Caspase-3在黄鳝性腺性逆转过程中的表达及定位分析

为探究黄鳝( Monopterus albus )Caspase-3在性腺性逆转发育过程中的功能和作用,实验扩增黄鳝 caspase-3 的部分序列,检测了其在不同发育时期性腺中mRNA表达水平、蛋白相对含量以及表达位置。通过PCR扩增获得黄鳝 caspase-3 基因cDNA序列,推导的氨基酸序列对比发现,黄鳝 caspase-3 与大黄鱼( Larimichthys crocea )和鳜( Siniperca chuatsi )亲缘关系最近。实时定量PCR ( qRT-PCR)结果显示, caspase-3 mRNA在黄鳝各发育时期性腺中均有表达,在卵黄生成期性腺中表达量最高,并伴随性腺向雄性发育表达量呈下降的趋势;而其蛋白相对含量在性腺性逆转过程中呈现逐渐上升的趋势,间性晚期最高。免疫组织化学染色结果显示,Caspase-3蛋白阳性信号定位于卵黄生成期卵母细胞细胞质、皮质小泡期卵母细胞的细胞核和颗粒细胞,以及各个发育时期性腺中初级生长期卵母细胞的细胞质和细胞核。综上,Caspase-3与黄鳝性逆转过程关系密切,推测其可能参与了性逆转过程中卵母细胞的凋亡过程。     

花䱻卵母细胞发育的组织学和超微结构观察

2015年7月至2017年10月在河南省驻马店宿鸭湖水库采集花?(Hemibarbus maculatus Bleeker)雌鱼样本190尾,体长7.12～32.21 cm,体重10.55～330.22 g,采用组织学和扫描电子显微镜技术观察了花?卵母细胞发育各时期的特征。结果表明,花?卵母细胞发育可分为5个时相,第I时相卵母细胞处于卵原细胞增殖阶段;第Ⅱ时相卵母细胞处于初级生长阶段,出现滤泡膜;第Ⅲ时相卵母细胞出现皮质液泡,细胞质膜之间形成放射带;第Ⅳ时相卵母细胞处于大生长后期,卵黄颗粒增多。电镜下观察发现放射带表面形成微孔状结构,核仁外排,可能与卵母细胞内营养物质积累有关;第Ⅴ时相卵母细胞中细胞核消失,卵母细胞发育为成熟卵子,与卵膜脱离,准备排卵。繁殖季节,花?卵巢成熟系数达到13.78%～17.04%。研究结果可为花?人工繁殖和育种工作提供参考。     

福建省东山岛西埔湾港养斑鰶的繁殖生物学

本文研究了福建省东山岛西埔湾港养斑鰶性腺周期发育、成熟系数周年变化、个体生殖力以及雌雄同体。港养斑鰶能在半封闭式湾内发育成熟而且自然产卵。其卵细胞发育分为6个时相,精巢发育分为6期。卵膜由透明带、外放射带和内放射带组成,内有孔道结构。第4时相中期卵母细胞的内放射带有7～8层纤维状片层。产卵后鞘膜细胞和颗粒细胞分裂增殖变成游走的吞噬细胞。西埔湾港养斑鰶Ⅰ龄鱼初次性成熟,生殖季节从12月下旬至翌年3月下旬,生殖盛期为2月,雌鱼成熟系数高达17.88％。个体绝对生殖力为7575～72621粒,平均 35310粒,系属于分批产卵类型,第1批生殖力大于第2批。     

东海鳓卵巢发育的组织学观察

于光镜于对东海鳓卵巢切片作了组织学观察，结果表明，5月－7月为东海鳓的繁殖期，其卵巢在Ⅳ期、Ⅴ期、Ⅵ－Ⅳ‘期和Ⅴ‘期，8月－9月鳓卵巢处在产后休整阶段；10月－翌年2月为越冬Ⅱ期卵巢；3月－4月卵巢处在产前发育阶段，在小生长期，卵核的体积较核质的体积增大快，核质比大，大小长期，随着卵黄物质的积累，卵母细胞体积快速增加, 比下降。在鳓卵母细胞发育中，曾见有两层卵膜结构，但接近成熟的卵母仅有一层卵膜。东海鳓的产卵方式为短期分批产卵类型。在一个生殖周期内，一般产卵2次。     

