牦牛卵泡液LDH同工酶及外源性激素对其表达模式的影响

采用比色法和不连续缓冲系统的聚丙烯酰胺凝胶电泳（ＰＡＧＥ）技术对未孕和怀孕初期牦牛卵泡液乳酸脱氢酶（ＬＤＨ）活性及同工酶进行了测定，并分析了促卵泡素３号（ＬＲＨ－Ａ３）、人绒毛膜促性腺激素（ｈＣＧ）和孕马血清促性腺激素（ＰＭＳＧ）对其表达模式的影响。结果表明，未孕和怀孕初期牦牛卵泡液ＬＤＨ活性分别为２００．４±３１．１，２１５．３±８６．６μｍｏｌ·ｓ－１·Ｌ－１，同工酶有５条谱带。３种外源性激素均使牦牛卵泡液ＬＤＨ活性显著升高（Ｐ＜０．０１），ＬＤＨ同工酶带型分布发生变化，降低了ＬＤＨ２／ＬＤＨ１比值，改变了同工酶中Ａ和Ｂ亚基所占比例     

Befruchtungs- und Entwicklungskompetenz nonovulatorischer Katzenoozyten nach Kokultivierung mit epididymalen Spermien und felinen Oviduktepithelzellen

Inhalt Follikulär eunreife Oozyten der Hauskatze wurden hinsichtlich ihrer Befruchtungsund Entwicklungskompetenz unter dem Einfluß von felinen Oviduktepithelzellen (FOEC) untersucht. Nonovulatorische Oozyten wurden durch Anschneidender Oberfläche ektomierter Ovarien und Ausspülen der Follikel gewonnen. Eine Klassffizierung der Oozyten erfolgte nach der optischen Dichte des Vitellus. Mit epididymalen Spermien, deren wichtigste Parameter ausgewiesen werden, wurde inseminiert. Oozyten mit optisch dichtem Vitellus zeigten eine viermal höhere Befruchtungs- und Teilungsrate als Oozyten mit transparentem Vitellus. Eine Kokultivierung von FOEC während des Inseminationsvorganges und der Embryokultivierung führte sowohl zu einer signffikanten Steigerung (p < 0,05) der Befruchtungsrate von 34,9% auf 49,5% als auch der Furchungsrate von 30,3% auf 48,6%.     

哺乳动物原始卵泡体外培养与激活调控研究进展

随着体外受精、克隆与转基因动物等基础研究快速发展,对卵母细胞的需求越来越多,科学家们不得不寻求能够获得大量优质卵母细胞的新途径。原始卵泡作为生长卵泡的来源,其初始容量影响哺乳动物的繁殖性能,是整个雌性生殖期中各阶段卵泡及卵子发育的源头。哺乳动物的卵巢在出生时由一定数量的卵泡组成,大多数原始卵泡处于休眠状态,只有极少数的原始卵泡被激活并进入生长卵泡池中。因此合理开发卵巢里尚未激活的原始卵泡对提高动物繁殖率、加速优良牛种的遗传改良、阐明动物卵泡发生的分子机理具有重要意义。本文将目前哺乳动物原始卵泡体外激活的国内外进展做一综述,为研究动物和人类卵泡激活的生物学过程提供理论基础。     

Acute stimulation of a smooth muscle constrictor by oestradiol‐17β via GPER1 in bovine oviducts

