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.     

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.     

青鳉(Oryzias latipes)性腺分化与发育的组织学观察

青鳉(Oryzias latipes)繁殖力强、繁殖周期短,为硬骨鱼类研究领域的模式生物。目前,尚无青鳉性腺分化与发育过程的系统资料。该文以青鳉孵化后不同时期的生殖腺为材料,经石蜡切片和HE染色后,于光学显微镜下观察其性腺的出现、分化及成熟过程。结果显示:孵化后5~10 d,仔鱼腹腔右侧已可见生殖腺,生殖细胞体积明显大于周围的体细胞;孵化后10 d,性腺开始分化,雌、雄性个体性腺出现结构上的差异,雌性个体内可明显观察到生殖包囊结构;随后,雌、雄性个体内先后可见卵子和精子的发生过程和组织学分期;孵化后50 d,首见卵巢腔结构和成熟精子。此外,还观察到性反转现象的特例,即两性特征共存的现象。该结果将为发育生物学、遗传生物学提供基础资料。     

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.     

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.     

鸡胚胎生殖细胞在鼠胚成纤维细胞饲养层上的生长

目的:探讨以鼠胚成纤维细胞为饲养层分离、培养鸡胚胎生殖细胞的方法和条件。方法:分离、培养12.5～13.5d鼠胚成纤维细胞。分离孵化5.5d鸡胚原始生殖细胞,原代培养时不使用饲养层,与性腺基质细胞共培养;继代培养时将其置于鼠胚成纤维细胞饲养层上,在含生长因子、分化抑制因子的培养体系中培养胚胎生殖细胞。结果:鼠胚成纤维细胞可连续传代18代以上(4个月),3～15代细胞可以用作饲养层细胞。分离的鸡胚胎生殖细胞在饲养层上可增殖形成典型胚胎生殖细胞集落,并能连续在体外培养超过9代。集落未分化标志高碘酸希夫反应(PAS)呈强阳性,体外分化实验表明胚胎生殖细胞具有多能性。结论:用鼠胚成纤维细胞作为饲养层能获得可连续增殖的胚胎生殖细胞。     

X射线照射成熟金鱼对于仔鱼胚胎发育的影响

成熟金鱼受1,000伦琴X射线照射后,所产的卵降低了受精率及孵化率,延长了孵化时间。在雌雄同时照射的一组中,孵化时胚胎全部是畸形,受照射雌鱼与正常雄鱼交配所产仔鱼孵化时有40%以上为畸形。畸形胚胎的外部变态为胸腔膨大,卵黄囊积聚体液,脊尾短或弯曲,头部分化不完整,眼分化不完整或缺少色素,下颚扩大等。X射线影响下,胚胎产生的主要缺陷为神经系统特别是脑部和尾部的组成物质受损伤,这些结果表明:胚胎的分化可能在生殖细胞时期已开始,生殖细胞受到了损伤,影响了某些控制以后器官分化的物质,使受精后细胞的分化发生了失调,得不到正常的发育所致。     

泽蛙的性腺分化及温度对性别决定的影响

通过组织学方法观察了泽蛙(Rana limnocharis)原始生殖细胞(PGCs)的迁移、原始性腺的形成和性腺分化,并且探讨在不同的培育温度条件下性腺分化的差异。泽蛙的性腺分化有其特殊性:生殖嵴形成时,其中既有体细胞,又有原生殖细胞;无论原始性腺是分化成为精巢还是卵巢,其中都出现一个初生性腔。蝌蚪孵化后的17-34d(Gosner 26-38期)为性腺分化的敏感时期。在蝌蚪孵化后的第2d(Gosner 25期),分别用不同水温18℃±1℃、30℃±1℃、32℃±1℃、34℃±1℃培育蝌蚪,直至完成变态幼蛙(Gosner 46期)形成。自然水温23℃-25℃为对照。对照组的雌、雄性比接近1∶1(1∶1.06);18℃±1℃实验组的雌、雄比例为1.83∶1,雄性率仅35.1%(P<0.01);从30℃±1℃实验组起,雄性率提高,34℃±1℃实验组的雄性率达74.0%(P<0.01)。较高的培育温度可使泽蛙蝌蚪性别分化趋向雄性,而较低的培育温度则使蝌蚪雌性化。泽蛙的性别分化属于温度依赖型性决定(TSD)。当前全球性气候变暖对两栖类性比的稳定存在着威胁。     

