鸡促性腺激素释放激素对卵泡膜分泌雌二醇的影响

本实验选用性成熟的京白种蛋鸡,从同一产蛋顺序中取其发育不同阶段的各级卵泡(F_1～F_4),分离膜层,消化为单个的膜细胞,进行短期细胞培养,着重观察鸡促性腺激素释放激素(GnRH)对培养过程中膜细胞雌二醇分泌的影响.用放射免疫法测定细胞培养液中雌二醇的含量,得到以下结果:①未加外源激素处理的对照组细胞,随着卵泡从小到大的发育成熟过程,膜细胞雌二醇的分泌量逐渐降低;②适当剂量的鸡 GnRH-Ⅱ对各级卵泡膜细胞雌二醇的分泌均有促进作用,其中 F_4,F_3,F_2比 F_1更敏感;③加前体物(孕酮或雄烯二酮)之后,再加鸡 GnRH-Ⅱ比单加前体物或单加鸡 GnRH-Ⅱ,膜细胞雌二醇的分泌量增加更明显.实验结果提示,在体外细胞培养的条件下,GnRH-Ⅱ对膜细胞雌二醇的分泌不仅有促进作用,还可能促进雌二醇的合成。     

The combined effects of temperature and GnRHa treatment on the final stages of sexual maturation in Atlantic salmon (Salmo salar L.) females

Atlantic salmon (Salmo salar L.) females (2 SW), maturing for the first time, were reared under one of three temperature regimes (high: 14.3 ± 0.5°C; natural: 10.6 ± 1.0°C; and cold: 6.9 ± 1.0°C) in combination with one of two experimental treatments; an injection of GnRH analogue (GnRHa) contained in biodegradable microspheres, or a sham injection (microspheres only). The six experimental groups were then reared under simulated natural photoperiod for 4 weeks. Blood samples were drawn for analysis of plasma steroid levels and the fish were inspected for ovulation weekly. Batches of stripped eggs were incubated in triplicate incubators in raceways until the eyed stage. Treatment with GnRHa resulted in a substantial advancement and synchronization of ovulation at all temperatures, while exposure to cold water also appeared to advance ovulation slightly. While 75% (warm and cold) to 90% (natural) of GnRHa fish ovulated during the 4-week trial, only 30% of sham-treated females exposed to cold water, and none of the sham-treated fish held at higher temperatures, ovulated during this period. Survival rates of embryos to the eyed-stage were significantly higher for broodstock exposed to cold water. Plasma levels of testosterone (T), 17β-oestradiol (E₂), and 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) were all significantly affected by treatment with GnRHa and, to a lesser extent, temperature. The efficiency of GnRHa in counteracting the negative effects of high temperature on ovulation and the associated changes in circulating sex steroids suggest that temperature inhibition operates at least in part at the brain or pituitary.     

奥利亚罗非鱼促性腺激素释放激素cDNA的克隆及原核表达

促性腺激素释放激素(gonadotropin-releasing hormone,GnRH)是下丘脑分泌产生的神经激素,对脊椎动物生殖的调控起重要作用。本研究通过RT-PCR方法从奥利亚罗非鱼丘脑中扩增出长约400 bp的目的序列GnRH,并将其克隆到T载体中,经酶切鉴定、序列测定分析后表明其与尼罗罗非鱼和乌颊海鲷具有较高的同源性,处于同一个进化分支上;而与日本青、金鱼、拟鲤、壁虎等的同源性较低。将此cDNA片段定向克隆到表达载体pMAL-c2x中构建重组表达质粒pMAL-GnRH,转化到大肠杆菌TB1中,0.3M IPTG诱导4 h后成功表达出与预期大小相符的约56 kD的融合蛋白。表明大肠杆菌表达系统可以成功地体外表达奥利亚罗非鱼GnRH,为进一步制备抗体了解其免疫调节作用奠定基础。     

促性腺激素释放激素在不同品种母牛垂体中的免疫组化定位

为了掌握促性腺激素释放激素（GnRH）及其受体在不同品种母牛垂体中的变化规律,采用免疫组化SP染色法分别对广西沼泽型水牛、摩拉杂交水牛、荷斯坦奶牛、广西本地黄牛垂体中GnRH的分布表达进行对比研究。结果表明,不同品种母牛垂体中均发现有呈棕黄色或黄褐色的GnRH阳性物质存在,经Image-Pro Plus 6.0软件统计发现,摩拉杂交母水牛的GnRH免疫阳性细胞较其他3个品种的大（P〈0.05）,而平均光密度值介于荷斯坦母奶牛和广西本地母黄牛之间（P〉0.05）。在神经垂体中仅发现GnRH免疫阳性纤维,而未发现GnRH免疫阳性细胞;在腺垂体远侧部的细胞均呈GnRH免疫反应阳性,阳性物质主要分布在腺垂体远侧部嫌色细胞和嗜色细胞胞质内。说明GnRH对腺垂体激素分泌的调节可能是直接通过神经调节,而神经垂体可能只是GnRH调节过程的通路。     

