Distribution of protein disulfide isomerase during maturation of pig oocytes

Oocyte maturation in mammals is characterized by a dramatic reorganization of the endoplasmic reticulum (ER). In mice, the ER forms accumulations in the germinal vesicle (GV) stage and distinctive cortical clusters in metaphase II (MII) of the oocyte. Multiple evidence suggests that this ER distribution is important in preparing the oocyte for Ca²⁺ oscillations, which trigger oocyte activation at fertilization. In this study, we investigated the time course and illustrated the possible functional role of ER distribution during maturation of porcine oocytes by immunostaining with protein disulfide isomerase (PDI). PDI forms clusters in the cytoplasm of oocytes. After immunostaining, PDI clusters were identified throughout the cytoplasm from the GV to metaphase I (MI) stage; however, at the MII stage, the PDI formed large clusters (1–2 µm) in the animal pole around the first polar body. PDI distribution was prevented by bacitracin, a PDI inhibitor. Our experiments indicated that, during porcine oocyte maturation, PDI undergoes a dramatic reorganization. This characteristic distribution is different from that in the mouse oocyte. Moreover, our study suggested that formation of PDI clusters in the animal pole is a specific characteristic of matured porcine oocytes.     

‘金二十世纪’梨果实ABA代谢酶基因克隆及其表达分析

为了解ABA对‘金二十世纪’梨果实成熟的调控作用,本研究采用高效液相色谱法(HPLC)分析测定了果实成熟过程中果皮、果肉和种子内源ABA含量变化,采用RT-PCR和RACE技术从该梨果实中克隆得到了2个ABA关键合成酶(NCED)和1个关键降解酶(CYP707A)基因,分别命名为PpNCED1、PpNCED2和PpCYP707A1,并对其进行了表达分析。结果显示:随着果实成熟,果皮中ABA含量持续增加,果肉和种子中ABA含量于采收前10d达到峰值后下降。果皮和果肉中,PpNCED2表达量显著高于PpNCED1,且与ABA含量变化趋势一致,是果皮和果肉中ABA生物合成的主效基因。种子中,PpNCED1和PpNCED2表达量均很高,是种子中ABA生物合成的主效基因。果皮、果肉和种子中PpCYP707A1表达模式与ABA含量变化趋势相反,是ABA含量变化的主要负调控基因。综上,ABA可能调控‘金二十世纪’梨果实成熟进程,而ABA含量水平是由合成酶和降解酶基因共同调控的,且不同组织中ABA生物合成的主效基因存在差异。     

猪卵巢卵母细胞的体外成熟和体外受精

将从屠宰母猪卵巢采得的卵母细胞-卵丘细胞复合体(Oocyte-cumulus Cell Complex.OCC)在含PMSG的M199培养40～44小时,卵丘细胞大部分扩散(86.4％)。48.1％(142/295)的卵母细胞排出第一极体(PBI)。将体外成熟的卵母细胞与体外获能精子授精后30～70小时,80.5％(103/128)的卵母细胞受精并可在体外发育到2～8细胞甚至桑椹胚。本文还对裸卵母细胞的体外成熟和体外受精进行了研究,对体外受精卵的早期发育作了观察。实验结果表明:PMSG对诱导卵丘细胞扩散及卵母细胞的全面成熟有重要作用,在OCC中的卵母细胞成熟率高于裸卵母细胞体外授精后8～10小时将受精卵放入改良KRB液培养可使卵裂比例明显提高。     

Periovulatory endocrinology and oocyte maturation in unmated mature blue fox vixens (Alopex lagopus)

Nine of 10 mature blue fox vixens (Alopex lagopus) in spontaneous oestrus ovulated approximately 2 days after the preovulatory increase in luteinizing hormone (LH). Plasma concentrations of follicle-stimulating hormone and progesterone increased simultaneously with the LH peak, whereas oestradiol-17 beta peaked 1 day previously. In the tenth vixen, an LH peak was not observed, and neither visible follicles nor corpora lutea were found in the ovaries 6 days after peak vaginal electrical resistance. Eggs were ovulated as primary oocytes, but oocyte maturation was initiated within the day of ovulation (2 days after the LH peak). Within the next 2 days (3-4 days after the LH peak) the first polar body was extruded, and the cumulus mass was completely dissociated from the zona pellucida. The interval between the preovulatory LH peak and initiation of the final oocyte maturation is thus considerably longer in the blue fox than for example in the cow (48-72 h compared with 9-12 h). This suggests that the relationship between these two events is somewhat different in the blue fox.     