温度对江黄颡鱼性分化的影响

通过组织学方法观察江黄颡鱼原始生殖细胞(PGCs)迁移、生殖嵴生成和性腺分化,并且探讨在不同温度培育下性腺分化的差异。实验结果显示:1日龄仔鱼PGCs位于鱼体中肠背方的脏壁中胚层中;5日龄时,PGCs迁移到背方的腹膜上皮;8日龄时,生殖嵴形成;14日龄时,原始性腺形成;23日龄时,性腺开始分化。从孵化后的第10天开始,分别用(20±0.5)、(24±1.0自然水温、对照组)、(30±0.5)和(34±0.5)℃4种水温培育仔鱼达25天。实验结束后统计结果显示:对照组和(20±0.5)℃组的雌、雄性比接近1∶1(分别为1∶1.09和1.22∶1);(30±0.5)℃组的为1∶4.89,雄性率达(83.3±0.7)%;(34±0.5)℃组的为2.85∶1,雄性率仅为(26.4±0.4)%。提示(30±0.5)℃可使幼鱼性腺发育趋向雄性,(34±0.5)℃则使幼鱼性腺发育趋向雌性。实验结果表明,江黄颡鱼的性分化是属于温度依赖型性别决定。     

温度对江黄颡鱼性分化的影响

通过组织学方法观察江黄颡鱼原始生殖细胞(PGCs)迁移、生殖嵴生成和性腺分化，并且探讨在不同温度培育下性腺分化的差异。实验结果显示：1日龄仔鱼PGCs位于鱼体中肠背方的脏壁中胚层中；5日龄时，PGCs迁移到背方的腹膜上皮；8日龄时，生殖嵴形成；14日龄时，原始性腺形成；23日龄时，性腺开始分化。从孵化后的第10天开始，分别用(20±0.5)、(24±1.0自然水温、对照组)、(30±0.5)和(34±0.5)℃4种水温培育仔鱼达25天。实验结束后统计结果显示：对照组和(20±0.5)℃组的雌、雄性比接近1∶1(分别为1∶1.09和1.22∶1)；(30±0.5)℃组的为1∶4.89，雄性率达(83.3±0.7)%；(34±0.5)℃组的为2.85∶1，雄性率仅为(26.4±0.4)%。提示(30±0.5)℃可使幼鱼性腺发育趋向雄性，(34±0.5)℃则使幼鱼性腺发育趋向雌性。实验结果表明，江黄颡鱼的性分化是属于温度依赖型性别决定。     

Efficacy of exemestane,a new generation of aromatase inhibitor,on sex differentiation in a gonochoristic fish

We report the first use of exemestane (EM), a steroidal aromatase inhibitor (AI) commercially known as aromasin, in studies of sex differentiation in fish. The effectiveness of EM was examined in two different age groups of the gonochoristic fish, Nile tilapia (Oreochromis niloticus). Untreated control fish (all female) showed normal ovarian differentiation through 120 days after hatching (dah), whereas fish treated with EM at 1000 and 2000 µg/g of feed from 9 dah through 35 dah, the critical period for sex differentiation, exhibited complete testicular differentiation; all stages of spermatogenic germ cells were evident and well developed efferent ducts were present. Fish treated with EM at 1000 µg/g of feed from 70 dah through 100 dah significantly suppressed plasma estradiol-17β level and increased level of 11-ketotestosterone. Furthermore, untreated control fish showed strong gonadal expression of the steroidogenic enzymes P450 cholesterol-side chain-cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 aromatase (P450arom). In contrast, EM-treated fish showed immunopositive reactions against P450scc and 3β-HSD but not against P450arom in interstitial Leydig cells. These results indicate that treatment of tilapia juveniles with EM during sex differentiation leads to the development of testes, apparently by a complete suppression of aromatase activity.     

