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.     

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.     

三倍体湘云鲫繁殖季节的性腺结构观察

在繁殖季节解剖３７尾龄和１６尾声龄湘云鲫,用光镜和电镜观察了湘云鲫的性腺结构。在湘鲫的性腺部位有三种类型的结构：（１）清巢型;精巢小叶内分布有精子细胞,精子细胞呈退化现象,没有发现有正常成熟精子。（２）卵巢型,该类型性腺的大部分为体积较小,分化程度较低的呈囊状排列的细胞,在这些小细胞中镶嵌着少量初级卵母细胞和体积较大的退化的卵母细胞。（３）脂肪型;该类型的性腺部位仅有两条脂肪组织,即没有卵巢结构,     

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%。       

鲤鱼雌雄同体现象的观察

雌雄同体存在于许多鱼类中,本文报道了鲤鱼的雌雄同体现象。作者在专题研究东江鲤鱼种群生态时,解剖测定了483尾鲤鱼,其中1尾有雌雄同体现象,这尾标本体长27厘米,体重699.2克,年龄3~+,成熟系数(性腺重/总体重× 100)为9.0。两侧性腺都由卵巢组织和精巢组织组成。肉眼观察可见卵巢部分呈桔黄色,精巢部分呈乳白色。对性腺进行了组织学检查,精巢组织中存在着大量成熟的精子和向精子变态的精子细胞,还有少量的精原细胞。初级精母细胞和次级精母细胞,卵巢组织中主要是第4时相的初级卵母细胞,少量是发育早期的卵母细胞。根据观察,精卵巢组织发育正常,精巢中已有成熟的精子,雄性生殖机能可能会发生。     

蓝太阳鱼第一次性周期性腺发育的组织学

蓝太阳鱼(Lepomis cyanellus)是广东省于1997年从北美引进的小型淡水鱼类,本文采用组织切片技术对蓝太阳鱼的性腺发育进行了研究。蓝太阳鱼的性腺发育程序可以分为5个时期。在1月龄,卵巢和精巢处于第Ⅰ期;2~3月龄时,卵巢和精巢发育到第Ⅱ期;4月龄时发育到第Ⅲ期;5~6月龄时发育到第Ⅳ期;7~8月龄时达到性成熟第Ⅴ期。过冬时,卵巢退化到第Ⅱ~Ⅲ期,而精巢仍停留在第Ⅴ期。成熟卵巢的成熟系数为2.80%~8.10%,成熟精巢的成熟系数为1.45%~3.45%。精巢和卵巢发育都为不完全同步型,且精巢发育比卵巢稍快。蓝太阳鱼的繁殖期在广州地区为3~11月,为多次产卵类型。本文从生殖和生长的关系上对蓝太阳鱼生长缓慢的原因进行了初步探讨,并将蓝太阳鱼作为一种鱼类实验动物的可行性进行了分析。     

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.     

西藏双须叶须鱼性腺发育的组织学观察（英文）

为丰富双须叶须鱼(Ptychobarbus dipogon)繁殖生物学,实验采用常规的石蜡切片及HE染色方法,对西藏双须叶须鱼的性腺发育及组织结构特征进行了研究,对双须叶须鱼人工繁殖成功具有重要的理论意义。结果表明:双须叶须鱼卵母细胞发育过程可分为5个时期,其卵巢发育可分为6个时期,在第Ⅴ期卵巢中,小卵和大卵数量比例为1.38鲶1,存在败育现象。雌性卵母细胞在第3时相卵黄颗粒和滤泡;第4时相卵母细胞中卵黄颗粒数量迅速增多,细胞核向动物极移动;第5时相卵母细胞中卵黄颗粒融合成片,卵细胞与滤泡膜分离并游离于卵巢腔中。双须叶须鱼精巢为小叶型,其生殖细胞可分为精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子,其精巢发育也分为6个时期。双须叶须鱼属于分批同步产卵鱼类。     

