Characterization of estrogen receptor beta2 and expression of the estrogen receptor subtypes alpha, beta1, and beta2 in the protandrous black porgy (Acanthopagrus schlegeli) during the sex change process

Estrogens play an important role in many physiological processes in both female and male vertebrates, mediated by specific nuclear receptor, estrogen receptors (ERs). We have isolated a third ER (ERbeta2), which was found to contain 2004 nucleotides including an open reading frame that encodes 667 amino acids. We have also cloned ERalpha and ERbeta1 from the published information (GenBank accession nos. AY074780 and AY074779) and investigated the expression pattern of these ER subtypes in the gonads during gonad sex change of black porgy by quantitative polymerase chain reaction. Maturity stages can be divided into five stages during the sex change process from immature male to female (immature male, mature male, male of mostly testis, male of mostly ovary and mature female). The expression of ERalpha mRNA was highest in the ovary of mature female, followed by the testis of mature male and testicular portion of mostly testis. ERbeta1 expression was higher in the mature testis and ovary than in the gonads of other maturity stages. In contrast to that, ERbeta2 was highest in the ovary of mature female, and significantly lower levels of ERbeta2 expression were observed in the gonads of the other maturity stages. The present study describes the molecular characterization of ERbeta2, and documents the expression changes of three ER subtypes during sex change process of the protandrous black porgy.     

四眼斑龟的泄殖系统解剖

对8只成体四眼斑龟(Sacalia quadriocllata)的泄殖系统进行解剖,结果表明：同一个体的左右肾的体积和重量无明显差异;膀胱呈Y型,体积小;左右各有一个副膀胱作为呼吸辅助器官。同一个体的左、右侧性腺大小和重量存在显著差异;在繁殖季节性腺有一定的变化,交配期精巢的重量可增加2倍;产卵期卵巢的卵球数增加。     

Estrogen synthesis in relation to gonadal development of Japanese scallop, Patinopecten yessoensis: gonadal profile and immunolocalization of P450 aromatase and estrogen

Aromatase activities and estrogen contents in the gonad of Japanese scallop, Patinopecten yessoensis, were determined during gonadal development and estrogenic cells in the testis were identified immunohistochemically. Ovaries and testes developed rapidly during January and February to reach the mature stage in March and the spawning stage in April. Increases in aromatase activities of the ovary and testis preceded the onset of the ovarian and testicular development. Aromatase activities reached the highest level at the growing stage in February and the mature stage in March, and showed a striking decrease at the spawning stage in April. Contents of ovarian and testicular estradiol-17beta changed similarly to the profile of aromatase activities in the ovary and testis, although estrone showed no change. Immunoreactivities against P450 aromatase and estradiol-17beta were detected in the cells along the inside of the acinar wall of the testis, whereas in the previous reports, the cells are distributed along the outside of the acinar wall in the ovary. This study thus suggests that estrogen is synthesized in the estrogenic cells of the ovary and testis through aromatization by P450 aromatase and that testicular estrogen may play a physiological role in spermatogenesis.     

赣昌鲫(日本白鲫♀兴国红鲤♂)的性腺结构观察

在繁殖季节与非繁殖季节采取解剖观察、组织切片等方法, 研究赣昌鲫(Carassius cuvierius♀Cyprinus carpio var. singuonensis♂)的性腺发育。经126尾鱼的解剖, 结果显示:赣昌鲫的性腺发育比较特殊, 性腺多为一侧发育或两侧不对称发育。在赣昌鲫的性腺部位大致有下列三个类型:(1)无性腺型(脂肪型):占24.6%, 为脂肪状, 没有性腺组织; (2)精巢型(包括以精巢为主体的两性嵌合体):占46.83%, 精巢小叶内分布有许多精子细胞, 或在曲细精管间隙中填充有多个不同发育阶级的卵母细胞, 大多数精子细胞发育异常、退化, 只有极少数正常成熟精子, 但挤压腹部, 始终没有精液流出, 显示为雄性不育; (3)卵巢型(包括以卵巢为主体的两性嵌合体):占28.57%, 卵细胞能发育到第Ⅲ时相, 到第Ⅳ时相时逐渐退化, 成熟的卵细胞少, 或在各发育时相卵细胞间隙中填充有精原细胞和败育的初级精母细胞, 解剖和挤压腹部始终观察不到排卵和卵粒流出, 同样表现为雌性不育。在精巢型与卵巢型的性腺中, 两性嵌合体型占总解剖数的36.51%, 其中以精巢为主的占25.4%、卵巢为主的占11.11%。       

