Meiotic competence of mouse oocytes reconstructed by replacing the germinal vesicle with a male pronucleus and somatic nucleus

The present study examines the contribution of the nucleus to meiotic competence in mouse oocytes that were reconstructed using nuclear transfer. Three types of reconstructed oocytes were produced: MP‐GV, by transplanting the male pronucleus (MP) into germinal vesicle (GV) stage oocytes; 3T3‐GV, by transplanting the nucleus of a National Institute of Health (NIH) 3T3 cell into a GV stage oocyte; and 3T3‐MII, by transplanting the nucleus of an NIH 3T3 cell into a metaphase II (MII) stage oocyte. The fusion rates differed, but not significantly, in the MP‐GV, 3T3‐GV, and 3T3‐MII groups (77, 63, 56%, respectively). Then, meiotic competence was compared in MP‐GV, 3T3‐GV and non‐manipulated GV stage oocytes as a control. Nuclear envelope breakdown occurred in all the reconstructed oocytes, as well as the control ones. The percentage of first polar body extrusion differed between the MP‐GV (100%), 3T3‐GV (72%), and control (67%) groups. DNA staining with Hoechst 33342 revealed that in the MP‐GV‐group oocytes that had reached MII stage, the chromosomes were condensed and aligned in a regular array similar to the normal metaphase plate. By contrast, in 3T3‐GV group oocytes, the condensed chromosomes were irregularly scattered in the cytoplasm. These results suggest that the donor nucleus affects meiotic competence in reconstructed oocytes.     

Effects of steroid hormones on in vitro oocyte maturation in white sturgeon (Acipenser transmontanus)

The in vitro effects of several steroids on the maturation of intact white sturgeon (Acipenser transmontanus) ovarian follicles were investigated. At the highest concentration (1024 ng ml^–1 for the C21 steroids and 1139 ng ml^–1 for the C19 steroids), all of the C21 steroids tested, progesterone (P4), 17-hydroxyprogesterone (17OHP), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,(20,21-trihydroxy-4-pregnen-3-one 20-S), 11-deoxycortisol (S) and cortisol (F), as well as testosterone (T) induced germinal vesicle breakdown (GVBD) at 14 and 22 h. At 6 h, only P4 and 17,20-P induced maturation at the highest concentration (1024 ng ml^–1). At 14 and 22 h, 11-deoxycortisol was the most potent steroid inducer of GVBD followed by P4, 17OHP, 17,20-P, and 20-S. The steroid 11-hydroxytestosterone (11OHT) was completely ineffective at all concentrations and exposure times. The C21 steroids induced oocyte maturation at concentrations ranging from 4 to 1024 ng ml^–1, whereas T induced GVBD at 225 to 1139 ng ml^–1. Calculation of the mean effective concentration that induced 50% GVBD (EC50) from the 22 h incubations revealed the following order of potencies: S > P4 > 17OHP > 17,20-P > 20-S >> F > T. These bioassay results, together with previous findings on the endogenous production of steroids by ovarian follicles from gonadotropin-primed females, indicate that more than one steroid has a biological role in the resumption of meiosis in sturgeon oocytes and provides empirical evidence for P4, 17OHP, S, 20-S, and 17,20-P as maturation-inducing steroids in white sturgeon.     

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。     

四氯联苯对鸡胚生殖新月原始生殖细胞分布的影响

为了探索四氯联苯(2,2′,5,5′)对鸡胚生殖新月原始生殖细胞(PGCs)分布的影响,本试验将四氯联苯注入种蛋的胚盘(第X期)处,在38℃,相对湿度60%,每2h45度转蛋的条件下孵化至原条期,通过不同方法检测原条期生殖新月明区、暗区及血液循环中的PGCs,探讨四氯联苯对胚盘生殖新月区PGCs分布的影响。结果表明,四氯联苯明显降低了原条期明区和血液中PGSs的数量(P<0.01),但对暗区PGCs的数量影响不明显。这说明四氯联苯对血液中PGCs的影响是由于降低了生殖新月明区PGCs的缘故,和暗区的PGCs无相关性。     

Fluctuations in gonadotropin and ovarian steroids during the annual cycle and spawning of the common carp

