Ets-1 and Ets-2 protooncogene expression in theca cells of the adult mouse ovary.

We have investigated the mRNA expression of the Ets-1 and Ets-2 genes in murine gonads and found expression in adult ovaries. In situ hybridization experiments show that the Ets genes are predominantly expressed in theca cells and cells of ovarian interstitium. By gel retardation experiments we detected DNA binding proteins in ovaries that specifically bind to the ETS motif, suggesting the expression of Ets or Ets-related proteins. Our results raise the possibility of Ets-2 involvement in ovarian pathology seen in patients with Down's syndrome.     

Expression of the Scavenger Receptor Class B type I (SR-BI) family in Drosophila melanogaster

In mammals, cholesterol is transformed into steroid hormones in the adrenal gland, the ovaries or the testes. The Scavenger Receptors Class B Type I (SR-BI) are membrane proteins that belong to the CD36 family and participate in the selective uptake of high density lipoprotein cholesteryl ester in the mammalian steroidogenic tissues. Fourteen members of the CD36 family have been identified in Diptera, although their expression patterns remain uncharacterized. Using in situ hybridization we have characterized the expression patterns of the fourteen SR-BIs in Drosophila melanogaster. We analyzed three different developmental larval stages prior to and during the peak of the insect steroid hormone ecdysone, which triggers the larval to pupal transition. We focused on the steroidogenic tissues, such as the prothoracic gland, the ovaries and the testes, and extended our analysis to non-steroidogenic tissues, such as the fat body, salivary glands, the gut, the gastric caeca or the central nervous system. Our results show highly regulated expression patterns, with three genes crq, pes and Snmp being upregulated in steroidogenic tissues at the onset of pupariation when steroidogenesis is crucial. This study underlines the importance of the transport of cholesterol and steroids in the process of ecdysone synthesis.     

Expression of the endothelial lipase gene in murine embryos and reproductive organs

Endothelial lipase (EL) is a recently discovered member of the triglyceride-lipase family that is involved in plasma HDL metabolism. In this study, we investigated the putative role of EL in mouse reproduction by studying EL gene expression in mouse embryos and adult reproductive organs. PCR analysis revealed that EL mRNA is expressed in mouse embryos on embryonic day 8.5 (E8.5) to E11.5, but not later in development. In situ hybridization studies on E10.5 whole embryos and embryonic sections showed expression of EL mRNA in multiple tissues, although of varying intensity. High expression was found in the neuroepithelium of the brain and the neural tube, the mesenchymal cells between organs, the optic lens and cup, and the otocyst. In adult mice, EL mRNA expression was high in ovaries from pregnant mice but low in ovaries from nonpregnant mice. EL mRNA was also highly expressed in placenta and testes. In situ hybridization studies demonstrated intense EL mRNA staining of lutein cells in corpora lutei in ovaries, of spermatocytes in the late pachytene and diplotene stages in testes, and of principal cells in epididymis. These results suggest that EL, in addition to its effects on plasma lipoprotein metabolism, plays a role in murine reproduction.     

The Dr-nanos gene is essential for germ cell specification in the planarian Dugesia ryukyuensis

Homologs of nanos are required for the formation and maintenance of germline stem cell (GSC) systems and for gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, alternating between asexual and sexual reproduction; they develop and maintain their somatic stem cells (SSCs) and GCSs from pluripotent stem cells known as neoblasts. We isolated a nanos homolog, Dr-nanos, from the expressed sequence tags (ESTs) of the sexualized form of Dugesia ryukyuensis. We examined the expression of Dr-nanos in asexual and sexualized planarians by in situ hybridization and analyzed its function using RNA interference (RNAi) together with a planarian sexualization assay. A nanos homolog, Dr-nanos, was identified in the planarian D. ryukyuensis. Dr-nanos expression was observed in the ovarian primordial cells of the asexual worms. This expression increased in proportion to sexualization and was localized in the early germline cells of the ovaries and testes. In X-ray-irradiated worms, the expression of Dr-nanos decreased to a large extent, indicating that Dr-nanos is expressed in some subpopulations of stem cells, especially in GSCs. During the sexualization process, worms in which Dr-nanos was knocked down by RNAi exhibited decreased numbers of oogonia in the ovaries and failed to develop testes, whereas the somatic sexual organs were not affected. We conclude that Dr-nanos is essential for the development of germ cells in the ovaries and testes and may have a function in the early stages of germ cell specification, but not in the development of somatic sexual organs.     