Germ line control of female sex determination in zebrafish

A major transition during development of the gonad is commitment from an undifferentiated “bi-potential” state to ovary or testis fate. In mammals, the oogonia of the developing ovary are known to be important for folliculogenesis. An additional role in promoting ovary fate or female sex determination has been suggested, however it remains unclear how the germ line might regulate this process. Here we show that the germ line is required for the ovary versus testis fate choice in zebrafish. When the germ line is absent, the gonad adopts testis fate. These germ line deficient testes have normal somatic structures indicating that the germ line influences fate determination of surrounding somatic tissues. In germ line deficient animals the expression of the ovary specific gene cyp19a1a fails to be maintained whereas the testis genes sox9a and amh remain expressed. Furthermore, we observed decreased levels of the ovary specific genes cyp19a1a and foxL2 in germ line deficient animals prior to morphological sex differentiation of the gonad. We propose that the germ line has a common role in female sex determination in fish and mammals. Additionally, we show that testis specification is sufficient for masculinization of the fish pointing to a direct role of hormone signaling from the gonad in directing sex differentiation of non-gonadal tissues.     

革胡子鲇原始生殖细胞的起源、迁移及性腺分化

革胡子鲇又称埃及胡子鲇,是一种多次产卵类型的硬骨鱼。作者用组织学、组织化学、电子显微镜等方法对革胡子鲇的原始生殖细胞(Primordial germ cells,PGCs)的起源、特征、迁移方式和性腺分化进行了研究。实验结果:PGCs来源于内胚层;PGCs的细胞质中存在着一种与生殖细胞有关的电子致密物--生殖质(Germ plasm);PGCs在迁移过程中有主动迁移的能力;PGCs到达生殖嵴的部位后,与生殖上皮细胞(Epithelisl cells)一起共同形成原始性腺;原始性腺分别逐步向精巢和卵巢分化;生殖质与性腺的分化有密切关系;卵巢的分化比精巢早。       

Role of Vasa, Piwi, and Myc-expressing coelomic cells in gonad regeneration of the colonial tunicate, Botryllus primigenus

In the colonial tunicate, Botryllus primigenus Oka, gonads consist of indifferent germline precursor cells, the primordial testis and ovary, and mature gonads, of which the immature gonad components can be reconstructed de novo in vascular buds that arise from the common vascular system, although the mechanism is uncertain. In this study, we investigated how and what kinds of cells regenerated the gonad components. We found that few Vasa-positive cells in the hemocoel entered the growing vascular bud, where their number increased, and finally developed exclusively into female germ cells. Simultaneously, small cell aggregates consisting of Vasa(-) and Vasa(±) cells appeared de novo in the lateral body cavity of developing vascular buds. Double fluorescent in situ hybridization showed that these cell aggregates were both Piwi- and Myc-positive. They could form germline precursor cells and a primordial testis and ovary that strongly expressed Vasa. Myc knockdown by RNA interference conspicuously lowered Piwi expression and resulted in the loss of germline precursor cells without affecting Vasa(+) oocyte formation. Myc may contribute to gonad tissue formation via Piwi maintenance. When human recombinant BMP 4 was injected in the test vessel, coelomic Piwi(+) cells were induced to express Vasa in the blood. We conclude, therefore, that in vascular buds of B. primigenus, female germ cells can develop from homing Vasa(+) cells in the blood, and that other gonad components can arise from coelomic Vasa(-)/Piwi(+)/Myc(+) cells.     

