A sex cord‐like structure and some remarkable features in early gonadal sex differentiation in the marine teleost Siganus guttatus(Bloch)

Several notable features of early gonadal sex differentiation in the golden rabbitfish Siganus guttatus are described including the first report among teleosts of a distinctive dual structure, consisting of somatic cells directly enclosing germ cells (sex cord‐like structure, SCS) and outer somatic tissue surrounding the SCS, in both undifferentiated and early differentiated gonads. Germ cells occurred and proliferated exclusively in the SCS during the process of ovarian and testicular differentiation. A second remarkable characteristic was the delayed germinal cell proliferation for oogenesis in the ovary, that commenced simultaneously with that in the testis, a relatively long time after the onset of somatic development. These observations suggest the possibility that sex differentiation of germ cells is preceded by some sex specific changes in somatic components of the SCS that are light‐microscopically indistinguishable between the sexes. The third unique feature was the detachment of gonadal tissue, including both somatic and germ cells, into the ovarian cavity in the ovary and into the seminiferous lobules and main seminal duct in the testis. This phenomenon occurred in the testis, forming the efferent duct network after 73 days post‐hatch (DPH), and in the ovaries, forming the ovigerous lamellae and regulating the number of oocytes attaining full maturation at c . 129 DPH.     

Expression of aromatase mRNA and effects of aromatase inhibitor during ovarian development in the medaka, Oryzias latipes

In teleost fish, several studies have implicated estrogens in the process of ovarian development, but the exact role of endogenous estrogen is still unclear. We examined the expression of aromatase mRNA with in situ hybridization, and the effects of Fadrozole, a nonsteroidal aromatase inhibitor (AI), during ovarian development in medaka Oryzias latipes. Medaka aromatase was first detected on the ventral side of ovaries from four to 10 days after hatching (dah), after occurrence of oogenesis. AI treatment after hatching suppressed the ovarian cavity formation from 30 dah but did not affect early oogenesis and folliculogenesis during ovarian development. These results suggest that endogenous estrogen is specifically required for formation of the ovarian cavity, but is not essential for early oogenesis and folliculogenesis in medaka.     

Induction of XY sex reversal by estrogen involves altered gene expression in a teleost, tilapia

To clarify the importance of endogenous estrogens during sex differentiation in a teleost fish, the Nile tilapia, we examined the target events for endogenous estrogens and their role during gonadal sex differentiation. The expression of CYP19a (P450arom) precedes any morphological gonadal sex differentiation. Further to these findings, the treatment of XX fry with non-steroidal aromatase inhibitor (AI), Fadrozole, from seven to 14 days after hatching caused complete sex reversal to functional males. The XX sex reversal induced by AI was rescued completely with simultaneous estrogen treatment. We also found that XY fry treated with estrogen, before the appearance of morphological sex differences, caused complete sex reversal from males to females. Taken together, these results suggest that endogenous estrogens are required for ovarian differentiation. To identify the down-stream gene products of estrogen during ovarian differentiation, we performed subtractive hybridization using mRNA derived from normal and estrogen treated XY gonads. Two out of ten gene products were expressed in germ cells, whereas the others were expressed in somatic cells.     

A very simple mode of follicular cell diversification in Euborellia fulviceps (Dermaptera, Anisolabididae) involves actively migrating cells

The ovaries of Euborellia fulviceps are composed of five elongated ovarioles of meroistic-polytrophic type. The individual ovariole has three discernible regions: the terminal filament, germarium, and vitellarium. The terminal filament is a stalk of flattened, disc-shaped somatic cells. In the germarium, germline cells in subsequent stages of differentiation are located, and the vitellarium comprises numerous ovarian follicles arranged linearly. The individual ovarian follicles within the vitellarium are separated by prominent interfollicular stalks. The follicles are composed by two germline cells only: an oocyte and a single, polyploid nurse cell, which are surrounded by a monolayer of somatic follicular cells (FCs). During subsequent stages of oogenesis, initially uniform follicular epithelium begins to diversify into morphologically and physiologically distinct subpopulations. In E. fulviceps, the FC diversification mode is rather simple and leads to the formation of only three different FC subpopulations: (1) cuboidal FCs covering the oocyte, (2) stretched FCs surrounding the nurse cell and (3) FCs actively migrating between oocyte and a nurse cell. We found that FCs from the latter subpopulation send long and thin filopodium-like and microtubule-rich processes penetrating between the oocyte and nurse cell membranes. This suggests that, in E. fulviceps, cells from at least one FCs subpopulation show the ability to change position within an ovarian follicle by means of active migration.     