虹鳟排卵前后血清中性类固醇激素浓度变化的研究

对虹鳟(Salmo gairdneri)排卵前后血清17β-雌二醇(17β-E_2),睾酮(T)和17α-羟-20β-双氢孕酮(17α20βP)的含量进行了测定。17β-E_2从排卵前15天的18.1ng/ml急骤下降到排卵前3—6天的2ng/ml,并继续下降,至排卵时仅为0.9ng/ml。睾酮在虹鳟雌鱼血清中的含量很高,血清浓度从排卵前9天的峰值水平143.3ng/ml缓慢下降,排卵时为24.3ng/ml。17α20βP的血清浓度变化明显,排卵前15天,该激素浓度接近于基线水平或甚至低得不能被检测,排卵前9天开始迅速上升,到排卵前3天达到峰值350.6ng/ml,排卵时为302.2ng/ml。本研究进一步证实了17α20βP在鲑鳟鱼类卵母细胞最后成熟过程中的生理作用,是一种诱发卵母细胞成熟的类固醇激素。临排卵前,血清17β-E_2浓度的下降,可能调节了17α20βP大量分泌的时间;17α20βP的大量分泌是卵母细胞达到最后成熟和排卵不可缺少的一环。注射合成的大麻哈鱼(Oncorhynchus keta)促性腺激素释放激素(s-GnRH)及其类似物(s-GnRH-A),诱发虹鳟血清类固醇激素17β-E_2,睾酮和17α20βP的变化趋势与自然排卵时的变化相类似。经注射药物诱发排卵的鱼,较对照组提早一周排卵,而且排卵较同步和集中。     

The maturation of the salmon GnRH system and its regulation by gonadal steroids in masu salmon

Pituitary gonadotropin (GTH) secreting cells and brain gonadotropin-releasing hormone (GnRH) secreting neurons are known to be subjected to feedback control by gonadal steroid in teleosts. In masu salmon, Oncorhynchus masou, salmon GnRH (sGnRH) neurons in the ventral telencephalon (VT) and the preoptic area (POA) are involved in the control of GTH cells because sGnRH synthesis in these areas is activated with gonadal maturation. In this study, we attempted to clarify mechanisms of feedback control of sGnRH neurons by gonadal steroids. We examined the effects of 17-methyltestosterone (MT) on sGnRH synthesis in yearling and 2-year-old female fish (which were immature during experimentation in May), and the effects of castration on sGnRH synthesis in underyearling precocious male fish in August. sGnRH synthesis in the POA, but not in the VT, was increased by MT administration in 2-year-old females only, indicating higher sensitivity to MT in the preoptic sGnRH neurons. Castration increased sGnRH synthesis in the VT but not in the POA. These results suggest that sGnRH neurons in the VT and those in the POA are differentially regulated by gonadal steroids.     

The duality of teleost gonadotropins

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.     

Differential distribution and response to experimental sexual maturation of two forms of brain gonadotropin-releasing hormone (GnRH) in the European eel,Anguilla anguilla

Using specific radioimmunoassays for the two GnRH molecular forms present in the European eel, Anguilla anguilla, (mGnRH and cGnRH II), we compared their distributions in the pituitary and different parts of the brain of female silver eels, as well as the modifications of their levels in experimentally matured female eels (treated with carp pituitary extract). In control eels, mGnRH levels were higher than cGnRH II levels in the pituitary, olfactory lobes and telencephalon, di- and mesencephalon, while the opposite was found in the posterior part of the brain (met- and myelencephalon). Experimental sexual maturation of the gonads significantly increased mGnRH levels in the pituitary and anterior parts of the brain; such a positive effect was not observed on the low cGnRH II levels, which were, in contrast, reduced. These data indicate that the positive feedback of gonadal hormones on GnRH, that we previously demonstrated, would specifically affect the mGnRH form. The differential distribution and control of mGnRH and cGnRH II suggest that these two forms have different physiological roles in the eel. The large increase in mGnRH during sexual maturation suggests the prime implication of this form in the neuroendocrine control of reproduction.     

The GnRH systems in the brain and pituitary of normal and hCG treated European silver eels

The distribution of immunoreactive GnRH was studied in the brain and pituitary gland of normal and human chorionic gonadotrophin (hCG) injected silver eels. It was found that the general organization of GnRH systems in this species is similar to that reported in other teleosts. Cell bodies were present in the olfactory bulbs, ventral telencephalon, periventricular hypothalamus and dorsal tegmentum. No positive perikarya could be detected in the preoptic region. Only scarce fibers were observed in the proximal neurohypophysis. Treatment with hCG does not modify the distribution of GnRH but it increases the density of positive structures, in particular at the level of the pituitary. The results are discussed in relation with the present status of knowledge of the mechanisms underlying the blockage of sexual maturation in the European eel at the silver stage.     

Pesticide—induced reproductive dysfunction in Indian fishes

Numerous studies are available reporting the effects of pesticides on reproductive activity in Indian fishes. The majority of these reports deals with histopathological changes in gonads and endocrine glands involved in the regulation of reproduction following treatment with different pesticides. Pesticides are reported to cause degenerative changes in gonads and arrest gametogenic processes either by acting directly on the gonads or by interfering with the secretory activity of the hypothalamo-hypophyseal-gonadal/thyroid axis that regulates various reproductive events. Secretion of hormones such as gonadotropin-releasing hormone (GnRH), gonadotropin, growth hormone, adrenocorticotropic hormone (ACTH), testosterone, estrogens, 17,20β-dihydroxyprogesterone and thyroid hormones are in general lowered, leading to cessation of gametogenesis, vitellogenesis, oocyte maturation, ovulation, spermiation, etc. Adverse effects of pesticides have also been demonstrated on fecundity, fertilization, hatching, and postembryonic development. The effects are highly variable and depend on the nature, dose, and mode of application of the pesticides.