黄颡鱼性腺分化的组织学观察

运用组织学方法,观察黄颡鱼(Pelteobagrus fulvidraco)性腺分化过程.结果表明,在水温(20±1)℃条件下,卵巢分化时间明显早于精巢.卵巢分化最早发生于孵出后13 d左右,其主要标志为原始性腺横切面上出现一个组织突起,进而形成卵巢腔;精巢分化的最早标志为精小叶和输精管的形成,开始于孵出后55 d.在发育早期,雌性生殖细胞的活动及分化明显早于雄性生殖细胞.孵出后25 d,卵原细胞开始通过有丝分裂快速增殖,孵出后34 d左右进入成熟分裂阶段;精原细胞在孵出后55 d时才开始大量增殖,成熟分裂最早发生于孵出后64 d.     

中华鳖性腺的发生与发育研究

为了揭示中华鳖性腺的发生与发育规律,在(32±0.5)℃温度下孵化鳖卵,72h时,从组织切片中可见原始生殖细胞移向中肠附近的内胚层;第5天生殖嵴形成;第13天性腺分化为皮质和髓质;第16天性腺开始分化为精巢或卵巢,至第22天时分化结束,形成精巢或卵巢.胚胎期、稚鳖和1龄鳖的性腺均呈短细管状,表面光滑,无色,胶状透明;2龄、3龄、4龄鳖的精巢呈长椭圆形.稚鳖的卵巢发育至卵原细胞期,组成精巢的曲细精管不明显,管内为精原细胞;1龄幼鳖的卵巢发育至初级卵泡期,精巢的曲细精管内精原细胞的数量增加;2龄和3龄鳖的卵巢处于生长卵泡期,3龄末的卵巢内可见到成熟卵泡,2龄鳖的曲细精管内出现初级精母细胞,3龄末的曲细精管开始出现精细胞和精子;4龄鳖的卵巢发育至成熟卵泡期,曲细精管内由管壁向管腔依次为精原细胞、初级精母细胞、次级精母细胞、精细胞和精子.结果表明,在(32±0.5)℃温度下,鳖胚发育至22d时,精巢或卵巢形成;在自然条件下,中华鳖至4龄时,性腺才完全发育成熟.     

中国大鲵幼体的性别分子鉴定与性腺形态

为探明中国大鲵(Andrias davidianus)雌雄幼体的性腺发育特征,确定适合的性别分子鉴定方法,对15尾5月龄和17尾17月龄养殖个体进行形态测量、解剖观察、性腺组织切片及PCR扩增雌性特异DNA片段。结果发现,引物adf225和adf340的扩增效果好,判定5月龄个体8雌7雄;17月龄个体8雌9雄,与依据性腺形态结构区分的结果一致。体视显微镜下5月龄幼体中肾腹侧有两条半透明细条状的原始生殖嵴;组织切片显示生殖细胞形态分化不明显。17月龄卵巢波浪状弯曲,有颗粒感,精巢呈光滑的白条状,形态分化明显;组织切片显示,卵巢分化出体积较大的卵母细胞,同时保留原始卵泡,精巢分化出生精小叶和精原细胞、支持细胞。外形测量显示,5月龄与17月龄性二型不明显,不能根据外形判断性别。本研究确定了大鲵幼体性别分子鉴定的最佳引物,可用于养殖过程中雌雄选配,以节约资源。     

黄鳝性腺自然逆转过程中vasa基因的表达分析

本研究采用RNA反义探针原位杂交技术,对vasa基因在黄鳝(Monopterusalbus)性腺发育过程中的表达情况进行了分析。结果表明:vasamRNA在Ⅰ、Ⅱ、Ⅲ期卵母细胞的胞质中均匀分布,在Ⅳ、Ⅴ期卵母细胞中vasamRNA有向胞质外周皮层迁移集中的趋势,但不明显;退化的卵粒也呈现vasamRNA阳性反应;在Ⅲ、Ⅳ期卵巢的被膜中检测到带有vasa阳性信号的细胞,这些细胞可能是待向精原细胞分化、迁移到卵巢被膜上的原始生殖细胞(Primordialgermcell,PGC),在性逆转过程中这些PGC可能由卵巢被膜迁移到精小叶中并发育成精子;在成熟精巢中,vasa在精原细胞和初级精母细胞中表达。进一步采用碱性磷酸酶染色法分析黄鳝卵巢及精巢后发现:在卵巢中,除了卵母细胞外,卵巢被膜中也检测到了带有碱性磷酸酶阳性信号的细胞;在成熟精巢中,只在生殖腺囊内的雄性生殖细胞中检测到碱性磷酸酶,而精巢被膜中没有检测到带有碱性磷酸酶阳性信号的细胞。本研究结果初步表明:黄鳝的雄性生殖细胞可能起源于雌性阶段卵巢被膜中的原始生殖细胞[动物学报51(3):469-475,2005]。     

