卵泡液对不同大小牛腔前卵泡体外培养过程中E2分泌的影响

Φ60~150μm牛腔前卵泡在有、无促性腺激素存在下,分别添加0、10%、15%、20%牛卵泡液(BFF)的McCoy′s 5 a无血清系统体外培养15 d,测定其雌二醇(E2)分泌量。结果显示:各添加卵泡液组在培养3、6、9、12 d的E2分泌量均显著高于对照组(0组)(P<0.01)。当培养液中有促性腺激素(FSH、LH)存在下,各添加卵泡液组的E2分泌量更高,其中添加10%牛卵泡液组中Φ>100μm腔前卵泡在培养6 d时E2分泌量达最高,为48.96±11.54 pg/mL。表明培养液中添加一定比例牛卵泡液(BFF)可显著刺激体外培养腔前卵泡E2的分泌,促性腺激素存在下更加强了卵泡液对E2分泌的刺激作用。     

牛卵泡卵体外成熟及其受精能力的研究

本试验主要研究了牛卵泡卵体外成熟、体外受精及其受精过程的发生。从屠宰场收集800枚牛卵泡卵进行体外成熟培养,并用体外获能的精子体外受精。在用m-TCM199培养21、22、24、26、28h后,达到中期Ⅱ的卵母细胞比例分别为33.89％、74.16％、77.78％、86.67％、85.71％;25h后,如果继续培养,成熟的卵母细胞比例不再显著增加。卵母细胞的自身形态显著地影响其成熟效果,A、B、C级卵培养24h后分别有94.60％、55.43％、31.25％的卵母细胞达到中期Ⅱ。卵泡卵在体外滞留8h后,其成熟率(47.11％)明显地低于45min(77.92％)或4h(74.60％)。肝素和钙离子载体A 23187都能诱发牛精子获能,二者在受精效报上差异不显著(P>0.05),并且都有多精入卵现象发生。授精后6h精子进入卵内,精子入卵后2～3h头部膨大,6h后出现早期原核,最早发生卵裂是在授精后30h。     

水牛健康与闭锁优势卵泡中颗粒细胞凋亡的生物学特征

采用组织学苏木精伊红(HE)染色与光镜检查、荧光显微镜检查和DNA电泳技术,检查了水牛健康优势卵泡(HDF)和闭锁优势卵泡(ADF)中颗粒细胞(GCs)凋亡的生物学特征。此外还应用孕酮处理早期的ADF,应用流式细胞术(FCM)方法检查该激素诱发GCs凋亡的情况。HE染色和荧光显微镜检查结果显示,HDF和ADF都含有凋亡的GCs,只是ADF中凋亡的GCs较多。凋亡GCs的HE染色表现细胞体积缩小,染色浓黑,荧光原位检测发现核浓缩。DNA电泳分析表明,在HDF中未发现GCs的DNA降解,而在ADF中因闭锁程度不同而表现不同程度的DNA降解,出现典型的"拖尾"带,但均未出现具有细胞凋亡特征的DNA"Ladder"。FCM检查结果见到早期的ADF存在着DNA的降解,孕酮处理使DNA降解碎片显著增加。上述结果提示,在水牛的HDF中,GCs的凋亡很少,在ADF中GCs的凋亡较多。水牛ADF中GCs的死亡,少量是以凋亡方式发生的,而坏死是主要方式。     

Autologous somatic cell nuclear transfer in pigs using recipient oocytes and donor cells from the same animal

The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p < 0.01) lower rate of fusion than the CCCs when two pulses of 170 V/mm DC were applied for 50 µsec (19 ± 2% vs. 77 ± 3%). The rate of DCC fusion with oocytes was increased by the application of two DC pulses of 190 V/mm for 30 µsec, although this was still lower than the rate of fusion in the CCCs (33 ± 1% vs. 80 ± 2%). The rates of cleavage (57 ± 5%) and blastocyst formation (1 ± 1%) in the DCC-derived embryos did not differ from those (55 ± 6% and 3 ± 1%, respectively) in the CCC-derived SCNT embryos. Autologous SCNT embryos derived from CFCs (5 ± 2%) showed higher levels of blastocyst formation (p < 0.01) than CCC-derived autologous SCNT embryos (1 ± 0%). In conclusion, the results of the present study show that culturing cumulus and follicular cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts.     