拟目乌贼卵子发生与卵巢发育

为了丰富拟目乌贼(Sepia lycidas)生物学资料, 为人工育苗与养殖提供理论依据, 采用解剖学和组织学的方法, 对水泥池养殖条件下拟目乌贼卵子发生和卵巢发育进行了研究。结果表明: 经过6个月水泥池养殖, 平均体重为256.34 g, 最大体重达到457.08 g, 个别发育成熟, 绝大部分未达性成熟。卵子发生不同步, 根据细胞形态、细胞大小、滤泡细胞形态和卵黄形成情况可分为卵原细胞阶段(卵原细胞期)、原生质生长阶段(无滤泡期、单层滤泡期和双层滤泡期)、间质生长阶段(滤泡内折早期、滤泡内折中期和滤泡内折晚期)和营养质生长阶段(卵黄发生早期、卵黄发生晚期和成熟期), 共4个阶段10个时期。卵巢发育根据外观形态、性腺指数变化和切面上各期细胞所占的比例, 可分为形成前期、形成期、小生长期、大生长期、成熟前期和成熟期6个时期。拟目乌贼繁殖周期为一年。       

An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal

Dietary administration of a cytochrome P450 aromatase (P450arom) inhibitor (fadrozole) in genetic female juveniles of zebrafish (Danio rerio) was performed at 15-40 days post-hatching. The percentage of gonadal masculinization in the genetic all-females at 40 days post-hatching, treated with 0, 10, 100 and 1000 microg fadrozole g(-1) diet(-1) were 0, 62.5, 100 and 100%, respectively. Rearing at high water temperature in genetic all-females was performed at 15-25 days post-hatching. The percentage of gonadal masculinization in the genetic all-females at 40 days post-hatching, at water temperatures of 28.5, 35 and 37 degrees C were 0, 68.8 and 100%, respectively. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive oocytes of early diplotene and perinucleolar stages in fadrozole-treated genetic females (1000 microg g(-1) diet(-1)) were observed at 15-40 days post-hatching during sex-reversal. In contrast, apoptotic oocytes of early diplotene stage in high temperature-treated genetic females (at 35 and 37 degrees C) during sex-reversal and presumptive males of wild-type fish during sex differentiation were found at 15-27 days post-hatching. Our findings indicate that oocyte apoptosis, depletion of P450arom activity and differentiation of spermatogonia during gonadal sex-reversal are caused by treatments of aromatase inhibitor or high water temperature.     

Differentiation and function of the ovarian somatic cells in the pseudoscorpion,Chelifer cancroides (Linnaeus, 1761) (Chelicerata: Arachnida: Pseudoscorpionida)

Pseudoscorpion females carry fertilized eggs and embryos in specialized brood sacs, where embryos are fed with a nutritive fluid produced and secreted by somatic ovarian cells. We used various microscopic techniques to analyze the organization of the somatic cells in the ovary of a pseudoscorpion, Chelifer cancroides. In young specimens, the ovary is a cylindrical mass of internally located germline cells (oogonia and early previtellogenic oocytes) and two types of somatic cells: the epithelial cells of the ovarian wall and the internal interstitial cells. In subsequent stages of the ovary development, the oocytes grow and protrude from the ovary into the hemocoel (opisthosomal cavity). At the same time the interstitial cells differentiate into the follicular cells that directly cover the oocyte surface, whereas some epithelial cells of the ovarian wall form the oocyte stalks – tubular structures that connect the oocytes with the ovarian tube. The follicular cells do not seem to participate in oogenesis. In contrast, the cells of the stalk presumably have a dual function. During ovulation the stalk cells appear to contribute to the formation of the external egg envelope (chorion), while in the post-ovulatory phase of ovary function they cooperate with the other cells of the ovarian wall in the production of the nutritive fluid for the developing embryos.     