The main objective of the paper is to describe the annual changes in hormones associated with reproduction in the female carp under the conditions prevailing in the Israeli fish culture. Fish were sampled monthly throughout 1984; gonadosomatic index (GSI) was calculated and the diameter of ovarian follicles was measured. Gonadotropin (GTH) content in the pituitary and the circulating GTH, estradiol, testosterone and 17, 20-dihydroxy-4-pregnen-3-one (17, 20-P) were determined by specific radioimmunoassays. GTH, estradiol and testosterone showed a bimodal annual pattern. The late summer peak was associated with initial vitellogenesis while the peak in spring occurred just before spawning, which took place in April-May. A resting phase in ovarian activity was noted in June and July. The levels of 17, 20-P were very low compared with those occurring during spawning induction. The paper summarizes a previous study by our laboratory on the changes in circulating hormones, as related to oocyte stages, in female carp induced to spawn by a GTH-calibrated pituitary extract. This study associates the short but prominent peak in 17, 20-P with the presence of follicles with maturing oocytes in the ovary. A correlation was found between the percentage of oocytes with eccentric germinal vesicle initially present in ovarian biopsies of females carp and their spawning success after hypophysation. The paper describes simple means to ensure successful induction of spawning in carp by utilizing a calibrated pituitary extract and prior selection of females that would respond to the induction treatment.     

日本鳗鲡排卵的人工诱导

对日本鳗鲡(Anguillajaponica)卵巢发育的时间与催产率的关系进行研究,对卵母细胞在体内胚泡破裂(GVBD)的发生过程、以及用17α-OHP和17α,20β-DHP诱导日本鳗鲡排卵的方式、时机和催产效果进行观察,结果表明,卵巢发育时间越长,催产率越低。造成催产率低的原因主要是由于日本鳗鲡在卵母细胞成熟后期,雌鳗个体对催产药物敏感性差异较大,催产时机难以掌握。通过采用适合日本鳗鲡卵母细胞的透明液观察GVBD的发生过程,并根据每个个体GVBD的发生速度,制订适宜的催产方案基本解决了上述难题。本实验采用CP、HCG、17α-OHP或17α,20β-DHP混合催产,使其平均催产率由35 4%分别提高到91 9%和93 0%。17α-OHP和17α,20β-DHP的催产剂量为5mg/(500g)和10mg/(500g)。在(21±0 5)℃水温条件下,催产效应时间分别为15～18h和13～16h。     

Presence of maturation-promoting factor in 17α,20β-dihydroxy-4-pregnen-3-one-induced oocytes of catfish,Clarias batrachus

Full-grown immature Clarias batrachus oocytes respond in vitro to exogenous 17,20-dihydroxy-4-preg-nen-3-one ( 17,20-DP) by undergoing germinal vesicle breakdown (GVBD). Cytosolic extract (CE) prepared from 17,20-DP-induced oocytes has been shown to produce similar effect when microinjected into unstimulated immature oocytes of the same fish. A dose of 50 nl is enough to cause 100% GVBD after 4 h. Maturation-promoting factor was investigated from 17,20-DP-induced, immature and cycloheximide treated oocytes incubated in presence of [³⁵S] methionine. When the proteins were extracted and analyzed on SDS-PAGE, two prominent bands corresponding to molecular weight 34- and 46-kDa were detected in the CE of mature oocytes. However, labelling of [³⁵S] methionine was observed mainly in the region of 46 kDa protein band indicating de novo synthesis of this particular protein during l7,20-DP-induction. Further, immunoblotting study by using rabbit anti-cyclin B₁ antibody has clearly demonstrated that the protein which is newly synthesized is highly homologous to Xenopus cyclin B₁ and goldfish cyclin B.     

补肾活血方对PC 0小鼠未成熟卵母细胞 体外核成熟的影响

〔摘要〕目的探讨补肾活血方对PCO小鼠未成熟卵母细胞体外核成熟的影响、方法(1)制备补肾活血方含药 血清;}2)制作PCO小鼠模型;}3)将PCO小鼠生发泡(germinal vesicle , GV)期卵母细胞分别在不同采血时间获取的 补肾活血方含药血清培养液中进行体外培养,观察补肾活血方含药血清对生发泡破裂(germinal vesicle break- down,GVBD)和第一极体(the first polar body,PBl)排出的时效关系结果(1)2 h和2.5 h药物血清组卵母细胞 GVBD的发生率高于正常血清组和对照组,培养后4 h差异有统计学意义(P<0.01) ; (2)2 h和2.5 h药物血清组卵 母细胞PB1的发生率高于正常血清组和对照组,培养后18 h差异有统计学意义(P<0.01)、结论2-2.5 h补肾活血 方含药血清对PCO小鼠未成熟卵母细胞体外核成熟具有明显促进作用、     

鸡包涵体肝炎的病理学研究

介绍了河北省中南部地区１９９４－－１９９５年间,８７个鸡场,３２００万只鸡发生鸡包涵体肝炎的系统病理学研究。结果表明,病鸡发生以肝脏严重变性、坏死,并在肝细胞核内形成核内包涵体的特征性病变。同时,机体免疫器官发生广泛损伤,产蛋鸡生殖器官发迟缓或停滞。