表皮生长因子对无透明带小鼠胚胎体外发育的影响

为了观察不同浓度的表皮生长因子对无透明带小鼠胚胎体外发育的影响,试验将小鼠原核期胚胎用链霉蛋白酶去除透明带后,分别置于含有不同浓度的表皮生长因子无血清胚胎体外培养液中培养,观察各期胚胎的发育情况.结果表明:除0.01 ng/mL表皮生长因子添加组胚胎的2细胞发育率和囊胚回收率与添加0.1 ng/mL表皮生长因子处理组差异不显著外(P>0.05),其体外胚胎各期的发育率、囊胚回收率和囊胚细胞数与其他处理组比较均差异显著(P<0.05).说明一定浓度的表皮生长因子可以提高无透明带小鼠胚胎体外培养各期的发育率,但是高浓度的表皮生长因子对无透明带小鼠胚胎的体外发育有一定的抑制作用.     

不同石蜡油对水牛孤雌激活胚胎体外发育的影响

水牛孤雌激活胚胎体外培养时采用石蜡油覆盖微滴培养,本文就几种不同石蜡油(协和油、Ameresco油、Sigma胚胎培养油和Sigma过滤油)对水牛孤雌激活胚胎体外发育的影响进行了研究。结果显示:在分裂率、孵化率上,不同石蜡油之间无明显差异;在7d囊胚率和总囊胚率上,分别为协和油32.1%、39.4%,Sigma胚胎培养油39.9%、42.8%,Sigma过滤油34.4%、40.2%,几组之间差异不显著;而Ameresco油7d囊胚率和总囊胚率分别为22.9%、28.8%,与Sigma胚胎培养油、Sigma过滤油之间差异显著(P<0.05)。     

表皮生长因子和胰岛素样生长因子对水牛卵母细胞胞质成熟的影响

以皮层颗粒（CG）单层分布于质膜下做为卵母细胞胞质成熟的标志,利用CG荧光染色法,研究了不同浓度表皮生长因子（EGF）和胰岛素样生长因子（IGF-1）及其组合对水牛卵母细胞胞质成熟的影响。结果发现：（1）添加不同浓度的EGF（10、20、30、50ng／mL）都可以提高胞质的成熟率,但是组间差异不显著（P〉0．05）;皮质颗粒的分布随着EGF浓度的升高逐步由中间分布向皮层分布转变;（2）在成熟液中添加IGF-130ng／mL时效果较好,能显著提高卵母细胞胞质的成熟率;皮质颗粒的分布随着IGF-1浓度的升高逐步由中间分布向皮层分布转变,在添加IGF一130ng／mL时,在皮层分布最好,随着IGF-1量的进一步增加,皮质颗粒又向中间分布转变;（3）添加20ng／mL EGF＋30ng／mL IGF-1组卵母细胞体外成熟率高于添加30ng／mL的IGF-1组,显著高于添加20ng／m LEGF组和对照组（P〈0.05）。由此表明,EGF和IGF-1对水牛卵母细胞体外成熟有协同作用。     

氨基酸、维生素对水牛体外受精胚胎体外发育的影响

通过在培养液中添加不同浓度的氨基酸或维生素,探讨其对水牛体外受精(IVF)胚胎体外发育的影响.结果表明:(1)非必需氨基酸可显著提高水牛卵母细胞IVF后胚胎的分裂率,但对囊胚发育率无显著影响;(2)低浓度的必需氨基酸对水牛IVF胚胎的发育具有一定促进作用,但高浓度则有抑制作用;(3)维生素对水牛IVF胚胎发育则有促进作用.在培养液中添加维生素,水牛IVF胚胎的分裂率和第7天囊胚发育率显著提高.     

表皮生长因子对成年水牛晶状体上皮细胞促增殖作用

为观察表皮生长因子 ( epidermalgrowth factor,EGF)对成年水牛晶状体上皮细胞增殖作用的影响。将不同浓度 EGF作用于体外培养的成年水牛晶状体上皮细胞 ,采用 MTT法测定细胞的增殖能力。结果 :EGF在浓度为 1ng/ m L和 1 0 ng/ m L作用的前 3 d促增殖作用逐渐加强 ,呈现时间依赖性 ,且在第 3 d达到最大促增殖效果。不同浓度的 EGF对晶状体上皮细胞的增殖作用不同 ,浓度在 1 0 ng/ m L作用 2 4h后即有明显的促增殖作用 ( P <0 .0 5) ,而作用72 h后最低的有效浓度为 1 ng/ m L,而且 2 50ng/ m L EGF有最大促增殖作用。结论 :EGF是诱导成年水牛晶状体上皮细胞增殖的重要因素。     