西藏双须叶须鱼性腺发育的组织学观察

为丰富双须叶须鱼 (Ptychobarbus dipogon) 繁殖生物学,实验采用常规的石蜡切片及HE染色方法,对西藏双须叶须鱼的性腺发育及组织结构特征进行了研究,对双须叶须鱼人工繁殖成功具有重要的理论意义。结果表明:双须叶须鱼卵母细胞发育过程可分为5个时期,其卵巢发育可分为6个时期,在第Ⅴ期卵巢中,小卵和大卵数量比例为1.38 鲶1,存在败育现象。雌性卵母细胞在第3时相卵黄颗粒和滤泡;第4时相卵母细胞中卵黄颗粒数量迅速增多,细胞核向动物极移动;第5时相卵母细胞中卵黄颗粒融合成片,卵细胞与滤泡膜分离并游离于卵巢腔中。双须叶须鱼精巢为小叶型,其生殖细胞可分为精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子,其精巢发育也分为6个时期。双须叶须鱼属于分批同步产卵鱼类。     

大弹涂鱼性腺发育的组织学观察

于光镜下对大弹涂鱼性腺切片作了组织学观察,对大弹涂鱼卵细胞和精子发育规律进行研究。结果表明:大弹涂鱼在一个生殖季节中只能产卵1次,大弹涂鱼属于一次性产卵类型。大弹涂鱼3月卵母细胞进入大生长期发育阶段,4—6月为繁殖盛期,7—8月为繁殖末期。10月卵巢基本修整完毕,进入Ⅱ′恢复期。卵细胞发育可分为6个时相:卵原细胞、卵母细胞单层滤泡、卵母细胞出现脂滴和卵黄、卵母细胞卵黄充满、卵母细胞核极化、卵母细胞退化时相。卵母细胞膜单层,由具有辐射纹的放射带构成,滤泡膜细胞分泌而成的次级卵膜成为成熟卵子的附着丝。大弹涂鱼2月精巢开始发育,5月GSI值达最高值,平均成熟系数达0.70%,排精量最旺盛,出现高峰。7—9月GSI值明显下降。11月至翌年2月GSI值波动于0.08%—0.20%之间,变辐小,此期间精巢处于静止发育状态。大弹涂鱼的精巢属于小叶型结构。精子发育分为6个时期:精原细胞期、精原细胞增殖期、精母细胞生长成熟期、精子细胞变态期、精子成熟期和退化吸收期。繁殖季节精小叶内充满精子,精小囊消失。       

人工诱导雌核发育日本白鲫

分别用遗传失活的散鳞镜鲤(Cyprinus carpio L.)、团头鲂(Megalobrama amblycephala)精子诱导日本白鲫(Carassius cuvieri)进行雌核发育.未经冷休克处理,用UV照射过的镜鲤的精子(其最佳UV照射剂量为4 200 mJ/cm2)与日本白鲫卵子受精获得(32.4±3.3)%雌核发育单倍体和(0.7±0.3)%杂交二倍体,而照射过的团头鲂精子(其最佳UV照射剂量为3 600mJ/cm2)与日本白鲫卵子受精得到了(33.8±1.4)%雌核发育单倍体和(0.5-±0.3)%杂交二倍体.日本白鲫卵子分别经灭活的镜鲤、团头鲂的精子激活(精子经各自最佳UV剂量处理),施以冷休克处理(受精后2min,0-4℃,40min),其孵化率分别为(27.8±2.1)%和(29.4±3.3)%,发育到摄食期,其正常的雌核发育二倍体鱼苗分别为(15.7±3.4)%和(23.6±4.1)%,在镜鲤实验组中,其正常的雌核发育二倍体鱼苗占孵化鱼苗总数的56%,这比团头鲂实验组中正常雌核发育二倍体鱼苗的比率(达到80%)低,结果表明诱导日本白鲫雌核发育,与母本遗传关系远的团头鲂精子较镜鲤的更有效.从染色体数目、外形特征和性腺结构等方面证实雌核发育后代的生物学特性.雌核发育日本白鲫全为雌性,这表明其雌性是同配XX型.雌核发育日本白鲫在生产上将有着潜在的价值,如比普通日本白鲫长得更快,抗病能力强等.所有的雌核发育日本白鲫的染色体数目都是100,而所有的团头鲂与日本白鲫的杂交后代都是三倍体(3n=124),新三倍体鱼在鱼类养殖和理论研究中将存在潜在的价值.     

