多刺裸腹溞孤雌溞的卵子发生

采用光镜和透射电镜技术观察多刺裸腹溞（Moina macrocopa）孤雌溞卵子的发生过程。根据卵母细胞细胞核的形态、卵黄的合成、积累情况及核物质的形态变化等观察结果,将卵子发生分为3个时期：卵原细胞期、卵母细胞期和成熟卵母细胞期。卵原细胞核大,胞质层薄,有少量线粒体分布,核物质浓缩为一团;卵母细胞发育的早期和中期细胞体积增大,细胞质层增厚,线粒体迅速增多,细胞核变圆,核物质分散成多块;后期卵母细胞胞质中开始出现大量卵黄颗粒和少量脂滴,细胞核边缘波浪化,线粒体空泡化,其中卵黄合成初期,核物质重新聚集在细胞核中央,在卵黄合成中期又分散开来;成熟卵母细胞质中充满卵黄颗粒,呈强嗜酸性,细胞核的形态呈不规则状,核物质分散。多刺裸腹溞生殖腺的外部形态随着生殖周期的变化而不同。在孤雌溞的整个生活史中生殖腺内卵子的发育经历了“同步一不同步一局部同步”的发育模式。发现1龄幼溞生殖腺中不存在卵原细胞,成龄溞的生殖腺有多个发育时期的生殖细胞并存。同时在亚显微水平上发现卵黄主要由线粒体演变而来,也通过吞饮作用吸收成熟的卵黄颗粒,内源与外源性共同作用形成卵黄。[中国水产科学,2006,13（4）：547—554]     

二硫苏糖醇对刺参卵母细胞体外成熟的影响

用海星鞘神经提取液和二硫苏糖醇对刺参卵母细胞进行了体外人工促熟研究。结果显示,二硫苏糖醇对刺参卵母细胞具有明显的促熟作用。刺参卵母细胞包被在滤泡中发育,卵母细胞通过动物极的卵突起连接到单层细胞构成滤泡膜上,充分发育的卵母细胞处于生发泡期,染色质凝集在核仁周围,处于生发泡期的卵母细胞在海水中不会自发成熟。用二硫苏糖醇处理充分发育的卵母细胞20min,卵母细胞的生发泡开始移动,并锚定在卵母细胞的动物极,继而出现生发泡破裂。此后,减数分裂重新启动,第一、二极体分别排出。但海星鞘神经提取液对刺参卵母细胞没有促熟作用。实验表明,二硫苏糖醇浓度在10-5～10-1mol/L范围内,均可促使刺参卵母细胞发生生发泡破裂,10-2mol/L时诱导卵母细胞成熟率可达90%以上。     

Nuclear transfer in loach (Paramisgurnus dabryanus Sauvage) by cell-to-cell electrofusion

To study nuclear transfer in the loach (Paramisgurnus dabryanus Sauvage), blastula and gastrula cells were fused with UV-inactivated oocytes by cell-to-cell electrofusion. To facilitate nuclear transfer, blastula and gastrula cells were cultured or incubated at 4 °C in different solutions. TC-199 medium supplemented with 20% calf serum was the best culture solution, and effectively retained the totipotence of blastula or gastrula cells for up to 10 days. It was found that gastrula cells incubated at 4 °C had the same totipotence as blastula cells. The optimal UV dosage for inactivation of the oocyte chromatin was 180–240 mJ cm⁻². Electrofusion was carried out in a cone-shaped fusion chamber, which permitted the recipient oocyte and the donor blastula cell to contact one another. The electrofusion procedure resulted in a 10% success rate of normal-appearing fish. Genetic analysis indicated that the nuclear material originated from the donor cell (blastomere) and the oocyte pronucleus did not take part in development.     

去眼柄后中华绒螯蟹法呢酸甲基转移酶表达和卵巢发育

为了解甲基法呢酯(methyl farnesoate,MF)在中华绒螯蟹(Eriocheir sinensis)卵巢发育中的调控作用,采用离体方法研究了中华绒螯蟹大颚器(mandibular organ,MO)以及X-器官窦腺复合体(X-organ-sinus gland,XO-SG)对卵巢发育的作用。将MO与离体卵母细胞共培养作为实验组1,将MO和XO-SG与离体卵母细胞共培养作为实验组2,仅添加培养液的卵母细胞作为对照组。实验发现,蟹去眼柄(eyestalk ablation,ESA)后的第1、3、6、14天,同对照组相比,MO对其卵母细胞的发育均有极显著促进作用。ESA处理第6天,MO对卵母细胞发育的促进作用最强。XO-SG能逆转ESA处理后MO对卵巢发育的促进作用(P<0.01)。荧光定量PCR技术检测结果显示,ESA处理后,MO中法呢酸甲基转移酶(farnesoic acid O-methyltransferase,FAMeT)mRNA相对表达量上升。同未去眼柄的对照组相比,第1、3天FAMeT mRNA表达丰度无明显变化(P>0.05),第6、14天表达丰度提高至265%左右(P<0.01)。结果表明,中华绒螯蟹MO功能性物质能够促进卵母细胞发育,且ESA处理后第6天MO生物效应最强。MO中FAMeT mRNA的合成受XO-SG的抑制调节。在离体条件下,中华绒螯蟹XO-SG能够抑制卵母细胞的发育。     