Ghrelin对水牛体外受精和孤雌激活胚胎体外发育的影响

本研究的目的是探讨Ghrelin对水牛体外受精和孤雌激活胚胎体外发育的影响.体外成熟的水牛卵母细胞经体外受精或离子霉素孤雌激活后.分别在舍0,0.5,5,50和500 μg/L Ghrelin的培养液中进行体外培养,观察各组胚胎的卵裂率和囊胚率.结果显示,在培养液中添加不同浓度的Ghrelin对体外受精和孤雌激活胚胎的卵裂率均无显著影响(P>0.05),但添加500 μg/L的Ghrelin显著提高体外受精胚胎的囊胚发育率(33.5% vs 13.7%,P<0.05),50 μg/L或500 μg/L的Ghrelin均显著提高孤雌激活胚胎的囊胚发育率(32.4%和34.6% vs 14.5%,P<0.05).结果表明,培养液中添加Ghrelin对胚胎的早期卵裂没有影响,但可促进水牛体外受精和孤雌激活胚胎囊胚的形成.     

氨基酸对水牛孤雌激活胚胎体外发育的研究

本研究主要探讨氨基酸对水牛孤雌激活(PA)胚胎体外发育的影响.在SOF液中添加1.5%的MEM非必需氨基酸(NEAA)显著提高PA(79.8%vs 67.7%)胚胎的分裂率(P＜0.05),囊胚发育率和囊胚孵化率无显著差异;添加1.0%的EAA显著提高PA胚胎的囊胚孵化率(77.3%vs 43.3%,P＜0.05),但各组间的分裂率和囊胚发育率无显著差异;在改良SOF基础液中联合添加1.0%NEAA和2.0?A可显著提高孤雌激活胚胎的分裂率(84.4%vs 68.8%,P＜0.05),但囊胚发育率和孵化率均无显著差异.     

血小板衍生生长因子和表皮生长因子对山羊SSCs凋亡相关基因的影响

为了研究表皮生长因子和血小板衍生生长因子-BB对山羊SSCs中凋亡相关基因的影响,本研究将60~75日龄的山羊睾丸(n=6)剪碎后,利用两步酶消化法得到睾丸单细胞悬液,然后通过两次差速贴壁法分离纯化山羊SSCs,并采用免疫荧光染色法对山羊SSCs进行鉴定。随后,单独或联合添加EGF和PDGF-BB,体外培养山羊SSCs 6d,运用real time PCR技术检测不同处理组中BAD、BCL2和BAX基因的相对表达量。结果表明,睾丸单细胞悬液经两次差速贴壁纯化后,能够获得纯度较高的SSCs。染色结果显示,纯化后的细胞大量表达SSCs的标记分子THY1和PLZF。与对照组相比,3个试验组的BCL2基因表达量均上调。其中,联合添加20ng/mL PDGF-BB+25ng/mL EGF处理组的中细胞的抗凋亡能力显著高于其他组(P0.05),25ng/mL EGF组的BCL2表达水平显著高于对照组(P0.05),然而20ng/mL PDGF-BB组中的抗凋亡基因BCL2表达水平与对照组相比差异不显著(P0.05);与其他所有试验组相比,对照组的促凋亡基因BAX和BAD的表达水平显著升高(P0.05),而试验组之间差异均不显著(P0.05)。     

血管内皮生长因子对牛胚胎体外发育影响的最适添加浓度筛选试验

为了探明血管内皮生长因子Vascular Endothelial Growth factor,VEGF）对牛卵母细胞体外发育影响的最适添加浓度，试验中应用了人类重组VEGF165。结果表明，添加1ng/ml、2ng/ml和5ng/ml VEGF均可显著提高胚胎的卵裂率和体外发育率，但以5ng/ml VEGF处理组可以得到最高的胚胎发育至4－8细胞期的发育率，因而选择5ng/ml VEGF浓度作为以后VEGF系列试验的添加浓度。     

氨基酸对胚胎体外发育的影响

1 前言 氨基酸是胚胎培养液的重要成分,在培养液中添加氨基酸可促进胚胎发育[1],提高囊胚率、孵化率和胚胎质量,使胚胎的细胞数增加[2].但不同氨基酸对胚胎发育的作用不同,不同添加方式其效果也不一样.Bavister和他的同事对小鼠的研究工作,第一次揭示了氨基酸在体外胚胎培养中的作用.     

