大鼠早期三级卵泡中的卵母细胞能在联合培养系统中成批适当成熟

我们建立了由离体同步未分化颗粒细胞、动情前期垂体前叶和△-4雄烯二酮组成的“联合培养系统”。该联合培养系统可模拟正常生理状态下各相关细胞间的协同作用。更有效地诱发经DES刺激后所获得的大量大鼠早期三级卵泡中的卵母细胞同步达到适当成熟。从而获得符合正常生理标准的成熟卵球。使卵巢内大量卵母细胞得到充分利用。我们的实验表明,经DES刺激,每侧幼龄大鼠卵巢中平均可取得处在早期三级卵泡中的189枚卵母细胞。在联合培养系统中培养。78％的卵母细胞排出第一极体达到成熟。这些成熟的卵母细胞中88％可正常受精,93％卵裂,经体外培养96h,有59％的受精卵可发育到桑椹或囊胚。2-细胞期胚胎移植后可发育为健康个体。因此,该联合培养系统为研究大鼠卵母细胞成熟、受精和早胚发育的分子机制提供了非常有效的实验模型。     

金黄仓鼠卵子冷冻及在人精子受精能力检测中的应用

通过对比试验,筛选出含３．０％牛血清白蛋白的ＢＷＷ培养基和２·０ｍｏｌ／Ｌ最终浓度的二甲基 亚砜（ＤＭＳＯ）冷冻保护剂配制成冷冻液,将金黄仓鼠卵以０．３－１·０℃／ｍｉｎ的冷冻速率分别降温至 －４０℃和－８０℃,再浸入液氮中冷冻保存（３～３１天）。结果表明：冷冻降温速率以０·３℃－０·５℃／ｍｉｎ 为宜,降温到一８０℃浸入液氮的冻卵存活率（６３％）比－４０℃（４６％）的高。将解冻后存活卵去除 透明带,与人获能精子进行体外穿透试验,穿卵率达３４． ４％,与新鲜卵对照组（５０％）相比,无统 计学差异（Ｐ＞０·０５）;扫描电镜观察结果与压片光镜观察相吻合。     

Perivitelline space of marsupial oocytes: Extracellular matrix of the unfertilized oocyte and formation of a cortical granule envelope following the cortical reaction

The purpose of this study was to characterize the structure of the vestments surrounding unfertilized and cortical granule-reacted oocytes from a marsupial, the grey short-tailed opossum Monodelphis domestica and to determine if a cortical granule envelope (CGE) forms in the perivitelline space (PVS) following the cortical reaction. Unfertilized oocytes collected from mature ovarian follicles and oviducal oocytes that had undergone a cortical reaction were fixed for electron microscopy in the presence of ruthenium red which stabilizes extracellular matrices (ECM) and facilitates demonstration of a CGE. Unfertilized oocytes were surrounded by a zona pellucida and had a PVS which contained a thick ECM comprised of granules and filaments. This matrix appeared to attach to the oolemma and was structurally similar to matrices reported previously in the PVS of unfertilized oocytes from eutherian mammals and two other marsupials, the Virginia opossum and the fat-tailed dunnart. The cortex of unfertilized oocytes contained cortical granules which were absent in oocytes recovered from the oviducts of mated females. Oviducal oocytes which lacked cortical granules exhibited a new coat within the PVS between the zona pellucida and the tips of the oocyte microvilli. This coat, the CGE, appeared structurally similar to CGEs described previously around fertilized eutherian oocytes. The CGE of the grey short-tailed opossum is approximately 1 μm thick and is made up of numerous small dense granules. The coats of the opossum oocyte are compared to those present around other marsupial and eutherian oocytes. © 1995 Wiley-Liss, Inc.     

小鼠卵慢速和快速降温及玻璃化冻存的比较研究

本文用４种冷冻模式：慢速降温（ＳＬ）,超快速降温（Ｑ１）和防冻剂组成不同的玻璃化法（Ｒ－ＦＶＭ和ＭＶＭ）对昆明小鼠卵进行冷冻保存的比较研究,并用体外受精技术检测冷冻复苏卵的受精能力。ＳＬ和Ｒ－ＦＶＭ的存活率分别为５５．１±１．２和６５．９±７．９,显著高于Ｑ１（２４．２±７．３）和ＭＶＭ（４．５±２．４）其受精率分别为７２．８±１．８和７３．９±０．４显著高于Ｑ１（５８．６±１１．２）和ＭＶＭ（４１．５±８．５）,而与对照组（７７．５±３．９）相似。结果表明：１）慢速降温程序（ＳＬ）和同时含有渗透性和非渗透性防冻剂的玻璃化法（Ｆ—ＲＶＭ）较适宜昆明小鼠卵的冻存;２）同时考虑冻存过程中的冰晶损伤和渗透压损伤是获得较好冻存结果的关键。     

α3, β2, and β4 Form Heterotrimeric Neuronal Nicotinic Acetylcholine Receptors in Xenopus Oocytes