Oocyte and embryo quality in Crassostrea gigas (Portuguese strain) during a spawning period in Algarve,South Portugal

Egg quality is one of the most important factors determining larval viability. The study of oocytes released by `wild' broodstock will contribute to the definition of quality criteria applied to hatcheries as well as being an indicator of the oyster reproduction potential and aquaculture ongrowing systems. The aim of this study was to determine the relationship between condition of Crassostrea gigas spp. angulata (Portuguese strain) from `wild' broodstock (condition index, gonadal maturity and number of oocyte released), oocyte biochemistry and hatching rate of eggs during the spring/summer of 1995. Stages of gonadal maturity and condition index indicated that natural spawning of this species occurred between April and August with the highest spawning intensity occurring in June. Analyses demonstrated that C. gigas oocyte organic matter consist of 44–74 % proteins, 16–38 % lipids and 7–12 % carbohydrates. A significant relationship was observed between some biochemical parameters – organic matter and lipid content – and broodstock condition index and hatching rate. It seems that these two oocyte parameters (organic matter and total lipids) could be used to define quality of oocyte and thus larval viability. The oocyte protein content was also found to be associated with broodstock and larval quality. In summary, these data are a contribution to the knowledge of the reproductive cycle of oysters in Europe and may be useful in improving hatchery management of this species.     

Histological examination of final oocyte maturation and atresia in wild and domesticated Penaeus monodon (Fabricius) broodstock

Oocyte maturation and gonadosomatic index (GSI) of eyestalk ablated Penaeus monodon females collected from the wild and from two first‐generation domesticated lines were assessed. Frequency and diameter of the different oocytes, and the intensity of oocyte atresia, were compared among groups through histological assessments of the sections of the middle ovarian lobe. Digitized images from ovary sections were used to record the frequency and diameter of different oocyte types. Spawning performance of the three groups were expressed in terms of the percentage of females that spawned at least once (productive females), time from eyestalk ablation to first spawning (latency period) and the number of spawnings per female stocked. Final ovarian maturation was attained in all groups, as indicated by the presence of mature oocytes with cortical rods (cortical oocytes), dark‐green ovarian colour and high GSI values (5.83–6.86%). However, domesticated groups presented significant larger immature oocyte types (previtellogenic and yolky oocytes) and smaller cortical oocytes compared with wild females, indicating a reduced vitellogenic activity during final oocyte maturation. Additionally, the frequency of atresia was comparatively higher for both domesticated groups, which could be related to their inferior spawning performance. The implications of these results on the reproductive potential and development of domesticated P. monodon are discussed.     

Induced spawning of hatchery-raised Brazilian catfish, cachara Pseudoplatystoma fasciatum (Linnaeus, 1766)

In the months of January 2001 and 2002, female cachara Pseudoplatystoma fasciatum were selected during their first and second gonadal maturation (2 years and 7 months old and 3 years and 7 months old, respectively) with an of oocyte diameter of 937.5 μm (82.5% with central nuclei and 17.5% with peripheral nuclei). Nine females in first maturation received two doses of carp pituitary extract (CPE), 0.5 mg/kg and 5.0 mg/kg; seven received two doses of human chorionic gonadotropin (hCG), 5 and 10 IU/g; five received doses of 0.5 CPE mg/kg and 5 hCG IU/g (CPE+hCG); and four received 0.9% saline (saline). Nine females from CPE and seven from hCG presented oocytes with the same diameter at the moment of oocyte release (100% with germinal vesicle breakdown and fertilization rate of 53.44±18.3 and 54.81±11.8%; larvae number of 165,330±94.1 and 158,570±20.6, respectively). The five females from CPE+hCG did not respond to the hormonal treatment. The four females from the saline group did not ovulate. In January 2002, 6 of 15 selected females that were going through the second reproductive cycle received CPE (five received hCG and four received saline), showing oocyte diameters similar to the ones in the first maturation. At stripping, CPE females had an oocyte diameter of 1062.5 μm (the hCG females had oocyte diameters ranging from 937.5 to 1125.0 μm; fertilization rates of 56.08±30.9 and 81.90±17.3%; 364,547±244 and 633,129±190, larvae, respectively). The fertilization rates and larvae number were higher in the second gonad maturation, both for CPE and hCG.     