水牛卵母细胞的体外受精及其胚胎发育的研究

本研究比较了不同来源的水牛卵母细胞的体外受精及其胚胎发育。对10头摩拉母水牛连续6周进行活体采卵,共采集292枚卵母细胞,头均回收卵母细胞4.87枚,头均A级卵数为3.07枚,从屠宰场收集74头水牛卵巢共采集559枚卵母细胞,头均回收卵母细胞和A级卵母细胞分别为7.55枚和5.20枚,均显著高于活体采集的水牛卵母细胞(P<0.01)。将收集的AB级水牛卵母细胞在相同的条件下进行体外成熟、体外受精和体外培养至囊胚。结果表明,活体采卵组和屠宰水牛卵母细胞组受精分裂率差异不显著,分别为54.81%和57.73%。屠宰水牛卵母细胞组的囊胚率则高于活体采卵组(28.78%vs21.34%,P<0.05)。利用两组的胚胎进行常规法冷冻保存,冻胚解冻后的存活率差异不显著(P>0.05)。     

Post-thaw development of in vitro produced buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification

The present study was conducted to examine post-thaw in vitro developmental competence of buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification. In vitro produced embryos were incubated with a medium containing cytochalasin-b (cyto-b) in a CO₂ incubator for 40 min for microfilament stabilization and were cryopreserved by a two-step vitrification method at 24℃ in the presence of cyto-b. Initially, the embryos were exposed to 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in a base medium for 4 min. After the initial exposure, the embryos were transferred to a 7 µl drop of 25% EG and 25% DMSO in base medium and 0.3 M sucrose for 45 sec. After warming, the embryos were cultured in vitro for 72 h. The post-thaw in vitro developmental competence of the cyto-b-treated embryos did not differ significantly from those vitrified without cyto-b treatment. The hatching rates of morulae vitrified without cyto-b treatment was significantly lower than the non-vitrified control. However, the hatching rate of cyto-b-treated vitrified morulae did not differ significantly from the non-vitrified control. This study demonstrates that freezing of buffalo embryos by cytoskeletal stabilization and vitrification is a reliable method for long-term preservation.     

Effect of melatonin on regulation of apoptosis and steroidogenesis in cultured buffalo granulosa cells

This study was aimed to address melatonin receptor expression, mRNA level of hypothalamus and hypophysis hormone receptors (GnRHR, FSHR, and LHR), steroidogenesis, cell cycle, apoptosis, and their regulatory factors after addition of melatonin for 24 hr in cultured buffalo granulosa cells (GCs). The results revealed that direct addition of different concentrations of melatonin (100 pM, 1 nM, and 100 nM) resulted in significant upregulation (p < 0.05) of mRNA level of melatonin receptor 1a (MT1) without affecting melatonin receptor 1b (MT2). Melatonin treatment significantly downregulated (p < 0.05) mRNA level of FSH and GnRH receptors, whereas 100 nM dose of melatonin significantly increased mRNA level of LH receptor. Treatment with 100 nM of melatonin significantly decreased the basal progesterone production with significant decrease (p < 0.05) in mRNA levels of StAR and p450ssc, and lower mRNA level of genes (Insig1, Lipe, and Scrab1) that affect cholesterol availability. Melatonin supplementation suppressed apoptosis (100 nM, p < 0.05) and enhanced G2/M phase (1 nM, 100 nM, p < 0.05) of cell cycle progression which was further corroborated by decrease in protein expression of caspase‐3, p21, and p27 and increase in bcl2. Our results demonstrate that melatonin regulates gonadotrophin receptors and ovarian steroidogenesis through MT1. Furthermore, the notion of its incorporation in apoptosis and proliferation of buffalo GCs extends its role in buffalo ovaries.     