史氏鲟Sox9基因cDNA的克隆及在早期发育过程不同组织中的表达

利用RT-PCR和RACE的方法从史氏鲟(Acipenser schrenchii)中克隆了Sox9基因cDNA的部分序列(1140bp)。组织特异性表达分析表明Sox9基因在1^ ～3^ 年龄史氏鲟的大脑、心脏、肝脏、眼睛、胰脏、肾脏、精巢和卵巢等8种组织中均有表达,只是表达量随发育阶段和组织的不同而稍有差异。刚孵出1日龄的史氏鲟鱼苗中Sox9微量表达,而孵出15日龄的鱼苗中表达量上升。1^ ～3^ 年龄的史氏鲟精巢和卵巢中Sox9基因均表达,说明Sox9基因在史氏鲟性别分化过程中所起的作用不明显。Sox9基因在史氏鲟不同发育时期的8种组织中广泛表达,这可能与Sox9基因在脊椎动物软骨分化过程中的功能保守性有关。     

Two distinct origins for Leydig cell progenitors in the fetal testis

During the differentiation of the mammalian embryonic testis, two compartments are defined: the testis cords and the interstitium. The testis cords give rise to the adult seminiferous tubules, whereas steroidogenic Leydig cells and other less well characterized cell types differentiate in the interstitium (the space between testis cords). Although the process of testis cord formation is essential for male development, it is not entirely understood. It has been viewed as a Sertoli-cell driven process, but growing evidence suggests that interstitial cells play an essential role during testis formation. However, little is known about the origin of the interstitium or the molecular and cellular diversity within this early stromal compartment. To better understand the process of mammalian gonad differentiation, we have undertaken an analysis of developing interstitial/stromal cells in the early mouse testis and ovary. We have discovered molecular heterogeneity in the interstitium and have characterized new markers of distinct cell types in the gonad: MAFB, C-MAF, and VCAM1. Our results show that at least two distinct progenitor lineages give rise to the interstitial/stromal compartment of the gonad: the coelomic epithelium and specialized cells along the gonad–mesonephros border. We demonstrate that both these populations give rise to interstitial precursors that can differentiate into fetal Leydig cells. Our analysis also reveals that perivascular cells migrate into the gonad from the mesonephric border along with endothelial cells and that these vessel-associated cells likely represent an interstitial precursor lineage. This study highlights the cellular diversity of the interstitial cell population and suggests that complex cell–cell interactions among cells in the interstitium are involved in testis morphogenesis.     

萍乡肉红鲫的性腺发育研究

萍乡肉红鲫(Pingxiang red-transparent crucian carp,Carassius auratus L.)是在江西省萍乡地区分布的天然三倍体鲫突变体经人工选育后获得的遗传性状基本稳定的后代,具有两性生殖和雌核生殖两种生殖方式.研究以F5代萍乡肉红鲫为材料,自孵化后每满1个月开始取性腺,观察了其卵巢1周年性成熟和精巢的发育过程,结果表明萍乡肉红鲫的性腺为1年成熟类型.卵巢发育进程町以分为6个时期,卵母细胞发育相应可分为6个时相.统计了卵巢成熟系数周年变化,体重为95 g左右的雌性萍乡肉红鲫,其成熟卵巢的成熟系数约为(11.73±2.8)%,成熟的卵母细胞内充满卵黄,相对怀卵量为(3018±310)粒/g.萍乡肉红鲫精巢属于小叶型,在精小叶中可观察到不同发育阶段的生殖细胞.由精原细胞分裂而来的仞级精母细胞经分裂增殖,产生次级精母细胞并最终发育成为精子.萍乡肉红鲫的精巢发育程序与普通鲫鱼和鲤鱼相似,卵巢和精巢的发育过程基本同步,孵化后50日龄内性腺分化不明显,到70日龄左右开始出现雌雄分化,3月龄发育为第1期,4-5月龄发育为第2期,6-7月龄发育至第3期,7-10月龄可见第4期卵巢,1年即可成熟产卵,精巢可排出精液.结果表明,该鲫鱼突变体的性腺发育与普通二倍体鲤(鲫)鱼的性腺发育方式类似.     

Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary

In the present study, two gonad cDNA libraries from zebrafish testes and ovaries were constructed and a total of 1025 expressed sequence tag (EST) clones were generated from the two libraries: 501 from the testis library and 524 from the ovary library. A total of 641 of the EST clones were identified to share significant sequence identity with known sequences in GenBank, representing at least 478 different zebrafish genes. In order to understand the molecular compositions of the two gonad organs, the expression profiles of the identified clones in these two gonad cDNA libraries were analyzed. Both gonad libraries have a higher portion of clones for nuclear proteins and a lower portion for proteins in translational machinery, cytoskeleton and mitochondria than our previously characterized whole-adult cDNA library. Most abundant cDNA clones in the two gonad libraries were identified and over 10% of ovary clones were found to encode egg membrane proteins (zona pellucida or ZP proteins). Furthermore, the testis library showed a more even distribution of cDNA clones with relatively fewer abundant clones that tend to contribute redundant clones in EST projects; thus, the testis library can supply more unique and novel cDNA sequences in a zebrafish EST project. Another aim of this study is to identify cDNA clones that can be used as molecular markers for the analysis of the gonad development in zebrafish. Eleven potential clones were selected to analyze their expression patterns by Northern blot hybridization. Most of them showed a specific or predominant expression in the expected testis or ovary tissue. At last, four of the clones were found, by section in situ hybridization, to be expressed specifically in the germ cells of the testis or ovary and thus they are suitable molecular markers for analyses of spermatogenesis and oogenesis.     

New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life.     

Ubiquitin C-terminal hydrolase-L1 (Uch-L1) correlates with gonadal transformation in the rice field eel

The ubiquitin-proteasome pathway is crucial for a variety of biological processes, including spermatogenesis. Ubiquitin C-terminal hydrolase-L1 (Uch-L1) is thought to associate with monoubiquitin to control ubiquitin levels. Here, we report the identification of Uch-L1 cDNA from the testis of the rice field eel, a natural sex reversal vertebrate, by using cDNA microarray analysis. Uch-L1 encodes a protein of 220 amino acids that shows high homology to Uch-L1 of vertebrates, especially fish species. Both mRNA and protein are mainly expressed in testis, ovotestis and ovary, as well as in the brain. Immunohistochemistry analysis revealed differential expression of Uch-L1 in three kinds of gonads. In the ovary, expression of Uch-L1 was observed mainly in the developing ovary and slightly in the mature ovary. In ovotestis during the intersex stage, Uch-L1 was expressed in the male gonad epithelium and degraded ovary. In testis, expression was observed in developing germ cells, including spermatogonia and spermatocytes. Furthermore, Uch-L1 was upregulated during gonadal transformation, especially from the beginning of the intersex stage onwards. Native-PAGE showed that Uch-L1 underwent dimerization and oligomerization in gonads, and that the relative level of dimerization/oligomerization decreased during gonadal transformation. Simultaneously, ubiquitin polypeptide expression was upregulated during this process. These results suggest that Uch-L1, via the ubiquitin-proteasome system, may play an important role not only in gametogenesis, but also in the gonadal transformation process in the rice field eel.     

Charting the course of ovarian development in vertebrates

The decision of the embryonic gonad to differentiate as either a testis or an ovary is a critical step in vertebrate development. The molecular basis of this decision has been the focus of much study, particularly over the past decade. Here we contrast the knowledge of early gonadal development and the switch to testis differentiation with the lack of molecular understanding of ovarian development at early stages. We review current knowledge regarding mechanisms of ovarian morphogenesis and propose a model for the hierarchical control of development of the fetal ovary, incorporating the few genes already known to be important and several signals or factors that are hypothesised to exist in the early ovary.