Oogenesis in the common Japanese conger Conger myriaster

ABSTRACT:    We investigated the process and characteristics of oogenesis in the common Japanese conger Conger myriaster . Young fish caught in November 1996 were reared for use in this experiment. Fish were sampled monthly from December 1997 to August 1998. Some were injected with human chorionic gonadotropin to stimulate ovarian maturation from May to August 1998. Oocytes from the chromatin nucleolus stage to the secondary yolk globule stage were obtained from non-hormone-treated fish; those of more advanced stages were obtained from hormone-treated ones. We divided oocyte development into eight stages from the chromatin nucleolus stage to the maturation stage. The yolk vesicle stage was not separated because yolk vesicles began to appear just after appearance of yolk globules. Oocyte, oil droplet, yolk globule and nucleus diameters all increased concomitant with oocyte development. Oil droplet and yolk globule diameters increased remarkably at the maturation stage. However, zona radiata thickness peaked at the secondary yolk globule stage, decreasing gradually thereafter. Increased gonadosomatic index was related to oocyte development as found in European and Japanese eels receiving hormone treatment to mature. The present study is the first report describing oogenesis characteristics in congrid eels. It indicates that oogenesis is almost identical to that of other anguillid eels.     

Patterns of oocyte development in natural habitat and captive <Emphasis Type="Italic">Salminus hilarii</Emphasis> Valenciennes, 1850 (Teleostei: Characidae)

Fecundity and oocyte development in Salminus hilarii female brood stock were analyzed with the aim of investigating the impact of migration impediment on oogenesis. Histological analyses of the ovaries were performed in adult females caught in two different environments—the Tietê River (natural) and captivity—and the gonadossomatic index, oocyte diameter and fecundity determined. Five germ cell development stages (oogonium, perinucleolar, cortical alveoli, vitellogenic, ripe) and two other structures (postovulatory follicles and atretic oocytes) were observed in females caught in the river. Captive animals lacked the ripe oocytes and postovulatory follicles and had a relatively higher number of atretic oocytes. Females in captivity are known to produce larger oocytes, and they release fewer eggs in each spawn (absolute fecundity) when compared with animals that are able to migrate. Our results suggest that the Tietê River is undergoing alterations which are being reflected in the reproductive performance of S. hilarii, mainly due to the presence of atretic oocytes in females caught in the river. The lack of postovulatory follicles and ripe oocytes in captive animals reveals that migratory impediment negatively impacts final oocyte maturation. However, the stage of maturation reached is adequate for ovulation induction with hormone manipulation.     

Changes in serum vitellogenin levels and immunohistochemical localization of vitellogenin in hepatic cells during ovarian development in the Japanese eel

ABSTRACT: The present study was designed to quantify changes in serum vitellogenin (VTG) levels and to localize VTG in hepatic cells immunohistochemically during artificial maturation of the Japanese eel Anguilla japonica . Hormone treatment resulted in a gradual rise in serum VTG levels, in which the highest values were attained at the migratory nucleus stage. Hepatocytes responded to hormone treatment by increased immunoreactivity; both staining area and intensity were enhanced with ongoing gonadal development. Moreover, cytoplasmic vacuoles increased in number during artificial maturation, occupying large areas inside hepatocytes. The unusual appearance of hepatocytes may reflect the effect of artificial maturation.     

Synthesis and possible function of 11-ketotestosterone during oogenesis in eel (Anguilla spp.)

The control of 11-ketotestosterone (11-KT) biosynthesis and its physiological roles were examined in female Japanese eel (Anguilla japonica) and New Zealand longfinned eel (Anguilla dieffenbachii). 11-KT was detected in serum of female eels of both species. Among various tissues from Japanese eel, the ovary had the greatest capacity to synthesize 11-KT in vitro. In addition, the oocyte diameters of eels treated with 11-KT had increased significantly. Furthermore, these oocytes were found to have an increased number of oil droplets. These findings suggest that 11-KT in female eels may be mostly of ovarian origin and that this androgen appears to play an important role in controlling pre-vitellogenic oocyte growth.     