性成熟雌性牙鲆(Paralichthys olivaceus)新型膜孕激素受体(mPRL)的定性定量表达分析

在牙鲆(Paralichthys olivaceus)繁殖周期,新型膜孕激素受体基因(mPR-Like,mPRL)在脑-垂体-性腺轴的表达变化规律与性腺发育成熟密切相关.为进一步研究牙鲆mPRL作用机制,本研究对其mRNA和蛋白的表达特征进行了分析.应用实时定量PCR方法分析mPRL mRNA在卵子形成过程中的时序表达,发现牙鲆mPRL mRNA相对表达量的最高值出现在性成熟阶段卵巢的Ⅴ时相卵母细胞.原位杂交分析mPRL mRNA在繁殖相关组织中的细胞学定位,发现mPRL mRNA主要分布在牙鲆性成熟阶段卵巢的卵母细胞膜上;在脑组织的神经元附近区域mPRL mRNA阳性信号也较强.制备牙鲆mPRL的多克隆抗体,采用Western blotting方法检测牙鲆mPRL蛋白在不同组织的表达特性,发现mPRL蛋白表达量在卵巢和脑组织中相对较高,在肝脏、头肾、肾脏中表达量相对较少.免疫组化结果显示,牙鲆mPRL蛋白在卵巢和脑组织的细胞学定位与mPRL mRNA定位一致.牙鲆mPRL在繁殖相关组织的表达特征说明其参与卵母细胞成熟的过程,并通过内分泌方式参与牙鲆的繁殖调控.     

贻贝雌性生殖系统的组织学研究

从组织学方面研究了贻贝雌性生殖系统的构造。卵巢壁薄、由外膜和内生殖上皮构成。外膜主要由结缔组绢,意志支上皮和薄层肌肉构成。由其产生的许多延伸物伸入卵巢中把卵巢分成一个个不规则小区。内生死上皮不断增殖平生生殖带,其中含卵原细胞阁阶段雌性生殖细胞和滤泡细胞。卵原细胞较小,核圆形,核仁不明显。初级卵母细胞体积增加,核呈生发泡状,核仁１个,明显。次级卵母细胞卵圆形、细胞体积更大,核仁１个,十分明显。当卵母     

孕酮受体膜组分1基因在性成熟雌性半滑舌鳎(Cynoglossus semilaevis)的组织学定位定量分析

运用Western blotting、免疫组化和原位杂交方法检测性成熟半滑舌鳎(Cynoglossus semilaevis)孕酮受体膜组分1(Progesterone receptor membrane component 1,PGRMC1)蛋白和mRNA在不同组织的分布和表达特征.原位杂交结果发现,PGRMC1 mRNA主要分布在成熟的卵母细胞膜上,在脑组织神经元和分散的垂体细胞中也有表达.利用制备的半滑舌鳎PGRMC1多克隆抗体,对不同组织中的PGRMC1蛋白表达量进行Western blotting检测,发现在半滑舌鳎卵巢、脑、肝脏中PGRMC1蛋白表达量相对较高,在垂体、头肾、肾也有表达,但表达量相对较少.免疫组化结果表明,半滑舌鳎PGRMC1蛋白在成熟的卵母细胞膜上显著表达,进一步证明PGRMC1为卵膜上的受体基因,推测其主要在卵膜上行使相关的生理功能.研究结果为探究PGRMC1在半滑舌鳎卵母细胞成熟过程中的生理功能提供了重要参考.     

促性腺激素调控半滑舌鳎(Cynoglossus semilaevis)卵母细胞孕酮受体膜组分1的表达特征

采用qRT-PCR方法分析性成熟半滑舌鳎(Cynoglossus semilaevis)不同发育时期卵巢卵母细胞的孕酮受体膜组分1 (PGRMC1)mRNA的表达,研究发现,在发育Ⅳ期的卵巢,处于第Ⅳ时相的卵母细胞PGRMC1 mRNA表达量最高(P＜0.05);在发育Ⅴ期的卵巢,成熟期卵母细胞的PGRMC1mRNA表达量最高(P＜0.05).采用不同浓度促性腺激素(HCG)处理卵巢发育Ⅴ期半滑舌鳎不同时相的卵母细胞,并通过qRT-PCR和Western blotting技术对其表达量变化进行检测.结果显示,20IU/mlHCG对PGRMC1 mRNA和蛋白表达的调控作用比10 IU/ml HCG作用更明显,表明PGRMC1 mRNA和蛋白表达对HCG调控作用存在剂量依存关系.HCG对半滑舌鳎不同时相的卵母细胞的调控作用效果不同,对第Ⅴ时相卵母细胞作用最明显(P＜0.05),表明PGRMC1主要在卵母细胞成熟阶段发挥作用.PGRMC1对HCG调控作用的正向应答效应预示其参与了卵母细胞成熟调控,为进一步探讨PGRMC1在半滑舌鳎繁殖过程的功能提供重要基础资料.     