牛卵母细胞体外成熟培养研究进展

作为胚胎生物工程技术研究的基础之一的牛卵母细胞体外成熟培养技术自上世纪30年代起至今,许多科学研究人员在牛卵母细胞体外生长、发育领域做了大量工作,对牛卵母细胞培养条件进行了广泛而深入地研究,并取得了较大的进展,目前牛卵母细胞体外成熟培养技术体系已建立,但在卵母细胞成熟机理、成熟调控方面尚存在许多尚未阐明的问题和一些亟待解决的问题。本文从卵母细胞的来源、卵母细胞体外培养条件、培养液的完善及卵母细胞成熟判定方法等方面详尽介绍了当前牛卵母细胞体外培养技术的研究进展。     

附睾功能及其对精子成熟的影响

哺乳动物的附睾为精子的成熟提供了特殊的微环境，是精子成熟、保护、运输和贮存的部位，在生殖过程中起着重要作用。附睾上皮细胞的合成、分泌、转运等功能使精子发生了一系列结构、生化和功能的改变．从而使精子获得受精和运动能力。作者对附睾的形态结构、功能和对精子成熟影响研究现状等作简要综述。     

海狸鼠卵母细胞体外成熟培养初探

实验用ＰＭＳＧ或ＰＭＳＧ＋ＨＣＧ处理或未经激素处理的海狸鼠８只，共获卵巢卵母细胞１３８枚。激素处理对获取卵巢卵母细胞的数量没有影响，而对体外成熟发育至卵丘扩展和半成熟阶段有促进作用。三种不同培养液（Ｗｈｉｔｅｎ＋ＦＣＳ；ＴＣＭ１９９＋ＰＭＳＧ＋ＦＣＳ；ＴＣＭ１９９＋ＨＣＧ＋ＦＣＳ）共培养１２５枚卵母细胞，培养后卵丘扩展率及半成熟率分别为５６．５％，４５．７％，４７．６％和２１．７％，１２．３％，９．５％，以Ｗｈｉｔｅｎ液较高（分别为５６．５％和２１．７％），但只有ＴＣＭ１９９＋ＰＭＳＧ＋ＦＣＳ组有２枚卵母细胞出现第一极体。结果表明海狸鼠卵母细胞与其它啮齿动物的卵母细胞一样，能够在体外培养成熟，完成第一次减数分裂，排出第一极体     

母源基因Gdf9、Zar1、Mater和Dnmt1mRNA在绵羊卵母细胞和早期胚胎中的表达

为探讨母源基因在绵羊卵母细胞和胚胎发育过程中的表达模式,运用Real-time PCR技术研究4种母源基因:Gdf9(生长分化因子9)、Zar1(合子阻泄因子)、Mater(胚胎必要的母体抗原)及Dnmt1(DNA甲基化转移酶1)的mRNAs在绵羊GV期卵母细胞,24h成熟卵母细胞,2-细胞、4-细胞、8-细胞、16-细胞胚胎以及囊胚中的含量变化情况。结果表明:在GV期卵中Zar1、Mater、Gdf9以及Dnmt1m RNA相对含量最高(P<0.05);从2-细胞胚胎开始,4种基因的mRNA相对含量显著降低(P<0.05),各基因mRNA的含量在不同发育阶段的卵母细胞和胚胎中存在动态变化,这对卵子生长和胚胎发育有重要意义。     

促黄体素对牛卵母细胞体外核成熟的影响

为了研究促黄体素(luteinizing hormone,LH)在牛卵母细胞成熟培养体系中的时间依从性,试验通过向牛卵母细胞体外成熟培养的不同时间段添加LH来观察其核成熟的情况。首先用无LH的基础成熟液I和添加LH的成熟液Ⅱ分别培养,在成熟的不同时间点(3、6、9h)用醋酸地衣红染色观察各自的生发泡破裂率。然后在体外成熟培养的不同时间段(0～3、0～6、0～9、0～24h)添加LH,24h后统计各自的第一极体率。结果表明,添加LH组在6h的生发泡破裂率显著低于对照1组(P0.05),24h添加LH组的第一极体率显著高于对照2组(P0.05)。因此,添加LH能延迟牛卵母细胞的核成熟,成熟培养中24h全程添加LH较不添加组能显著提高核成熟率。     

Intra‐annual changes in reproductive indices of male and female Himalayan snow trout,Schizothorax richardsonii (Gray, 1832)

Periodic changes in reproductive hormone levels, gonadal histology and gonadosomatic index (GSI) of snow trout, Schizothorax richardsonii, were examined to ascertain annual cycle of gonadal development and reproductive status in their natural habitat. In females, there were coherent changes in plasma 17β‐oestradiol and vitellogenin along with GSI, oocyte maturation and vitellogenic progression, collectively indicating two distinct maturation peaks during the months of September and February. Coinciding with this, in males, plasma 11‐keto testosterone was also noticeably higher during September and February, with highest GSI values in September. However, plasma 17α, 20β‐dihydroxyprogesterone levels in males were found to be persistently high from September to February. This observation suggests the potential presence of matured oozing males over a longer period, unlike in females. Overall, the close association between reproductive hormone levels, GSI and gonadal maturation stages in males and females (particularly, the presence of postovulatory follicle complexes) with apparent natural synchronization clearly indicates that S. richardsonii breeds twice in a year, possibly during late September to early November and late February to early April in the coldwater riverine habitats of the Indian Himalayan region.