江黄颡鱼卵细胞膜的结构特征

Using conventional histochemical methods and scanning electron microscope, the egg surface of mature oocyte of Pelteobagrus vachelli was observed. The micropyle is funnel-like and consists of micropylar vestibule and micropylar canal. The opening of micropylar vestibule is about 11.35μm in diameter while entrance of the micropyle to the oocyte is only about 2.6 μm. There are plenty of micropores on the surface near the micropyle. Micropyle might act as an apparatus leading spermatozoa to penetrating an oocyte. Regular cord-like structures between basal membrane (the outermost layer of egg surface) and plasmic membrane (the inner layer of egg surface) are found. These cord-like structures, each about 16.75 to 18.50μm in length, 5μm in diameter, comprised small balls whose diameters vary from 2.18 to 2.5μm and are arranged cornpactly under the basal membrane. These kinds of structures are zona radiata and act as special eggshell. After activation of the oocytes, the cord-like structures would partially transform into mucoprotein. To our best knowledge, this paper is the fast report on stereo view of the zona radiata of teleost oocyte.     

半滑舌鳎卵巢发育的组织学和形态数量特征研究

采用组织学方法系统研究了人工养殖条件下半滑舌鳎亲鱼卵巢的组织发育周年变化特征.结果表明,卵母细胞发育可分为6个时相,卵巢发育分为6期.卵巢不同发育时期都由不同类型的卵母细胞组成,半滑舌鳎为非同步分批多次产卵类型.周年发育过程中,6月龄前的鱼卵巢发育处于第Ⅰ期;9～12月龄卵巢可发育至第Ⅱ期;12月龄半滑舌鳎性腺可发育达到Ⅲ期,并一直保持至24月龄.3龄雌鱼达到性成熟,卵巢可发育至第Ⅳ期;随着水温和光周期的调控,卵巢发育进入成熟期(Ⅴ期),发生水合现象是卵母细胞成熟并准备排卵的信号;产卵结束后卵巢退化至第Ⅵ期,排出的卵母细胞退化吸收,以Ⅲ期卵巢越冬后,卵巢退化至Ⅱ期并持续至下一个繁殖周期.人工养殖条件下,卵巢内存在卵母细胞闭锁现象.性腺周年发育过程中,性腺指数(GSI)在性腺达到成熟期时达到峰值,产卵结束后性腺快速退化,GSI值各月份差异显著(P<0.05),亲鱼性腺重量在卵巢发育成熟过程中一直处于生长状态.亲鱼肝脏指数(HSI)在产卵前7～8月份(性腺达到成熟期前30 d左右)达到最大,表明7～8月份是性腺进行卵子发育、卵黄能量储备的重要时期,亲鱼在产卵前通过积极摄食储备体内能量,肝脏在卵母细胞卵黄积累方面具有重要的生理作用.肥满度(CF)值在繁殖盛期10～11月份达到最大,表现出与GSI类似的变化趋势.     

鲤脑垂体匀浆液和人绒毛膜促性腺激_省略_性腺激素及性类固醇激素含量的影响

雄鳗注射５－６次、雌鳗注射９－１０次鲤脑体匀浆液（ＣＰＥ）＋人绒毛膜促性腺激素（ＨＣＧ）能分别诱导精巢和卵巢发育成熟。在雌雄鳗鲡,注射ＣＰＥ＋ＨＣＧ可显著增加端脑、间脑、中脑和下丘脑ｍＧｎＲＨ的含量,而对后脑和延髓ｍＧｎＲＨ的影响较小;注射ＣＰＥ＋ＨＣＧ增加雄鳗后脑和延髓ｃＧｎＲＨ－Ⅱ含量,对雌鳗脑区ｃＧｎＲＨ－Ⅱ则无显著影响。雌雄鳗鲡每次注射ＣＰＥ＋ＨＣＧ后１天,血清促性腺激素（ＧｔＨ）急剧上升     