Oviducts play roles in reproductive processes, including gametes transport, fertilization and early embryo development. Oviductal transport is controlled by various factors such as endothelins (EDNs) and nitric oxide (NO), smooth muscle contracting and relaxing factor, respectively. EDNs and NO production depend on an ovarian steroid hormone, oestradiol‐17β (E2) and E2 quickly exerts their biological functions through G protein‐coupled oestrogen receptor 1 (GPER1), which mediates rapid intracellular signalling. Because follicular fluid which contains a high concentration of E2 enters the oviduct, we hypothesized that E2 in the follicular fluid participates via GPER1 in producing EDNs and NO. To test this hypothesis, we investigated 1) the expression and localization of GPER1 in bovine oviductal tissues and 2) rapid effects of E2 via GPER1 on EDN1, EDN2 and inducible NO synthase (iNOS) expression in cultured bovine oviductal isthmic epithelial cells. GPER1 was observed in the oviductal epithelium, stroma and smooth muscle, and its expression was highest in the isthmus. Short‐term treatments (≤1 hr) of E2 increased EDN2 mRNA expression in the isthmic epithelial cells, although E2 did not affect EDN1 and iNOS mRNA expressions. Results of GPER1‐selective agonist G‐1 and GPER1‐selective antagonist G‐15 treatments revealed acute stimulation by E2, which is mediated via GPER1. The overall findings suggested that E2 in follicular fluid rapidly stimulates EDN2 expression via GPER1 in the isthmic epithelial cells. Follicular fluid may play a role in retention of the ovulated oocyte in the end of ampulla by contracting the isthmus for successful fertilization.     

Co-culture model reveals the characteristics of theca cells and the effect of granulosa cells on theca cells at different stages of follicular development

Theca cells (TCs) play an important role in follicular development, which cannot be separated from granulosa cells (GCs). However, compared with mammals, the TCs and the effects of GCs on TCs at different follicular development stages (FDSs) have specific characteristics in avian species, but none of them have been clearly defined. In this study, we established an in vitro co-culture (with GC at the corresponding stage) model of goose TCs at different FDSs (pre-hierarchical, hierarchical and F1) by using a transwell system. The properties of TCs in co-culture at the three FDSs, including cell morphology, activity and intracellular lipid content, as well as the expression of key genes involved in de novo lipogenesis, steroidogenesis, proliferation and apoptosis, were examined and defined. We further compared the mono-culture and co-culture groups. After co-culture, the activity of TCs showed significant (p < .01) increases in all stages; moreover, in pre-hierarchical TCs, the expression levels of FAS, SREBP, 3β-HSD and CCND1 were promoted, and PPARγ, CYP19, BCL2 and CAS3 were inhibited (p < .05); in the hierarchical TCs, the expression levels of PPARγ, FAS, CYP19, CCND1 and BCL2 were promoted, and SREBP, STAR, 3β-HSD and CAS3 were inhibited (p < .05), whereas in the F1 TCs, the expression levels of PPARγ, FAS, 3β-HSD, CYP19 and CCND1 were promoted, and STAR and CAS3 were inhibited (p < .05). These results suggested that GCs at the three FDSs have dynamic and complex influences on the physiological characteristics of TCs, and the influences on TCs at the three FDSs were varied.     

牛卵泡AGTR2序列结构及表达特性分析

【目的】研究牛卵泡发育过程中关键调控蛋白CART的候选受体AGTR2的分子特征和立体结构,并结合AGTR2在不同生理状态牛卵泡的表达特性分析其功能。【方法】 同期发情处理后,经B超声波连续监测（每12 h 一次）采集牛双侧卵巢,分离优势卵泡（dominant follicles, DF）和从属卵泡（subordinate follicles, SF）;其中3头牛DF和SF分别分离颗粒细胞（granulosa cells, GCs）,抽提总RNA后,经反转录、引物特异性扩增、胶回收及测序,获得AGTR2基因全CDS区;生物信息学方法对其序列结构、亲缘关系及立体结构进行分析;设计AGTR2和内参基因RPLP0 qRT-PCR引物,分析AGTR2在牛DF和SF的差异表达情况;另1头牛DF和SF经4%多聚甲醛固定后,设定阳性、阴性对照组和试验组,应用免疫组织化学技术对AGTR2进行表达和定位分析。【结果】 AGTR2 CDS区全长1 089 bp,编码362个氨基酸;牛卵泡AGTR2氨基酸序列与NCBI数据库获得的其他24种动物对应的氨基酸序列BLAST分析表明,该序列与印度水牛序列相似性最高（99.4%）,与其他动物序列相似性为92.0%—98.9%;立体结构和功能域分析表明,AGTR2立体结构中包含有7个横跨细胞膜的平行的α螺旋结构,符合G蛋白偶联受体（G-protein-coupled receptors, GPCRs）的典型特征;qRT-PCR分析结果表明AGTR2 mRNA在DF的表达量极显著高于SF（P<0.01）,且差异表达倍数达7.47倍;免疫组织化学分析结果表明AGTR2在牛DF和SF颗粒层、膜层细胞均有表达,特异性显色强度表明AGTR2在SF膜层细胞表达量高于DF。【结论】 AGTR2属于G蛋白偶联受体,符合神经肽CART受体的基本特征;本试验为进一步研究AGTR2在牛卵泡发育过程中调控信号通路和激素分泌的机理奠定基础,同时,对后期鉴定CART的受体、深入阐明CART调控牛卵泡发育的作用机理具有重要意义。     