无尾两栖类性腺发育研究进展

脊椎动物的性腺发育一直是生物学领域研究的热点,无尾两栖动物因其胚胎发育的独立性和易观察性而成为发育生物学研究领域的良好材料,并取得了许多成果.本文综述了无尾两栖类原始性腺形成、性腺分化、精巢和卵巢的发育,以及配子发生等方面的研究进展.无尾两栖类原始性腺形成主要发生在鳃盖褶和后肢芽形成时期,不同物种略有不同;性腺分化通常...     

Aromatase inhibitor induces complete sex change in the protogynous honeycomb grouper (Epinephelus merra)

The protogynous hermaphrodite fish change sex from female to male at the certain stages of life cycle. The endocrine mechanisms involved in gonadal restructuring throughout protogynous sex change are not clearly understood. In the present study, we implanted maturing female honeycomb groupers with nonsteroidal aromatase inhibitor (AI), Fadrozole (0, 1, and 10 mg/fish) and examined changes in gonadal structures and serum levels of sex steroid hormones 2(1/2) months after implantation. The ovaries of control females had oocytes undergoing active vitellogenesis, whereas AI caused females to develop into functional males. These males had testes, which were indistinguishable in structure from those of normal males, but bigger in size, and completed all stages of spermatogenesis including accumulation of large amount of sperm in the seminiferous tubules. AI significantly reduced the serum levels of estradiol-17beta (E2) and increased levels of testosterone (T), 11-ketotestosterone (11-KT), and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP). Further, AI suppressed in vitro production of E2, and stimulated the production of T and 11-KT in the ovarian fragments of mature female. In the honeycomb grouper, suppression of both in vitro and in vivo production of E2 and degeneration of oocytes by AI suggests that AI induces complete sex change through inhibition of estrogen biosynthesis, and perhaps, subsequent induction of androgen function.     

Aromatase is expressed and active in the rainbow trout oocyte during final oocyte maturation

While it is generally well accepted that the ovarian follicular sites of estradiol-17β (E2) synthesis are restricted to somatic cells, the possible contribution of the germinal compartment has received little or no attention in teleosts. In order to demonstrate the expression of ovarian aromatase in the oocyte, cyp19a1a mRNA was studied in ovarian follicles by in situ hybridization. In addition, the expression of cyp19a1a was studied in both somatic and germinal compartments of the ovarian follicle in rainbow trout (Oncorhynchus mykiss) during final oocyte maturation (i.e., maturational competence acquisition and subsequent meiosis resumption) by real-time PCR. The enzymatic activity of ovarian aromatase was also studied in both somatic and germinal compartments of the ovarian follicle. Finally, E2 levels were monitored in follicle-enclosed oocytes throughout the pre-ovulatory period. We were able to demonstrate a significant ovarian aromatase expression and activity in the late vitellogenic oocyte. Furthermore, a dramatic decrease in aromatase expression and activity occurs in the oocyte during late oogenesis, concomitantly with the trend observed in surrounding follicular layers. We also report an unexpected increase of E2 levels in the oocyte during the pre-ovulatory period. To our knowledge, these observations are reported for the first time in any teleost species. Together, our data support the hypothesis of the participation of the germinal compartment in follicular estrogen synthesis and a biological role of E2 during oocyte and/or early embryo development.     

Some data on the structure of the gonads of manybar goatfish Parupeneus multifasciatus (Mullidae) from the Nha Trang Bay, South China Sea

Gonadal differentiation and development of sex cells are described in the tropical representative of the family Mullidae, manybar goatfish Parupeneus multifasciatus, using histological methods and observations of oocytes in vivo. Anatomical differentiation of the gonads is registered by approximately 5 cm fork length (FL) and 1.5 g body weight of the fish, and cytological differentiation (in the females) occurs by approximately 6 cm body length and 2.6 g body weight. In the ovaries of sexually mature females before spawning, oocytes of all phases and periods of development can be found. Based on the gonadal structure and frequency distribution of oocyte diameter, the type of oogenesis is continuous.     