泥鳅性腺发生和分化的组织学研究

通过人工繁殖技术获得泥鳅幼体,采用石蜡显微切片技术对幼体性腺发生和分化的组织学特征进行了系统观察.结果表明泥鳅性腺发生于出膜后12 d,40日龄卵巢开始分化,至55日龄卵巢分化完全;精巢于出膜后55 d左右开始分化,100 d左右分化完全.卵巢分化早于精巢.从性腺分化开始,将要发育为卵巢的性腺还表现为体积快速增大,向体腔中间靠拢,横截面变宽;而将要发育为精巢的性腺则呈两端尖中间稍突的梭形,增生并不明显.这些特征可能与雌雄性腺的发育及生殖细胞的分化速度有关,可以作为泥鳅性腺早期分化的形态特征.     

中国林蛙性腺的发育及温度对其性别分化的影响

为探讨幼蛙性别分化与温度的关系,在恒温和变温条件下培养中国林蛙（Rana chensinensis)受精卵至变态完成,结果表明：（1）胚胎发育到24期时生殖嵴开始出现,25期个别原始生殖细胞（PGCs)已迁移到生殖嵴中,生殖细胞与生殖嵴共同发育成生殖腺;（2）胚胎发育到31期生殖腺出现性别分化,卵巢分化初期较易识别,而精巢分化不明显;…（3）卵巢分化完成于37期,精巢分化完成于变态之后,两侧生殖腺等大;（4）胚胎发育从30期开始,性别分化对温度较为敏感,低温利于雌性化,高温利于雄性化;（5）15－25℃为变温培养时性比发生变化的敏感温度区,缓慢升温雄性比较显著增加,缓慢降温雌性比例显著增加。     

中国大鲵胃肠道胚后发育的解剖学与组织学观察

为了探讨中国大鲵(Andrias davidianus)胃肠道形成、分化及发育的基本特征,采用解剖学和组织学方法对其胃肠道胚后发育的形态结构变化进行了观察.结果表明,出膜第7天时的大鲵,胃肠道尚未分化,为一直管;出膜第21天时,已有胃和肠的分化;出膜第35天时,分化出了胃、小肠和大肠.出膜第7天时,胃肠道壁仅由黏膜上皮...     

多普勒B超鉴定中国大鲵性别

首次报道了从影像学水平上鉴定中国大鲵性别的研究结果。采用多普勒B超,常规方法直接扫描雌雄中国大鲵腹部,并记录大鲵的精巢或卵巢的形状、内部回声。实验结果表明,雌雄中国大鲵的检测结果存在明显的差畀,在雄性中国大鲵的腹部,可检测到精巢组织;在雌性中国大鲵的腹部,可检测到为卵巢组织和卵泡。初步分析了实验结果,并认为将多普勒B超鉴定法与泄殖孔法鉴定相结合可以作为中国大鲵性别鉴定的可靠方法。     

高温和皮质醇对黄颡鱼性别分化的影响

研究以性染色体类型已确定且已有性别特异分子标记的黄颡鱼为研究对象,开展高温与皮质醇诱导黄颡鱼(Tachysurus fulvidraco Richardson)XX个体雄性化组织学进程研究,以期为环境应激诱导鱼类雄性化提供研究基础。通过对每尾鱼采用性别特异性标记鉴定遗传性别(XX或XY)及组织学鉴定生理型性别,仅经过24d的处理(12—35日龄),高温或皮质醇便能诱导XX遗传型个体雄性化。在此过程中,部分XX遗传型个体卵母细胞受到抑制,之后发育成带有卵巢腔的精巢结构。62日龄时, XX伪雄鱼性腺较正常XY雄鱼大, XX伪雄鱼体重与正常XY雄鱼相近,而显著大于未发生性逆转的XX雌鱼。122日龄时, XX伪雄鱼从62日龄带有卵巢腔的精巢结构发育成具有典型的精小叶结构样精巢,且都具有生理性雄鱼特有的生殖突,推测这些雄鱼可能具有与正常雄鱼类似的生殖能力。部分XX个体对高温处理不敏感,没有发生性逆转,温度处理反而加快了卵巢发育的进程,这些个体对高温的耐受性和另外一些发生性逆转的个体对温度的敏感性值得进一步研究。     