应用体细胞克隆技术和胚胎移植技术生产克隆水牛

为了解决克隆水牛技术在供核细胞、受体细胞中的选用问题,探索不同条件对移植受孕的影响,完善克隆技术的理论体系。试验以摩拉奶水牛的耳成纤维细胞作为供核细胞,本地水牛卵母细胞为受体胞质进行细胞克隆构建重组胚胎;将发育5～10 d的重组胚胎移植于本地水牛、杂交水牛的子宫内,观察不同发情方式、胚胎类型、胚龄及季节对移植受孕的影响。结果表明:水牛的克隆胚胎妊娠率较低,分别为12.05%、12.04%、13.95%。自然发情和同期发情对克隆胚胎移植受胎率的影响差异不显著;鲜胚胎移植186头,受胎22头,受胎率为11.83%(22/186);冻胚胎移植174头,受胎23头,受胎率为13.22%(23/174)。克隆水牛从出生至12月龄体尺增长明显高于本地水牛;水牛克隆胚胎移植受胎在秋季最好,秋季受胎数占全年的53.33%,显著高于其他季节。     

AQP8 participates in oestrogen-mediated buffalo follicular development by regulating apoptosis of granulosa cells

Aquaporins (AQPs), a family of small membrane-spanning proteins, are involved in fluid transport, cell signalling and reproduction. Regulating AQP8 expression influences apoptosis of granulosa cells (GCs), ovarian folliculogenesis, oogenesis and early embryonic development in mice, but its role has never been investigated in other species. The aim of the present study was to characterize the AQP8 function in buffalo follicular development. The expression pattern of AQP8 in buffalo follicle was analysed by immunohistochemistry method. 17β-Estradiol (E2) or oestrogen receptor antagonist ICI182780 was used to treat GCs cultured in vitro, and the expression of AQP8 was detected using qRT-PCR. Its roles in apoptosis of buffalo GCs were investigated by shRNA technology. AQP8 was found to be expressed higher in secondary follicles (p < .05), and its mRNA level in GCs was upregulated by E2 via receptor-mediated mechanism in a dose-dependent manner. A 732-bp buffalo AQP8 coding region was obtained, which was highly conserved at the amino acid level among different species. AQP8-shRNA2 had more effective inhibition on target gene than AQP8-shRNA1 (66.49% vs. 58.31%) (p < .05). Knockdown of AQP8 induced GCs arrested at G2/M stage and occurred apoptosis. Compared with the control group, higher Caspase9 expression were observed in AQP8-shRNA2 lentivirus infected GCs (p < .05), while Bcl-2 and Bax expression levels had no obvious change (p > .05). Altogether, the above results indicate that AQP8 is involved in oestrogen-mediated regulation of buffalo follicular development by regulating cell cycle progression and apoptosis of GCs.     

Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 µm) were isolated by micro-dissection and cultured in 0 (control), 10^-3, 10^-5, 10^-7, and 10^-9 M SNP. To examine the reversible effect of SNP, PFs were cultured with 10^-5 M SNP + 1 mM N^ω-nitro-L-arginine methyl ester (L-NAME) or 1.0 µg hemoglobin (Hb). The results showed that greater concentrations of SNP (10^-3, 10^-5, 10^-7 M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10^-9 M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.     

Oocyte holding in the Iberian red deer (Cervus elaphus hispanicus): Effect of initial oocyte quality and epidermal growth factor addition on in vitro maturation

Current in vitro embryo production protocols in the Iberian red deer (Cervus elaphus hispanicus) need to be optimized; oocyte harvesting in situ followed by overnight holding could reduce the human effort and shipping costs. In our work, post‐mortem ovaries were retrieved, and the oocytes were harvested and allocated to G1 group (good quality) or G2 + G3 group (low quality). The oocytes were separately subjected to immediate in vitro maturation (IVM) or held overnight in a holding medium composed of 40% of TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts and 20% fetal bovine serum (FBS), at room temperature (16 hr). In vitro maturation was carried out in a basal medium supplemented or not with 50 ng/ml of epidermal growth factor (EGF). Our data showed that addition of EGF to the maturation medium increases the percentage of G1 oocytes reaching metaphase II (3.9% vs. 50%, basal vs. EGF; p < .001) and decreased their degeneration rate (69.9% vs. 22.2%, basal vs. EGF; p < .01) when oocytes were immediately matured. Overnight holding increased the meiotic competence of G1 oocytes (37.5% matured in basal medium) and EGF increased prophase arrest in G2 + G3 oocytes (16.1% vs. 38.8% in germinal vesicle [GV] stage in basal medium vs. EGF added medium; p < .05). Our data demonstrate that oocyte holding can be used in Iberian red deer oocytes. Interestingly, EGF addition increases the oocytes' meiotic competence in immediately matured oocytes but not after oocyte holding depending upon initial oocyte quality.