半滑舌鳎(Cynoglossus semilaevis)新型膜孕激素受体基因(mPRL)在卵母细胞成熟过程中的表达特征

为进一步提高半滑舌鳎(Cynoglossus semilaevis)人工育苗技术的水平,对半滑舌鳎新型膜孕激素受体(mPR-like,mPRL)基因的表达特征和作用机制进行了研究.应用实时定量PCR方法分析mPRL mRNA在卵子形成过程中的时序表达,发现半滑舌鳎mPRL mRNA相对表达量的最高值出现在性成熟阶段卵巢的Ⅴ时相卵母细胞.原位杂交分析mPRL mRNA在繁殖相关组织的细胞学定位,发现mPRL mRNA分布在半滑舌鳎性成熟阶段卵巢的卵母细胞膜上;在脑的神经元和垂体内分散的细胞中,mPRL mRNA阳性信号较强.制备半滑舌鳎mPRL的多克隆抗体,采用Westem blotting方法检测半滑舌鳎mPRL蛋白在不同组织的表达特征,发现mPRL蛋白的表达量在卵巢、脑、垂体中相对较高,在肝脏、头肾、肾脏中表达量相对较少.免疫组化结果显示,半滑舌鳎mPRL蛋白在卵巢、脑和垂体的细胞学定位与mPRL mRNA定位一致.应用实时定量PCR和Westem blotting方法检测促性腺激素调控下半滑舌鳎不同时相卵母细胞mPRL基因mRNA和蛋白的表达变化,结果显示,促性腺激素对半滑舌鳎卵母细胞中mPRL基因mRNA和蛋白表达都有一定的上调作用,特别是对Ⅴ时相卵母细胞中mPRL mRNA和蛋白的表达量提升明显;发现表达量与促性腺激素调控作用具有剂量依存关系.半滑舌鳎mPRL在繁殖相关组织的表达特征表明其通过脑-垂体-卵巢轴参与繁殖调控,同时也揭示了mPRL介导卵母细胞成熟机制.     

Stimulation of spermiation and induction of ovulation in pike (Esox lucius)

Mature northern pike were given various hormonal treatments in March or April in order to stimulate spermiation or to induce ovulation. In males the total amount of sperm collected after treatment increased, in comparison with saline-injected males, by 3–11 times with partially purified salmon gonadotropin (PPSG-activity: half of the highly purified s-GTH; injected at doses of between 5 and 100 μg/kg body weight); 3–6 times with crude carp pituitary extract (0.5–3 mg/kg body weight); and 3–7 times with fresh pike pituitaries (14 and 1.2 mg wet weight/kg body weight). The sperm obtained after hormonal treatment was of good quality. Intracardiac injection of superactive LRH analogue had no effect. In females, PPSG induced 90 and 100% ovulation at the doses of 50 and 25 μg/kg body weight. Dried salmon pituitaries (2.5 mg/kg, equivalent to 50 μg of PPSG) gave 25% ovulation; at 10 mg/kg, 25% complete ovulation was again recorded, but in addition 70% of the females showed oocyte maturation and partial ovulation. Similarly, dried carp pituitary (3 mg/kg) induced only oocyte maturation but no ovulation. The oocytes obtained after hormonal treatment were in general fertile. Intraperitoneal injection of LRH in an emulsified form induced neither oocyte maturation nor ovulation. The lack of effect of LRH analogue is discussed and shows that the use of this compound as a substitute for pituitary preparation is not very promising.     