Abstract: One of the problems faced when using heterologous expression systems to study receptors is that the pharmacological and physiological properties of expressed receptors often differ from those of native receptors. In the case of neuronal nicotinic receptors, one or two subunit cDNAs are sufficient for expression of functional receptors in Xenopus oocytes. However, the stoichiometries of nicotinic receptors in neurons are not known and expression patterns of mRNA coding for different nicotinic receptor subunits often overlap. Consequently, one explanation for the discrepancy between properties of native versus heterologously expressed nicotinic receptors is that more than two types of subunit are necessary for correctly functioning receptors. The Xenopus oocyte expression system was used to test the hypothesis that more than two types of subunit can coassemble; specifically, can two different β subunits assemble with an α subunit forming a receptor with unique pharmacological properties? We expressed combinations of cDNA coding for α3, β2, and β4 subunits. β2 and β4, in pairwise combination with α3, are differentially sensitive to cytisine and neuronal bungarotoxin (nBTX). α3β4 receptors are activated by cytisine and are not blocked by low concentrations of nBTX; acetylcholine-evoked currents through α3β2 receptors are blocked by both cytisine and low concentrations of nBTX. Coinjection of cDNA coding for α3, β2, and β4 into oocytes resulted in receptors that were activated by cytisine and blocked by nBTX, thus demonstrating inclusion of both β2 and β4 subunits in functional receptors.     

低温保存的小鼠卵母细胞的体外授精研究

采用75分钟和150分钟两种精卵作用时间,对经6℃低温处理2小时和6小时的小鼠卵母细胞进行体外受精。精卵作用75分钟后,经6℃处理的卵子无一受精,而对照组的受精率为30.7％。作用150分钟后,低温处理2小时、6小时和对照组的受精率分别为62.0％,36.55％和76.0％。并对试验所出现的现象作了讨论。     

两种发育型鱼类卵母细胞成熟过程中促精核发育现象的研究

采用实验细胞学技术,比较研究了红鲤精核在雌核发育鱼类(银鲫)和两性融合发育鱼类(红鲤、彩鲫)卵母细胞成熟各阶段中的发育状况,结果表明:在两性融合发育鱼类中,当卵母细胞发育到胚泡破裂期吋,精核开始解凝发育,及至第一次成熟分裂中期后阶段,高度解凝的精核才开始原核化,相应地,在雌核发育银鲫中,精核也只有在与胚泡物质接触后,才能开始解凝,并且在三极纺锤体形成后期初步原核化,但与两性融合发育鱼类相比,精核的原核化程度明显降低。这些实验结果初步揭示,在两性融合和雌核发育鱼类卵中均可产生促精核解凝和原核化的两类因子(SNDFs和SPDFs),其中SNDFs的活性表达起始于卵母细胞胚泡破裂时期,而SPDFs活性则产生于第一次成熟分裂中期后或三极纺锤体形成后阶段,并且两性融合发育鱼类卵中的SPDFs活性远远高于雌核发育鱼类卵母细胞。     

Subcellular distribution of ribosomal proteins S6 and eL12

Summary The process of ribosome assembly in eukaryotes was studied by injecting tritium-labeled ribosomal proteins S6 and eL12 into oocytes of Xenopus laevis. The subcellular distribution of the two proteins was visualized by means of autoradiography in sections of oocytes. Protein S6 but not eL12 was found in the nucleus where it accumulated at the nucleoli. In the presence of actinomycin D the accumulation of S6 at the nucleoli was reduced. In-situ immunofluorescence studies indicated that S6 is located at the nucleoli and eL12 exclusively in the cytoplasm. It appears that S6 is involved in the early ribosomal assembly process at the nucleoli, whereas eL12 is restricted to the cytoplasm where it is incorporated into 60S ribosomal subunits in a late assembly step.     

Effect of “restricted ovulator” gene on uptake of yolk-precursor protein

Summary The bulk of the yolk proteins and lipoproteins constituting the yolks of mature oocytes in birds are synthesized by the liver and transported via the plasma to the oocytes where they are incorporated by micropinocytosis. Evidence is presented indicating that oocytes of hens possessing a mutation identified by Jones, Briles, and Schjeide as a restricted ovulator gene fail to incorporate normal amounts and proportions of low density lipoproteins, lipovitellin and possibly other proteins making up the bulk of the yolk material. Plasma albumin is taken into the yolks but the other proteins synthesized by the liver for deposition within the oocytes accumulate in the plasma, attaining very high levels. The possible nature of the lock preventing normal deposition of the excluded yolk proteins is discussed.     

In vitro induced pinocytotic activity by a juvenile hormone analogue in oocytes of Drosophila melanogaster

Summary Pinocytotic activity has been analyzed in Drosophila oocytes following either in vivo or in vitro exposure to horseradish peroxidase. The enzyme tracer gains access to the yolk spheres only when supplied to the oocyte in vivo. In oocytes cultured in vitro, peroxidase remains restricted to the residual coated vesicles and to the tubular profiles formed in excess in the cortical ooplasm.In an attempt to induce peroxidase uptake by oocytes cultured in vitro, various incubations were tested. Among these, hemolymph from both sexes is capable of promoting peroxidase uptake up to a level comparable to that detectable in vivo. On the other hand, fat body extracts fail to promote such cellular activity. Finally, the juvenile hormone analogue ZR-515 is shown to be the only factor required to promote pinocytotic activity under the experimental conditions tested. The observations are interpreted to indicate that vitellogenin has no inductive role on pinocytosis but simply acts by adhering to the forming coated vesicles which in turn are produced by the oolemma in response to the action of juvenile hormone.