斑马鱼性腺发育的组织学观察

在过去几十年,斑马鱼(Danio rerio)由于其发育周期短且速度快,胚胎发育透明,已经成为众多研究领域的典型模式生物.斑马鱼的性腺发育和分化非常特殊,雄性和雌性幼鱼的性腺在早期全部发育成"类卵巢"结构.目前,对于斑马鱼的性别分化和性腺分化机制还不清楚.本文以孵化后不同时期的斑马鱼仔鱼和幼鱼的生殖腺为材料,经石蜡切片和苏木精染色后,荧光显微镜下观察了斑马鱼仔鱼性腺从出现、分化到成熟的发育过程.结果发现:孵化后5～10日龄仔鱼腹腔两侧可以观察到没有分化的生殖腺,其中的生殖细胞明显比周围的体细胞大;孵化后14～24日龄仔鱼的生殖腺中可见由卵原细胞分裂形成的生殖包囊,其中的生殖细胞进一步分化、分裂形成体积更大、数量更多的卵母细胞;25日龄左右的仔鱼,其性腺成为在腹腔两侧对称,而且在组织结构上也较为典型的卵巢样结构.到35日龄前后可见一部分仔鱼的性腺逐步由卵巢样结构向精巢结构转变的过程.我们在2周左右的仔鱼的性腺中观察到了生殖包囊存在,这一现象还未见有前人报道.在本试验中,我们不仅清楚地观察到类似卵巢的性腺中"卵母细胞"逐渐凋亡消失的过程,还观察到性腺由最初的类似卵巢样结构逐渐变成典型的精巢结构的整个过程.这些研究成果将为发育生物学提供有价值的信息和第一手资料.     

Differentiation and development of testis in the oviparous lizard, Calotes versicolor (Daud.)

The differentiation and development of the testis in the lizard Calotes versicolor was studied histologically and histoenzymatically from the day of oviposition (stage 27) to 2 months after hatching. The study reveals the appearance of the gonadal component as a genital ridge at stage 27. The first sign of testis differentiation is observed at stage 33, which displays a well-developed medulla consisting of seminiferous cords comprising Pre-Sertoli cells. The sex differentiation of the embryonic gonads occurs at stage 34. At this stage, seminiferous cords of the testis are prominent and extensive with many pre-Sertoli cells and few spermatogonia. The interstitial space consists of immature fibroblast-type Leydig cells. Pre-Sertoli cells of the seminiferous cords differentiate into Sertoli cells with a triangular nucleus becoming apparent around stages 36-37. The fibroblast-like Leydig cells differentiate into round matured Leydig cells at stage 40. Quantitative estimation of germ cells reveals that the number of germ cells increases in individual gonads, and in 5-day-old hatchling's, this number multiplies by manifold. Spermatogonia show reductional division in the testis of 1-day-old hatchlings.Histochemical localization of Delta5-3beta-HSDH and G-6-PDH activity appears in the seminiferous cords (medulla) of the testis after sexual differentiation (stage 36), indicating that the embryonic medulla is the site of steroidogenesis and not the cortex in C. versicolor. This study also suggests that morphological differentiation of the gonad precedes detectable steroidogenesis in this species. In 10-day-old hatchling's, Delta5-3beta-HSDH activity is seen in the interstitial cells of the testis, which, however, is not detected in the seminiferous tubules. The intensity of the enzyme activity remains more or less the same in the testis up to 10 days after hatching and begins to increase thereafter. The increase in steroidogenesis parallels the progressive post-hatching increase of the interstitial/Leydig cells.     

Sex reversal and aromatase in chicken.