Efficacy of human chorionic gonadotropin (hCG) and crude pituitary extract of fish and frog in oocyte maturation and ovulation in African catfish, Clarias gariepinus Burchell, 1822 and Clarias anguillaris L., 1762

Abstract. The African catfish, Clarias gariepinus Burchell and Clarias anguillaris L., were induced to ovulate in aquaria by the administration of human chorionic gonadotropin (hCG) and pituitary extracts of Clarias albopunctatus (Nichols & Lamonte) and frog, Rana elegans. For final maturation and ovulation in both species of Clarias , the total dosage of each hormone was inversely proportional to the initial oocyte diameters of the recipient females. The time of ovulation after the last injection (latent period) was significantly shorter (11–13h) in females with large oocyte diameters (>920μm) than in those whose mean oocyte diameters were less than 920 μm (13–16h) ( P < 0·05). The mean dosage of each test hormone required to induce ovulation was lower in females with large oocyte diameters than in those with smaller oocyte diameters (<920μm). The mean percentage hatch did not depend significantly on the nature of the treatment ( P > 0μ05) and was about 86%. Clarias albopunclatus and R. elegans , which are economically unimportant and abundant in Anambra River basin, Nigeria, could be used as effective sources of hypophyseal factors in Clarias breeding programmes.     

Influence of size, age and maternal condition on the oocyte dry weight of Argentine hake (Merluccius hubbsi)

Effects of maternal age–size and condition on oocyte dry weight were analysed for Merluccius hubbsi. Samples of mature females were collected in the Patagonian spawning area during 10 research surveys carried out between 2000 and 2005. Dry weight of 50 hydrated oocytes ranged from 0.80 to 1.90 mg. Relationships of this variable with total length and age were best described by a logarithmic model during the spawning peak. At the beginning and at the end of the reproductive season no significant relationship between these variables was observed, but a decrease in egg dry weight was detected during the spawning period. In general, young spawners (≤5-year-old) showed more variability in egg dry weight, which was associated with a reproductive season shorter than that of old females. Relative condition factor did not shows a significant relationship with oocyte dry weight, and mean values of this index were similar for different months of the spawning seasons analysed.     

Early development of the silver catfish Rhamdia quelen (Quoy & Gaimard, 1824) (Pisces:Heptapteridae) from the São Francisco River Basin, Brazil

The silver catfish, Rhamdia quelen , is endemic to North, Central and South America with high aquaculture potential and wide acceptance in the market. Breeder fish were subjected to induced reproduction through hypophysation using a crude common carp pituitary extract. Egg characteristics, oocyte surface ultrastructure and histology of larval ontogenesis until whole yolk resorption were described for the first time for this species. Oocytes and semen were obtained by manual extrusion, and fertilization was conducted using the dry method. After fertilization, eggs were kept in incubators at 24 °C. The embryonic development was monitored using a stereomicroscope every 10 min until hatching. To analyse the larval development, larvae samples were collected from incubators daily until the fifth day, fixed in Bouin's fluid and subjected to routine histological techniques. The oocyte extrusion occurred 8 h after the second hormone dose at 26 °C. The oocytes were spherical, non-adhesive and yellow, with a diameter of 1471.75±47.63 μm. Scanning electron microscopy revealed a thin jelly coat covering the zona radiata in the animal pole around the micropyle. The blastopore closure occurred within 8 h after fertilization, and the fertilization rate was 79.9±5.2% at 24 °C. Embryonic development was completed within 25 h 30 min after fertilization. The complete resorption of the yolk and the formation of the digestive system organs and the mouth opening occurred on the fifth day, indicating a need for exogenous feeding. The results of this study provide information important for improvement in R. quelen culture and management.     

北太平洋长鳍金枪鱼卵巢的发育特征

根据2013年10月–2014年2月在北太平洋海域(29°08′~41°08′N,163°50′~144°19′W)采集的364尾长鳍金枪鱼的卵巢样本,利用组织学分析,详细描述了长鳍金枪鱼卵巢、卵细胞的发育阶段。结果显示,北太平洋长鳍金枪鱼卵巢内同时存在不同时相的卵细胞,为分批产卵类型;组织学上,长鳍金枪鱼的卵细胞发育过程分为6个时相,卵巢发育过程分为6个时期;卵巢成熟指数在成熟期为Ⅰ~Ⅴ期时逐渐增大,在Ⅵ期时减小;北太平洋长鳍金枪鱼产卵高峰为12月中旬和1月初,其卵巢成熟指数随纬度的升高呈递减趋势,随经度变化规律不明显。研究表明,通过对北太平洋长鳍金枪鱼卵巢的发育特征的分析与探讨,可为北太平洋长鳍金枪鱼的资源状况评估及渔业可持续发展提供生物学信息。     

Expression of a glycoprotein hormone receptor gene in the ovary of the bullseye puffer (Sphoeroides annulatus)

A fragment of a glycoprotein hormone receptor (GpHR) gene was isolated from the ovary of the bullseye puffer. This cDNA fragment showed similarity to the thyrotropin (TSH) and gonadotropin (LH and FSH) receptors. GpHR mRNA expression in the ovary gradually increased during oocyte maturation.