Induced Gynogenesis in Grass Carp (Ctenopharyngodon idellus) Using Irradiated Sperm of Allotetraploid Hybrids

Grass carp (Ctenopharyngodon idellus) eggs were activated by UV-irradiated diploid sperm of allotetraploid hybrids derived from red crucian carp (♀)?×?common carp (♂) and then duplicated by cold shock in 4-6°C water for 10-12 min. Different cold shock initiation times resulted in two types of diploid gynogenetic grass carp: meiotic gynogenetic (meiG) and mitotic gynogenetic (mitG). Over a 5-year period, a total of 17,170 meiG and 1,080 mitG fry were produced and 6,862 meiG and 372 mitG grass carp survived. The gynogenetic fish were confirmed by morphological characteristics, chromosome examination, and microsatellite DNA analysis. The morphological traits of the gynogenetic grass carp were similar to those of wild diploid grass carp. Normal gynogenetic fish were identified as diploid with 48 chromosomes by chromosomal metaphases examination, while nonviable abnormal embryos were detected as haploid with 24 chromosomes. Microsatellite DNA analysis indicated that after one generation of gynogenesis, the genetic purity of meiG and mitG grass carp was significantly increased over that of wild grass carp. In addition, both meiG and mitG grass carp groups were 100% female, and 88% of these showed normal ovary development. Thus, the sex determination mechanism in female grass carp was homogamety. The ability to establish pure all-female groups of meiG and mitG grass carp should be a valuable contribution to both fish genetics and grass carp breeding.     

Induction of Gynogenesis in Japanese Crucian Carp (Carassius cuvieri)

Diploid gynogenesis was induced in Japanese crucian carp (Carassius cuvieri) eggs using UV-irradiated genetically inactive spermatozoa from mirror carp (Cyprinus carpio L.) or blunt snout bream (Megalobrama amblycephala), with or without cold shock. The optimal radiation dosage was 4200 mJ/cm² and 3600 mJ/cm² for mirror carp and blunt snout bream sperm, respectively. At this dosage and without cold shock, the yields were (32.4±3.3)% vs. (33.8±1.4)% gynogenetic haploids and (0.7±0.3)% vs. (0.5±0.3)% hybrid diploids, respectively. At the optimal UV dosage but with cold shock (2 min after fertilization, 0-4°C for 40 min), the hatching rates were (27.8±2.1)% and (29.4±3.3)%, respectively. From hatching to feeding, (15.7±3.4)% and (23.6±4.1)% normal gynogenetic diploids were recorded, respectively. Survival of normal gynogenetic diploids was 56% out of the hatched fry when using irradiated spermatozoa of mirror carp, which was lower than that (up to 80%) when using irradiated spermatozoa of blunt snout bream. This indicated that the sperm of blunt snout bream, with distant genetic relation to the maternal Japanese crucian carp, was more effective than that of mirror carp to induce diploid gynogenesis. The nature of the gynogenetic progeny was identified with external appearance, chromosome number and gonad structure. The presence of only females in gynogenetic progeny probably suggested XX genotype in the female Japanese crucian carp. The gynogenetic diploids have potential values such as faster growth and stronger disease resistance than the normal Japanese crucian carp. All gynogenetic progeny possessed 100 chromosomes whereas all J x B crosses were triploid with 124 chromosomes. The formation of the new triploid hybrids in J x B crosses may be useful in aquaculture.     