Aromatase inhibitors administered before sexual differentiation of the gonads can induce sex reversal in female chickens. To analyze the process of sex reversal, we have followed for several months the changes induced by Fadrozole, a nonsteroidal aromatase inhibitor, in gonadal aromatase activity and in morphology and structure of the female genital system. Fadrozole was injected into eggs on day four of incubation, and its effects were examined during the embryonic development and for eight months after hatching. In control females, aromatase activity in the right and the left gonad was high in the middle third of embryonic development, and then decreased up to hatching. After hatching, aromatase activity increased in the left ovary, in particular during folliculogenesis, whereas in the right regressing gonad, it continued to decrease to reach testicular levels at one month. In treated females, masculinization of the genital system was characterized by the maintenance of the right gonad and its differentiation into a testis, and by the differentiation of the left gonad into an ovotestis or a testis; however, in all individuals, the left Müllerian duct and the posterior part of the right Müllerian duct were maintained. In testes and ovotestes, aromatase activity was lower than in gonads of control females (except in the right gonad as of one month after hatching) but remained higher than in testes of control and treated males. Moreover, in ovotestes, aromatase activity was higher in parts displaying follicles than in parts devoid of follicles. The main structural changes in the gonads during sex reversal were partial (in ovotestes) or complete (in testes) degeneration of the cortex in the left gonad, and formation of an albuginea and differentiation of testicular cords/tubes in the two gonads. Testicular cords/tubes transdifferentiated from ovarian medullary cords and lacunae whose epithelium thickened and became Sertolian. Transdifferentiation occurred all along embryonic and postnatal development; thus, new testicular cords/tubes were continuously formed while others degenerated. The sex reversed gonads were also characterized by an abundant fibrous interstitial tissue and abnormal medullary condensations of lymphoid-like cells; in the persisting testicular cords/tubes, spermatogenesis was delayed and impaired. Related to aromatase activity, persistence of too high levels of estrogens can explain the presence of oviducts, gonadal abnormalities and infertility in sex reversed females.     

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.     

Production of chicken progeny (Gallus gallus domesticus) from interspecies germline chimeric duck (Anas domesticus) by primordial germ cell transfer

The present study aimed to investigate the differentiation of chicken (Gallus gallus domesticus) primordial germ cells (PGCs) in duck (Anas domesticus) gonads. Chimeric ducks were produced by transferring chicken PGCs into duck embryos. Transfer of 200 and 400 PGCs resulted in the detection of a total number of 63.0 ± 54.3 and 116.8 ± 47.1 chicken PGCs in the gonads of 7-day-old duck embryos, respectively. The chimeric rate of ducks prior to hatching was 52.9% and 90.9%, respectively. Chicken germ cells were assessed in the gonad of chimeric ducks with chicken-specific DNA probes. Chicken spermatogonia were detected in the seminiferous tubules of duck testis. Chicken oogonia, primitive and primary follicles, and chicken-derived oocytes were also found in the ovaries of chimeric ducks, indicating that chicken PGCs are able to migrate, proliferate, and differentiate in duck ovaries and participate in the progression of duck ovarian folliculogenesis. Chicken DNA was detected using PCR from the semen of chimeric ducks. A total number of 1057 chicken eggs were laid by Barred Rock hens after they were inseminated with chimeric duck semen, of which four chicken offspring hatched and one chicken embryo did not hatch. Female chimeric ducks were inseminated with chicken semen; however, no fertile eggs were obtained. In conclusion, these results demonstrated that chicken PGCs could interact with duck germinal epithelium and complete spermatogenesis and eventually give rise to functional sperm. The PGC-mediated germline chimera technology may provide a novel system for conserving endangered avian species.     

Sexual differentiation in Salaria (=Blennius) pavo

The sexual differentiation of Salaria (= Blennius ) pavo is described from the stage of hatching to a body length of 35 mm. At hatching, the primordial germ cells (PGCs) can be recognized clearly. At a standard body length of 5 mm, they begin to protrude into the peritoneal cavity and at 14 mm they transform to oogonia. At 17 mm length, the first oocytes can be observed. In males at a standard length of 16–17 mm, the first signs of a differentiation into a testis can be recognized. Shortly after the differentiation of the male sex, the division of the male gonad into a testis and a testicular gland can be seen. The fine structural characteristics of the PGCs and of differentiation stages are presented.     