褪黑素对体外培养绵羊卵泡颗粒细胞分泌雌二醇和孕酮的影响

采集绵羊卵泡颗粒细胞进行体外培养,根据试验设计加入褪黑素(MLT)和HCG,分别于不同培养时间收集细胞培养液,用放射免疫测定法(RIA)测定细胞培养液中雌二醇(17β-E2)和孕酮(P4)含量。结果表明:卵泡颗粒细胞在无血清培养体系中可体外培养,并能够分泌相应17β-E2;用MLT或MLT与HCG共同处理卵泡颗粒细胞,能促进细胞分泌17β-E2,并呈时间和剂量的依赖性,但对P4的分泌没有影响。     

天祝白牦牛超排过程中卵泡闭锁的初步研究

[目的]全面监测天祝母白牦牛超排过程中卵巢的变化情况。[方法]对12头健康成年天祝母白牦牛采用CIDR+FSH+PG的超数排卵处理,并运用超声探测技术对超排过程中卵巢的变化情况做了全程监测。[结果]在处理的12头牛中有5头监测到明显的卵泡闭锁现象。在超排处理前期,这5头的卵巢直径逐渐增大,到第10天达到最大值,平均(3.39±0.65)cm,之后又开始逐渐缩小;卵巢上最大卵泡的直径也在第10天达到最大值,平均13.6±2.6 mm;优势卵泡从第8天开始大量出现,到第10天达到最多,平均每个卵巢有6.5±2.3个;此后这些优势卵泡的直径逐渐缩小,在B超图上未看到黄体影像,说明这类卵泡没有正常排卵,而是最终走向了闭锁。[结论]该研究为评价及优化超排处理方案提供准确可靠的依据。     

细胞自噬与多囊卵巢综合征研究进展

多囊卵巢综合征（Polycystic ovary syndrome,PCOS）是导致女性月经不调、排卵障碍性不孕的主要原因,确切发病机制尚未阐明。自噬可以发生在卵泡发育的各个阶段,卵泡颗粒细胞自噬相对活跃,具有促性腺激素依赖性和细胞特异性。卵泡颗粒细胞自噬稳态平衡以维持卵巢功能,调控卵泡发育,影响胚胎质量和妊娠结局。PCOS卵泡颗粒细胞自噬活性过高,自噬相关基因表达过强,与PCOS卵泡发育异常、内分泌代谢紊乱、临床自然流产率高和辅助生殖中发生卵巢过度刺激综合征风险增高密切相关,可能是PCOS发病的重要机制。补肾中药通过下调PCOS卵泡颗粒细胞过度自噬,改善卵巢功能,维持机体阴阳平衡,但具体作用机制亟待进一步阐明,为PCOS、不孕症等妇科疑难病症临床诊疗提供新思路。     

弥散性神经内分泌细胞标志物在不同生殖周期小鼠卵巢中的表达

【目的】研究卵巢内弥散性神经内分泌系统细胞的分布情况。【方法】采用间接免疫荧光染色法检测弥散性神经内分泌细胞广谱、共同的标志物S100蛋白和神经元特异性烯醇化酶(NSE)在不同生殖周期小鼠卵巢中的表达情况。【结果】S100蛋白和NSE在动情周期次级卵泡和囊状卵泡外膜细胞、妊娠期囊状卵泡外膜细胞和部分黄体细胞中表达,阳性细胞散在或成簇分布,一些细胞具有明显的突起,细胞核不着色。【结论】小鼠卵巢中存在弥散性神经内分泌细胞;弥散性神经内分泌物质在卵泡不同发育阶段发挥着不同的调控作用;在卵巢的发育过程中伴随着部分细胞神经内分泌化。     

Bovine ovarian stem cells differentiate into germ cells and oocyte‐like structures after culture in vitro

Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte‐like structures. The ovarian stem cells were isolated and cultured in α‐MEM⁺ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p < .05). The results showed that at day 0, ovarian stem cells expressed specific markers of pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase, which is a marker commonly used to identify primordial germ cells (PGCs). After the period of differentiation, cells had morphological features that resemble PGCs and oocyte‐like cells (OLCs). An increase, ranging from five to 14 times, in the expression of VASA was observed in cells cultured in medium supplemented with BMPs and follicular fluid, while the increase in DAZL expression ranged from four to six times. In addition, OLCs had an increase in expression of mRNAs for GDF9, ZPA and SCP3 that ranged from two to eight times. In conclusion, OLCs can be differentiated in vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.