鲫鱼的人工和天然雌核发育

用照射处理的草鱼或华南鲤的精液授精的鲫鱼卵为单倍体雌核发育;所有单倍体胚胎都是畸形,并在孵化前死亡。如果用照射精液授精的鲫鱼卵在18—19℃的水中放置2分钟,然后在0—3℃的冰水中放置20分钟,再放回室温水中孵化,可以从每百颗受精卵中得到4.5—11.9(平均8.34)尾成活的二倍体仔鱼。根据38尾银鲫(其中33尾捕自黑龙江省方正县双凤水库)的细胞学和胚胎学检查,证实它们是雌核发育繁殖的。       

雌核发育银鲫的受精生物学研究——天然雌核发育银鲫繁殖方式的讨论

本文研究了同源雌核发育银鲫精子在4种类型的雌核发育银鲫卵中的发育特征。初步揭示了天然雌核发育银鲫根据精子的来源不同而分别具有二种不同的繁殖方式,对其在维持雌核发育银鲫种群生存,促使克隆分化等方面的独特的生物学意义进行了讨论。     

Genetic Evidence for Gonochoristic Reproduction in Gynogenetic Silver Crucian Carp (Carassius auratus gibelio Bloch) as Revealed by RAPD Assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp. Received: 5 January 2000 / Accepted: 3 August 2000     

Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp.MPF activity was measured by using histone H1 as phosphorylation substrate.There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes,but there existed some subtle difference between them.The subtle difference was that the first peak of MPF kinase activity was kept to a longerlasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp.It was suggested that the difference may be related to the spindle behavior changes,such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.     

雌核发育银鲫与两性生殖彩鲫卵壳蛋白组分的比较分析及其受精前后的变化

以雌核发育银鲫和两性生殖彩鲫的成熟卵为材料,分离卵壳,经处理得到卵壳可溶性蛋白组分。SDS－PAGE梯度凝胶电泳分析在雌核发育银鲫中揭示出3条较明显的差异蛋白带。同时,采用相同处理方法对受精前后卵壳蛋白组分进行比较分析后发现,这些差异蛋白带在受精后发生了变化,其带纹表现为减弱或消失,表明这些差异蛋白可能与受精过程相关。 Abstracts:Egg chorions were isolated from unfertilized and fertilized eggs of gynogenetic silver crucian carp and gonochoristic color crucian carp by homogenization and further purification techniques.Then,soluble proteins were extracted from the isolated egg chorions,and were analyzed by gradient SDS-PAGE.Three differential protein bands were revealed between the gynogenetic silver crucian carp and gonochoristic color crucian carp.Furthermore,these differential proteins were demonstrated to undergo obvious changes during fertilization.It was suggested that these differential proteins should be related to the special fertilization process in gynogenetic silver crucian carp.     

雌核发育和两性融合发育鱼卵调控精核受精发育的生化特性研究

两性融合生殖的鱼卵受精后,精核能疏松、解凝,形成雄性原核：雌核发育银鲫卵子受精后,精核发育受到抑制,无法形成原核。采用显微注射去膜精核以及细胞学和电镜观察的方法,本文对两类鱼卵受精后精核早期发育的生化性质进行了初步探讨,并着重研究了雌核发育银鲫卵子控制精核发育的生化特征。实验结果显示,两性融合生殖鱼类卵质中,一定量的Ｃａ２＋的存在,二硫键的还原作用对于精核的发育显然是必要的;而在雌核发育银鲫卵中,Ｃａ２＋的功能和二硫键的还原作用与精核发育受到抑制之间并无直接联系。银鲫卵质中似乎显示出异常的磷酸酶脂解活性,导致磷酸化过程无法进行,使精核解凝受到阻碍。另外,两性融合生殖的鱼卵重质层中具有大量诱导精核原核化的有关因子,而银鲫卵质中则缺少该因子（或活性极低）。银鲫卵质中还可能缺乏某些与雄性原核的核膜重组装有关的大分子物质。