Histological and ultrastructural investigation of early gonad development and sex differentiation in Adriatic sturgeon (Acipenser naccarii,Acipenseriformes, Chondrostei)

Gonad development and sex differentiation from embryos to 594‐day‐old individuals were investigated in farmed Acipenser naccarii using light and transmission electron microscopy. The migrating primordial germ cells first appear along the dorsal wall of the body cavity in embryos 1.5 days before hatching. The gonadal ridge, containing a few primary primordial germ cells (PGC‐1) surrounded by enveloping cells, appears in 16‐day‐old larvae. At 60 days, the undifferentiated gonad is lamellar and PGC‐1 multiply, producing PGC‐2. In 105‐day‐old juveniles, a distinct germinal area with advanced PGC‐2 appears on the lateral side near the mesogonium and the first blood vessels are visible. At 180 days, putative ovaries with a notched gonadal epithelium and putative testes with a smooth one appear, together with adipose tissue on the distal side. In 210‐day‐old juveniles, active proliferation of germ cells begins in the putative ovaries, whereas putative testes still contain only a few germ cells. The onset of meiosis and reorganization of stromal tissue occurs in ovaries of 292‐day‐old individuals. Ovaries with developed lamellae enclosing early oocyte clusters and follicles with perinucleolar oocytes occur at 594 days. Meiotic stages are never found, even in anastomozing tubular testes of 594‐day‐old individuals. Steroid producing cells are detected in the undifferentiated gonad and in the differentiated ones of both sexes. Anatomical differentiation of the gonad precedes cytological differentiation and female differentiation largely precedes that of the male. Gonad development and differentiation are also associated with structural changes of connective tissue, viz. collagen‐rich areas are massive in developing testes and reduced in ovaries. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Gonadal development and differentiation in Alligator mississippiensis at male and female producing incubation temperatures

The development and differentiation of the gonads of embryonic alligators incubated at 30 °C (100% female producing) and 33 °C (100% male producing) was investigated histologically. The stage of development of the gonad and differentiation into an ovary or a testis occurred at essentially the same time at both temperatures. This contrasts with the overall development of the embryos which was slower at the lower temperature. A few days prior to differentiation, gonads grew more quickly at 33 °C than they did at 30 °C. However, once differentiated into a presumptive testis, gonads reduced in volume so that at hatching presumptive testes were smaller than presumptive ovaries. It is hypothesized that synchrony/asynchrony of development of the gonad and the rest of the embryo may account for temperature-dependent sex determination.     

Ultrastructural Observations on the Origin and Differentiation of Somatic Cells during Gonadal Development in the Frog Rana nigromaculata

The process of gonad development in the frog Rana nigromaculata was observed using the electron microscope. The gonadal medulla was formed by the proliferation and displacement of the epithelial cells within the primordial gonad, and a distinct continuity was observed between the cortical and medullary cells. Sex differentiation of the gonad occurred directly from the sexually indifferent primordial gonads. In the rudimentary testes, the continuity between the cortical and medullary regions increased closer, and the intermingling of cortical and medullary cells was evident. The inner region of the cortex developed into a cord-like structure and subsequently differentiated into rudimentary seminiferous tubules. The medulla differentiated into the testicular rete and efferent duct. In the rudimentary ovaries, the cortex and medulla were separated and the ovarian cavity was formed in the medullary region. In the cortex, the cortical cells surrounding oocytes which had reached the diplotene stage, differentiated into follicular cells. The intrusion of mesenchymal or blastemal cells derived from extragonadal regions into the cortex or medulla was never observed. These findings do not support Witschi's cortico-medullary antagonistic theory of sex differentiation.     

Gonadal dysgenesis induced by prenatal exposure to ethinyl estradiol in mice

Daily oral administration of ethinyl estradiol (0.02, 0.2, or 2.0 mg/kg of body weight) to pregnant Jc1:ICR mice resulted in ovotestis and intra-abdominal testis with persistent Müllerian duct and Wolffian duct in male fetuses and ovarian hypoplasia in female fetuses when it was given from day 11 through day 17 of gestation (before gonadal differentiation in the fetus). The ovotestis consisted of testicular and ovarian portions. In the testicular portion, a few solid seminiferous tubules containing spermatogonia, some with pachytene nuclei with Sertoli cells and compact interstitial tissue including Leydig cells, were seen. In the ovarian portion, pachytene nuclei were seen. The intra-abdominal testis was smaller and contained more spermatogonia per tubule in cross section than the control testis. These findings suggest that in male fetuses ethinyl estradiol affects Sertoli cell differentiation resulting in suppression of Müllerian inhibiting factor. On the other hand, in the ovarian hypoplasia, the primordial follicles and follicular cells in a primordial follicle were significantly decreased in number, and the number of the degenerated primordial follicles was significantly increased. It seems likely that ethinyl estradiol affects the intimate contact between follicular cells and oocytes to cause degeneration of primordial follicles.