聚乙烯吡咯酮与促卵泡素对奶牛活体采卵效率的影响

采用4×4拉丁方设计,研究聚乙烯吡咯酮(PVP)溶解促卵泡素(FSH)一次肌肉注射对奶牛活体采卵效率和体内血清FSH含量的影响。将8头荷斯坦空怀母奶牛随机分为4组,每组2头,分别用5mgFSH 5mL15%PVP(15%PVP组)、5mgFSH 5mL30%PVP(30%PVP组)、5mgFSH 5mL生理盐水(对照组)和常规注射FSH(常规组)处理供体奶牛。试验期4个月,每月处理1次,各组均为处理后4d应用B型超声波导引采卵。结果如下:30%PVP组每头次平均可采卵泡9个,获可用卵母细胞4枚,与常规组比较差异不显著(P>0.05),但与对照组比较差异显著(P<0.05);15%PVP组每头次平均可采卵泡6个,获可用卵母细胞2.25枚,与对照组比较差异显著(P<0.05);体内血清FSH浓度,30%PVP组与常规组变化趋势相似,维持适宜FSH浓度时间较长,15%PVP组次之,而对照组注射后4~12h血清FSH浓度较高,以后急剧下降,表明PVP可延长FSH在体内存留时间,但以30%PVP效果最佳。     

水牛有腔卵泡颗粒细胞凋亡的生物形态学特征

为了解水牛有腔卵泡闭锁的起因和有腔卵泡中是否存在卵泡颗粒细胞凋亡，本研究选择青春期前水牛和成年（发情间期和发情期）水牛的卵巢，采用石蜡组织切片、HE染色技术进行光镜观察，并采用DNA原位末端标记（TUNEL）技术和透射电镜观察研究了卵泡颗粒细胞凋亡的特征。结果表明：青春期前水牛的健康有腔卵泡上无或仅有极个别凋亡的卵泡颗粒细胞（GCs），而闭锁有腔卵泡上存在有多量凋亡的GCs，凋亡的形式：GCs层内出现单个或多个凋亡细胞，卵泡腔内出现凋亡小体群。在成年水牛发情间期的闭锁大卵泡上，GCs层弯曲皱折，存在多量凋亡的GCs。发情期水牛成熟卵泡的GCs层也存在GCs凋亡，排卵前的GCs层细胞全部脱落到卵泡腔内。本研究首次在同一组织切片上先后分别应用HE染色和TUNEL检查细胞凋亡，且证实了水牛有腔卵泡GCs凋亡的存在。超微结构显示了GCs凋亡的核边集化、核浓缩及凋亡小体的形态。上述结果提示：在水牛闭锁有腔卵泡中存在着GCs凋亡，GCs凋亡是启动水牛有腔卵泡闭锁的潜在机理。     

发育阶段对牛卵泡卵母细胞冷冻效果的影响

研究了用3种不同方法（A法：程序冷冻法：B法：超快速冷冻法；C法：玻璃化冷冻法）冷冻保存的牛GV期和IVM卵母细胞的发育潜力，结果表明，无论采取哪种方法。GV期卵母细胞的发育潜力均显著低于IVM卵母细胞。三种冷冻方法中，以玻璃化冷冻为最佳。     

生殖激素与卵巢卵泡颗粒细胞凋亡

本文综述了促性腺激素释放激素、促卵泡激素、马绒毛膜促性腺激素、雌激素、雄激素、生长激素等生殖激素对动物卵巢卵泡颗粒细胞凋亡的影响。