Cloning of estrogen receptor-β cDNA of common Japanese conger Conger myriaster and expression of the gene during oocyte development

ABSTRACT:   Estrogen receptor (ER) cDNA was cloned from the liver of common Japanese conger Conger myriaster . Common Japanese conger ER cDNA contains 2543 nucleotides including an open reading frame that encodes 596 amino acids, with a molecular weight of 66.1 kDa. The deduced amino-acid sequence of the common Japanese conger ER shares 85% identity with that of eel Anguilla japonica ER-β, and 39–46% and 53–59% identity with the deduced sequences of ER-α and ER-β of other species, respectively. In a phylogenetic analysis, the common Japanese conger ER was found to cluster with the ER-β of other species. The common Japanese conger ER-β also has six domains (A–F), which are conserved in the nuclear receptor superfamily. The amino-acid sequences in the C and E domains were highly conserved between common Japanese conger ER-β and other ER. However, the other domains showed low homology. In the female common Japanese conger, ER-β mRNA was highly expressed in the liver and pituitary gland. The expression levels of the ER-β gene increased from the oil droplet stage to the primary yolk globule stage and were maintained until the migratory nucleus stage.     

Maturation and reproductive cycle of female armorhead <Emphasis Type="Italic">Pseudopentaceros wheeleri</Emphasis> from the southern Emperor—northern Hawaiian Ridge Seamounts

ABSTRACT:   The stage of ovarian development and annual reproductive cycle of female armorhead Pseudopentaceros wheeleri collected from seamounts of the southern Emperor–northern Hawaiian Ridge in 1993 and 1995–1996 was investigated through use of the gonadal somatic index ( GSI ) and ovarian histology. The stages of oocyte development were classified into eight developmental stages (perinucleolus, cortical alveolus, oil droplet, early yolk globule, late yolk globule, migratory nucleolus, prematuration, and maturation) based on histological characteristics. Oocytes of different size and developmental stage were present within ovaries during the spawning season. Oocyte development appears to be asynchronous and armorhead apparently spawn several times during the spawning season. The stage of the most developed oocyte was used to classify the stage of ovarian maturation. The GSI increased gradually with ovarian development. Oocytes in the perinucleolus, cortical alveolus, and oil droplet stages were present in ovaries throughout the year. Early and late yolk globule stages appeared during August–September indicating that yolk formation begins during late summer. The migratory nucleolus through maturation stages were detected during November–January. Post ovulatory follicles were present during November–March, and GSI values rapidly decreased in February. Results indicate that spawning occurs during November–February and peaks during December–January.     

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica

Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g^-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish^-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g^-1 BW week^-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3^rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.     

The first production of glass eel in captivity: fish reproductive physiology facilitates great progress in aquaculture

Weekly injections of salmon pituitary extracts (SPE) were administered to female Japanese eel, Anguilla japonica at a dose of 20 mg/fish. This induced vitellogenesis and caused oocytes to reach the migratory nucleus stage. Later, a majority of the females that received an injection of SPE at a priming dose, followed 24 h later by 17,20β-dihydroxy-4-pregnen-3-one (DHP), ovulated 15 to 18 h after the final injection. In cultivated males, repeated injections of human chorionic gonadotropin (hCG) at a dose of 1 IU/g BW/week induced spermatogenesis and spermiation. Since potassium ions were revealed to be an essential constituent for the maintenance of motility in the eel spermatozoa, artificial seminal plasma containing KCl was designed as a diluent of milt, and enabled the preservation of milt for several weeks in refrigeration. As a result, artificial fertilization performed immediately after ovulation with pre-diluted and stocked milt consistently resulted in the production of high-quality gametes. Recently, a slurry-type diet made from shark egg yolk has been found to be a suitable feed for captive-bred eel larvae. Although preleptocephalus larvae can be reared with this diet beyond the depletion of their yolk and oil droplet stores, it remains inadequate because larvae reared under this way cannot be raised to the following stage. Therefore, the diet was improved by supplements of krill hydrolysate, soybean peptide, vitamins and minerals. Larvae fed on this new diet have grown to 50 to 60 mm in total length (TL), and have begun to metamorphose into glass eels approximately 250 days after hatching.     