银鲫种系细胞标记分子Vasa: cDNA克隆及其抗体制备

种系细胞始自胚胎发育早期,是动物生殖及生殖工程的基础。为研究鱼类的种系细胞提供标记分子,我们克隆并鉴定了银鲫的vasacDNA即Cagvasa。CagvasacDNA全长2771碱基(nt),编码的蛋白为银鲫Vasa即CagVasa,全长701个氨基酸(aa)。CagVasa蛋白与已知Vasa蛋白的结构特征一致:在N端有14个RGG重复序列,在C端Vasa所特有的8个功能域俱全。银鲫Vasa与鲤鱼、斑马鱼、陆生脊椎动物和果蝇的Vasa蛋白分别有95%,89%,61%-66%和50%的同源性。卵巢切片的RNA原位杂交揭示,Cagvasa限于种系细胞,且表达水平呈现出低-高-低的动态变化:即两头低(卵原细胞跟Ⅳ期成熟卵子),中间高(Ⅱ-Ⅲ期卵子)。为分析鱼类种系细胞提供手段,我们用310aa的N端序列产生细菌的重组蛋白来免疫大白兔,获得了抗Vasa的多克隆抗体αVasa。Western免疫印迹表明,αVasa特异性地识别一个鱼类性腺的蛋白,该蛋白的分子量为75kD,仅见于银鲫的性腺和卵子。卵巢切片的组织免疫荧光共聚焦显微分析表明,抗体αVasa只对种系细胞染色:卵原细胞着色最深,卵母细胞和早期的卵子都浓染,成熟卵则浅染。类似情况亦见之于精子发生早期阶段的雄性种系细胞。卵巢和精巢的体细胞则不着色。因此,Cagvasa编码的当是Vasa同源蛋白,为银鲫种系细胞的第一个标记分子。我们的研究表明,抗体αVasa染色灵敏度高,特异性好,当是鉴别银鲫及其它鲤科鱼类的种系细胞的有效手段     

Identification and lineage tracing of two populations of somatic gonadal precursors in medaka embryos

The gonad contains two major cell lineages, germline and somatic cells. Little is known, however, about the somatic gonadal cell lineage in vertebrates. Using fate mapping studies and ablation experiments in medaka fish (Oryzias latipes), we determined that somatic gonadal precursors arise from the most posterior part of the sdf-1a expression domain in the lateral plate mesoderm at the early segmentation stage; this region has the properties of a gonadal field. Somatic gonadal precursors in this field, which continuously express sdf-1a, move anteriorly and medially to the prospective gonadal area by convergent movement. By the stage at which these somatic gonadal precursors have become located adjacent to the embryonic body, the precursors no longer replace the surrounding lateral plate mesoderm, becoming spatially organized into two distinct populations. We further show that, prior to reaching the prospective gonadal area, these populations can be distinguished by expression of either ftz-f1 or sox9b. These results clearly indicate that different populations of gonadal precursors are present before the formation of a single gonadal primordium, shedding new light on the developmental processes of somatic gonadal cell and subsequent sex differentiation.     

PROCESS OF FUNCTIONAL SEX REVERSAL OF THE GONAD IN THE FEMALE GUPPY, POECILIA RETICULATA, TREATED WITH ANDROGEN BEFORE BIRTH

In the guppy, Poecilia reticulata , ovarian differentiation occurs during the embryonal life by 14 days after the preceding parturition. Testicular differentiation begins with the appearance of prominent aggregations of stroma cells in the gonadal hilus occurring by 18 days following the last parturition.
Oral administration of methyltestosterone (400 μ/g diet) to gravid guppies, begun 13–15 days after the preceding parturition and continued until the end of gestation, induced a male-type aggregation of somatic cells in the hilus of ovaries of female embryos. Gonads of newly born, androgenized females still had developing oocytes but were always provided with atypical clusters of stroma cells in their hilus. The gonads of affected female offspring developed successively into definite testes within 20 days after birth, displaying a precocious differentiation of the hilar stroma into sperm ducts and testicular interstitium, a concomitant initiation of spermatogenesis, and a conspicuous degeneration of oocytes. A successful masculinization of the somatic element, which may occur prior to that of the germ cells, in androgen-affected embryonic ovaries seems to be essential for the functional sex reversal of genetic females in the guppy.     