Seabream GnRH immunoreactivity in brain and pituitary of XX and XY Nile tilapia, Oreochromis niloticus during early development

Seabream gonadotropin-releasing hormone (sbGnRH)-the chief preoptic area-hypothalamus (POA-H) form of GnRH in tilapia is involved in sexual maturation. In this study, we investigated the qualitative changes in ontogeny of sbGnRH immunoreactivity (ir-), between sexes to understand its impending role during sex differentiation. For this, the differences in immunocytochemical localization of sbGnRH in genetically male (XY) and female (XX) fish were studied from 1 day after hatching (dah), through the critical period of sex differentiation (7-21 dah) to 40 dah and mature Nile tilapia. Specific antisera against sbGnRH were used for immunolocalization. SbGnRH ir- neurons were observed in POA-H as early as 5 and 15 dah in XY fish and XX fish, respectively. Higher ir- was detected in the POA-H of XY tilapia compared with XX population till 10 dah. There was a qualitative drop in sbGnRH ir- neurons/cell bodies in POA-H around 20 dah till 30 dah in XY population compared with other durations. SbGnRH ir- cells were detected in pituitary of XX fish by 15 dah and in XY fish around 10 dah but seemed to drop down by 20 dah in XY whereas it continued to remain steady in XX fish. The sbGnRH ir- in XY fish showed a rise from 35 dah and thence till 40 dah. This study revealed subtle differences in POA-H and pituitary sbGnRH ir- during early development between genetic male and female fish with possible implications in sex differentiation.     

Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17beta (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down.     

Estrogen-induced gonadal sex reversal in the tammar wallaby

Estrogens have a feminizing effect on gonadal differentiation in fish, amphibians, reptiles, and birds. However, the role of estrogen during gonadal differentiation in mammals is less clear. We investigated the effect of estrogen on gonadal differentiation of male tammar wallabies. Male pouch young were treated orally with estradiol benzoate or oil from the day of birth, before seminiferous cords develop, to Day 25 postpartum and were killed at Day 50 postpartum. In all estrogen-treated neonates, a decrease in gonadal volume, volume of the seminiferous cords, thickness of the tunica albuginea, and number of germ cells was found. The stage of treatment affected the magnitude of the response. Two of three male young born prematurely after 25 days of gestation and treated subsequently with estradiol had ovary-like gonads, with well-developed cortical and medullary regions and primordial follicle formation. Furthermore, at Day 50 postpartum, many (21%) of the germ cells in these sex-reversed ovaries were in the leptotene and zygotene stages of meiosis, similar to female germ cells at the same stage of development. In the other males born on Day 26 of gestation or later, estradiol treatment from the day of birth caused development of dysgenetic testes, with abnormal Sertoli cells, atrophy of the seminiferous tubules and tunica albuginea, and absence of meiotic germ cells. In this marsupial, therefore, estradiol can induce either partial or complete transformation of the male gonads into an ovary with meiotic germ cells. These results confirm that estrogen can inhibit early testicular development, and that testis determination occurs during a narrow window of time.     

Proliferation of germ cells during gonadal sex differentiation in medaka: Insights from germ cell-depleted mutant zenzai

The proliferation of germ cells becomes sexually dimorphic during gonadal sex differentiation, although the underlying dynamics of this are not well understood in vertebrates. By tracing GFP-labeled germ cells in vivo and analyzing the germ cell-depleted mutant, zenzai, we show that the proliferation and differentiation of germ cells are regulated in a sexually dimorphic manner in the teleost fish medaka. In the undifferentiated gonads, germ cells resume proliferation by slow intermittent division (type I), producing isolated daughter cells. While germ cells in the male gonads continue this mode of proliferation, some germ cell fractions in the female gonads initiate two to four rounds of continuous division (type II), forming cysts of four, eight, or sixteen cells, which subsequently enter meiosis synchronously. Thus, female germ cells become differentiated much earlier than do male germ cells. In the zenzai mutant, a defect in slow intermittent division eventually leads to the depletion of germ cells in the adult gonads in both sexes, despite the fact that cyst-forming division is unaffected. This argues that slow intermittent division is essential for the maintenance of germ cells. The proliferation and differentiation of germ cells are thus important components of gonadal sex differentiation in vertebrates.     

Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus

Vas (a Drosophila vasa homologue) gene expression pattern in germ cells during oogenesis and spermatogenesis was examined using all genetic females and males of a teleost fish, tilapia. Primordial germ cells (PGC) reach the gonadal anlagen 3 days after hatching (7 days after fertilization), the time when the gonadal anlagen was first formed. Prior to meiosis, no differences in vas RNA are observed in male and female germ cells. In the ovary, vas is expressed strongly in oogonia to diplotene oocytes and becomes localized as patches in auxocytes and then strong signals are uniformly distributed in the cytoplasm of previtellogenic oocytes, followed by a decrease from vitellogenic to postvitellogenic oocytes. In the testis, vas signals are strong in spermatogonia and decrease in early primary spermatocytes. No vas RNA expression is evident in either diplotene primary spermatocytes, secondary spermatocytes, spermatids or spermatozoa. The observed differences in vas RNA expression suggest a differential function of vas in the regulation of meiotic progression of female and male germ cells.     

Gonadal sex reversal in triploid rainbow trout (Oncorhynchus mykiss).

Sex determination in salmonids is primarily governed by sex chromosomes; however, phenotypic expression and successful development of the gonads may be influenced by additional factors. Exposure to exogenous steroids during the critical period of gonadal differentiation will reverse the expected phenotypic sex of both female and male trout. Triploidy, a viable condition in rainbow trout (RBT), alters the degree of gonadal development in a gender-specific manner. Males produce testes with similar morphology and function as diploid fish, but females produce underdeveloped ovaries devoid of growing oocytes. One possible explanation for this observed gender difference is that the timing of meiotic initiation may influence ovarian/testicular development in triploid RBT. To determine whether the early entrance of germ cells into meiosis results in the lack of ovarian development in triploid females, the objective of this study was to sex-reverse genotypic triploid female RBT (XXX) into phenotypic males and genotypic triploid male RBT (XXY) into phenotypic females. Male fish were exposed to estradiol-17beta (E(2)) and females were exposed to the non-aromatizable androgen 17alpha-methyldihydrotestosterone (MDHT). Over 90% of the male fish treated with exogenous E(2) developed gonadal structures indistinguishable from the gonads of triploid females. Triploid female RBT treated with MDHT developed testes; however, not all fish treated with this androgen were completely sex reversed. The results of this investigation are consistent with the hypothesis that the failure of ovarian development in triploid RBT is due to the early onset of meiosis and does not appear to be due to genotypic sex. J. Exp. Zool. 284:466-472, 1999.     

Effects of estradiol-17beta on germ cell proliferation and DMY expression during early sexual differentiation of the medaka Oryzias latipes

Sex reversal of XY male to functional females was induced by estrogen treatment during the embryonic period in the medaka Oryzias latipes. The present study aimed to examine whether exogenous estrogen (estradiol-17beta; E(2)) affects early sex differentiation, paying particular attention to DMY expression and proliferation activity of germ cells in estrogen treated XY individuals. Our results showed that germ cell number was not affected by E(2) treatment at hatching, and that DMY expression was not suppressed under conditions of sex reversal. Therefore, male differentiation of germ cells, which is triggered by the expression of DMY in the supporting cell lineage, proceeds even in E(2) treated XY individuals until hatching, and early sex differentiation is not altered by estrogen. However, sex reversal occurred after hatching probably because of estrogen remaining in the yolk. Interestingly, DMY expression was also detected in the large follicle layer of E(2 )treated XY ovary. These results suggested that DMY regulates male determination in early embryonic stage but does not suppress female follicle development.