyclinA8和Oct4的转录,这些结果说明小鼠骨髓间充质细胞经视黄酸的诱导可以向生殖细胞方向分化。     

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.     

Stem cell protein Piwil2 modulates expression of murine spermatogonial stem cell expressed genes

The piwi family genes are highly conserved during evolution and play essential roles in stem cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Arabidopsis to human. Piwil2, known also as Mili gene, is one of three mouse homologues of piwi. Piwil2 was found in germ cells of adult testis, suggesting that this gene functions in spermatogonial stem cell self-renewal. In order to find molecular mechanisms underlying stem cell activity mediated by Piwil2 gene, an in vitro gain of function cell culture model was established. Messenger RNAs isolated from cells expressing Piwil2 and mRNAs isolated from cells without Piwil2 expression were compared using a stem cell array technique. It was shown that Piwil2 modulates expression of stem cell specific genes, including platelet-derived growth factor receptor, beta polypeptide (Pdgfrb), solute carrier family 2 member 1 (Slc2a1), gap junction membrane channel protein alpha 7 (Gja7), and spermatogonial cell surface markers Thy-1 (CD90), integrin alpha 6 (Itga6), CD9, and spermatogonia specific markers heat shock protein 90 alpha (Hsp90a), and stimulated by retinoic acid gene 8 (Stra8). These molecules play essential role in stem cells proliferation (Pdgfrb), energy metabolism (Slc2a1), cell adhesion, cell-cell interaction (Itga6, Gja7, Thy-1, and CD9), and germ cell differentiation (Stra8). The expression of these markers in spermatogonial stem cells and other nongerminal stem cells suggests that these cells share elements of common molecular machinery with stem cells in other tissues which are modulated by stem cell protein Piwil2.     

Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene

The full-length cDNA corresponding to Stra8, a novel gene inducible by retinoic acid (RA) in P19 embryonal carcinoma cells, has been isolated and shown to encode a 45-kD protein. Both Stra8 mRNA and protein were induced in cells treated by all-trans and 9-cis retinoic acids. Two- dimensional gel analysis and dephosphorylation experiments revealed that the two stereoisomers of RA differentially regulate the phosphorylation status of the Stra8 protein, which was shown to exist in differently phosphorylated forms. Subcellular fractionation and immunocytochemistry studies showed that the Stra8 protein is cytoplasmic. During mouse embryogenesis, Stra8 expression was restricted to the male developing gonads, and in adult mice, the expression of Stra8 was restricted to the premeiotic germ cells. Thus, Stra8 protein may play a role in the premeiotic phase of spermatogenesis.     

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.