Diurnal rhythm of serum steroid hormone levels in the Japanese whiting,Sillago japonica,a daily-spawning teleost

Ovarian developmental stages and serum steroid hormone levels were examined at six different times of day (0100, 0600, 1000, 1300, 1600, 2000 h) in a marine teleost, the Japanese whiting Sillago japonica, which has an asynchronous-type ovary containing oocytes at various stages of development and spawns every day during a period ranging up to three months. The largest oocytes in the ovaries at the active vitellogenic or post-vitellogenic stages were found between 0100 and 1300 h. Oocyte maturation indicated by germinal vesicle breakdown (GVBD) occurred at 1600 h, and ovulated oocytes were observed in the ovaries collected at 2000 h. These processes were accompanied by a significant daily change in serum steroid hormone levels. The serum level of estradiol-17β showed a peak in fish with mature oocytes sampled at 1600 h. In these fish, the second-largest oocytes in the ovaries were at the initial stage of vigorous vitellogenesis, the secondary yolk stage. Therefore the highest level of serum estradiol-17β was considered to be due to the second-largest oocytes. Testosterone levels remained low and constant throughout the experimental period. The serum levels of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog) peaked at 1600 h at which time all fish had mature oocytes. These results indicate that the Japanese whiting possesses a diurnal rhythm of oocyte development including vitellogenesis, oocyte maturation and ovulation, and further suggest that daily cycles in oocyte growth and maturation which simultaneously take place in an ovary are regulated by diurnal secretions of estradiol-17β and the maturation-inducing steroid, 17α,20β-diOHprog.     

凡纳滨对虾卵子发生的超微结构

利用透射电镜研究凡纳滨对虾卵子发生过程中细胞内部结构的变化。结果显示,卵原细胞结构简单,代谢水平低,核孔稀少,通过核周池来完成核、质之间的物质交换。卵黄发生前晚期和卵黄发生初期的卵母细胞变化显著：核膜凹凸不平,核仁数量多,核孔密集,大量核仁外排物经核孔输送到卵质中;卵质中胞器极为丰富和发达,代谢活性极强。卵黄发生旺盛期是卵黄大量形成的阶段,卵质边缘还呈辐射状排列了一圈椭圆形皮质棒,细胞出现微吞饮活动并形成卵黄膜。卵黄发生晚期卵质中充满了粗大的卵黄粒和脂滴,胞器锐减。另外,探讨了卵细胞内部结构的变化和卯黄形成的关系以及皮质棒的来源与功能。     

Temporal progression in migratory status and sexual maturation in European silver eels during downstream migration

The onset of downstream migration of European eels is accompanied by a cessation of feeding and the start of sexual maturation which stresses the link between metabolism and sexual maturation, also suggesting an important role for exercise. Exercise has been tested with eels in swim tunnels and was found to stimulate the onset of sexual maturation. In this study, we have investigated the interplay between migration and maturation in the field during the downstream migration of female silver eels. Temporal changes in migratory status and sexual maturation among silver eels of the upstream Rhine River system over 3 months of the migration season (August, September and October) were determined in biometrical parameters, plasma 17β-estradiol and calcium levels, oocyte histology and gonadal fat levels. Furthermore, the ecological relevant parameters age as determined by otolithometry and health aspects indicated by haematocrit, haemoglobin and swim-bladder parasite load were measured. Silver eels were estimated to be 14 years old. A strong temporal progression in migratory stage was shown over the months of downstream migration. Catches probably represented a mix of reproductive migrants and feeding migrants of which the ratio increased over time. Furthermore, this study confirmed our hypothesis linking the migratory stage to early maturation as indicated by enlargement of the eyes, oocyte growth and fat deposition in the oocytes, exactly the same changes as found induced by exercise but not ruling out environmental influences. Migrants show extensive fat uptake by the oocytes, probably stimulated by the swimming exercise. In addition, at least 83% of the silver eels in this spawning run may have suffered from negative effects of swim-bladder parasites on their swimming performance.