Gonadal morphogenesis and sex differentiation in intraovarian embryos of the viviparous fish Zoarces viviparus (Teleostei, Perciformes, Zoarcidae): a histological and ultrastructural study

It is essential to know the timing and process of normal gonadal differentiation and development in the specific species being investigated in order to evaluate the effect of exposure to endocrine-disrupting chemicals on these processes. In the present study gonadal sex differentiation and development were investigated in embryos of a viviparous species of marine fish, the eelpout, Zoarces viviparus, during their intraovarian development (early September to January) using light and electron microscopy. In both sexes of the embryos at the time of hatching (September 20) the initially undifferentiated paired bilobed gonad contains primordial germ cells. In the female embryos, ovarian differentiation, initiated 14 days posthatch (dph), is characterized by the initial formation of the endoovarian cavity of the single ovary as well as by the presence of some early meiotic oocytes in a chromatin-nucleolus stage. By 30 dph, the endoovarian cavity has formed. By 44 dph and onward, the ovary and the oocytes grow in size and at 134 dph, just prior to birth, the majority of the oocytes are at the perinucleolar stage of primary growth and definitive follicles have formed. In the presumptive bilobed testis of the male embryos, the germ cells (spermatogonia), in contrast to the germ cells of the ovary, remain quiescent and do not enter meiosis during intraovarian development. However, other structural (somatic) changes, such as the initial formation of the sperm duct (30 dph), the presence of blood vessels in the stromal areas of the testis (30 dph), and the appearance of developing testicular lobules (102 dph), indicate testicular differentiation. Ultrastructually, the features of the primordial germ cells, oogonia, and spermatogonia are similar, including nuage, mitochondria, endoplasmic reticulum, and Golgi complexes.     

雌激素对中华鳖性腺分化及DMRT1、SOX9基因表达的影响

中华鳖（Pelodiscus sinensis）性别决定的方式一直存在较大的争议,分子机制更是不清楚。在大部分脊椎动物中,雌激素在性别决定和性腺分化中扮演重要的调控作用。实验通过对性别分化前胚胎进行雌二醇（E2）和芳香化酶抑制剂（AI）处理,研究雌激素在中华鳖性腺分化中的作用及机理。实验结果显示,与对照组（雌性比例49%）相比,E2处理组中雌性中华鳖仔鳖比例显著增加,高达92.3%;而在AI处理组中,雌性比例显著下调至13.1%。HE染色分析表明,ZZ（雄性）和ZW（雌性）胚胎分别经过E2和AI处理后,ZZ和ZW性腺结构呈现明显的雌性化和雄性化特征。同时,通过RT-PCR和免疫荧光染色发现,E2能显著降低雄性性别关键因子DMRT1和SOX9 mRNA和蛋白表达水平;AI则表现相反的调节作用。综上所述,雌激素通过抑制雄性性别关键因子DMRT1和SOX9的表达来抑制雄性分化,促进雌性分化,揭示雌激素在中华鳖雌性性别分化中起着重要的调控作用。       

Effects of ethynylestradiol on vitellogenin synthesis and sex differentiation in juvenile grey mullet (Mugil cephalus) persist after long-term exposure to a clean environment

We investigated the continuing effects of exposure to ethynylestradiol (EE₂) in juvenile grey mullet after transfer to a clean environment. Eleven-month-old juvenile fish containing immature phenotype gonad were fed dry diets; the low and high EE₂-treated groups were fed diets with 0.04 and 4 μg EE₂/g body weight for 4 weeks, respectively. After treatment, they were transferred to clean seawater, and reared with an EE₂ free diet for 350 days. Vitellogenin (VTG) was not detected in the serum of the control group throughout the experimental period. However, in both treatment groups, abnormal values of serum VTG were detected until approximately 100 days after transfer to a clean environment. In the control group, sex differentiation was not confirmed until 206 days after transfer to a clean environment. However, some of the fish in the 0.04 μg EE₂-treated group had ovarian cavity and oocytes at 26 days. In most of the fish in the 4 μg EE₂-treated group, the ovarian cavity had already appeared at the end of EE₂ treatment (0 day), and oocytes were observed at 26 days, suggesting that EE₂ accelerates ovarian differentiation. These results suggest that previous exposure to EE₂ has long-term effects on VTG synthesis and gonadal development.