Molecular analysis shows differential expression of <Emphasis Type="Italic">R</Emphasis>-<Emphasis Type="Italic">spondin1</Emphasis> in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) gonads

R-spondin1 (RSPO1) is a potential female-determining gene in human (Homo sapiens) and mouse (Mus musculus). Its differential expression in these mammals is correlated with signaling for sex determination. As a way of studying sex determination in fish we cloned and analyzed a RSPO1 gene in zebrafish (Danio rerio). Using real-time PCR, we observed that RSPO1 is expressed more strongly in ovaries than in testes, suggesting that RSPO1 may have a role in gonad differentiation. High RSPO1 expression was detected in some non-gonadal organs like muscle and kidneys. In situ hybridization results demonstrate that RSPO1 is expressed in premature germ cells, in oogonia and primary oocytes in ovaries and in spermatogonia and spermatocytes in testes. It is also expressed in gonad somatic cells during gonadal development: in granulosa cells and theca cells of early and late cortical-alveolar stage follicles in ovaries, and in Leydig cells in testes. This differential expression may indicate that RSPO1 has a role(s) in zebrafish gonad development and differentiation. By fusing zebrafish RSPO1 with a green fluorescent protein gene, we found that RSPO1 is located in the cytosol and Golgi apparatus but not the nucleus of fish epithelioma papulosum cyprinid (EPC) cells. These preliminary findings suggest some aspects of RSPO1 like differential expression linked to sex determination may be conserved in fish while other aspects like subcellular localization differ from the mammalian RSPO1.     

Expression of murine Asb-9 during mouse spermatogenesis

We previously showed that Asb-4 and Asb-17 is uniquely expressed in developing male germ cells. A recent report showed that Asb-9 is specifically expressed in the kidney and testes; however, detailed expression patterns in developing germ cells have not been shown. Northern blot analysis in various tissues demonstrated that mAsb-9 was strongly expressed in the testes. Expression analysis by RT-PCR and Northern blot in developing mouse testes indicates that mAsb-9 is expressed from the fourth week after birth to adulthood, with the highest expression in round spermatids. Expression sites were further localized by in situ hybridization in the testes. Pachytene spermatocytes and spermatids expressed mAsb-9 but spermatogonia and generated spermatozoa did not. This study reveals that mAsb-9 could be a specific marker of active spermatogenesis and would be useful for studies of male germ cell development.     

人、鼠原肠形成蛋白基因的克隆和初步分析

目的：分析原肠形成蛋白（TSG）在几个发育阶段的人和小鼠睾丸中的差异表达。方法：应用cDNA微阵列技术对成人和胚胎睾丸进行基因表达图谱分析,获得在成人高表达、胚胎低表达的人TSG的全长基因;利用RT-PCR,从4周龄小鼠睾丸中克隆出小鼠TSG基因,用地高辛标记的探针进行原位杂交,检测小鼠TSG在睾丸中的表达。结果：人TSG在成人睾丸中高表达,在胚胎中低表达;小鼠TSG仅在小鼠睾丸生殖细胞中表达,在间质细胞中不表达。结论：小鼠睾丸TSG与骨形态发生蛋白（BMP）可能同步表达,提示在小鼠精子发生中也可能存在一条由TSG信号激活BMP的传导途径。     

Mouse granulosa cells contribute more to the mRNA synthesis of mZP2 than oocyte does

In order to elucidate the biogenesis of mouse zona pellucida 2 (mZP2) protein, RT-PCR, and in situ hybridization were carried out to localize the expression of mouse ZP2 mRNA. Cumulus cells of the OCC (Oocyte-Cumulus cell Complex) were isolated from the oocytes after superovulation for the RNA extraction. The frozen sections of ovaries from adolescent and aged mice were prepared to hybridize with RNA probe of mouse ZP2. mRNA of ZP2 was detected in isolated cumulus cells by RT-PCR. Results of in situ hybridization showed that the mRNA of ZP2 was synthesized in both oocyte and granulosa cells at different folliculogenesis stages; and the expression of ZP2 mRNA in granulosa cells was stronger than that in oocyte; much weaker expression of mZP2 was detected in the follicles of aged mouse. These suggest that the entire amount of ZP2 mRNA generated in the granulosa cells layer should be much more than that in oocyte. Therefore, we think that the granulosa cells contribute more to the mZP2 mRNA synthesis than oocyte does.