Butyrolactone-I (BL-I) which is a selective inhibitor of cyclin-dependent kinases, competitively binds p34(cdc2) and inhibits meiotic resumption. However, the function of BL-I in improving the synchronization of nuclear and cytoplasmic maturation of mammalian oocytes in vitro remains unknown In the present study, the effects of different concentrations and durations of BL-I treatment on the In vitro Maturation (IVM) of sheep oocytes were assessed together with the effects after removal of inhibition for different durations. The developmental competence of ovine oocytes was also determined based on the maturation rate and blastocyst rate of ovine parthenogenetic and in vitro fertilized embryos. The ultrastructure of oocytes treated by BL-I was also observed and analyzed by transmission electron microscopy. The germinal vesicle rate was arrested in 62.9% of oocytes treated by in vitro culture for 24 h with 150 mu mol L-1 BL-I. The maturation and blastocyst rates of the treatment groups were 70.83 and 20.33% vs. 63.44 and 24.39% in the control but these differences were not significant. The number of blastocysts produced by IVC for 24 h after removal of inhibition was significantly higher than that for the control (71.26 +/- 32.68% vs. 66.73 +/- 21.64%, p>0.05). Furthermore, electron micrography analysis also showed some differences with regard to the structure of microvilli, mitochondria and granular cells. The BL-I-induced inhibition of nuclear maturation in ovine oocytes did not appear to play a role in improving their IVM and embryonic development via enhancement of the synchronization of nuclear and cytoplasmic maturation but cytoplasmic maturation was still promoted. In conclusion, BL-I reversibly inhibits IVM of ovine oocytes but does not improve their maturation and subsequent in vitro development.

To find out the effects of ovine sperm acrosome reaction induced by zone pellucid,three experiments were designed:first,co-incubation non-capacited sperm with zone pellucid;second,co-incubation capacitation sperm with zone pellucid;third,adding with calcium ionophore A23187 after co-incubation non-capacited sperm with zone pellucid.The results showed that the rates of acrosome reaction induced by zone pellucid are not significant difference whether the spermatozoa were capacited,IA23187 don ' t cooperate with zone pellucide to improve the rate of acrosome reaction.

It was aimed to investigate the effects of different concentrations(0.05,0.1,0.2,0.5,1,5,10 μmol/L) of calcium ionophore(A23187,IA) and 2 mmol/L caffeine on the ovine sperm capacitation in vitro.The results showed that the higher rate of capacitation in vitro,lower the rate of acrosome reaction(AR),and the lower sperm motilily were induced by the high concentration of IA,but the dead sperm could be increased.The capacitation and AR of ovine sperm in vitro were not affected even delaying time.Caffeine could promote IA to induce AR.Collectively,this might be recommol/Lended that it be applicable for improving ovine capacitation in vitro with 0.05 to 0.2 μmol/L IA for 1 min.

In order to find out some modified methods of ovine sperm capacitation in vitro,the effects of their sperm capacitation,motility,enduring durations and fertilization after respectively adding with 0,5,10,20,50 μg/mL heparin at different time (0,30,45,60,90,120 min),were compared and analyzed.The results showed that ovine sperm capacitation rate was highest (40.92%),acrosome reaction was lowest (16.59%),enduring durations were longest (18 h),and the blastula rate was highest,when adding with 10 μg/mL heparin in the capacitation medium for 45 min.The effects of capacitation when adding with caffeine or caffeine and 10 μg/mL heparin,were not as good as the effects adding with 10 μg/mL heparin alone.It can be recommended that it is applicable for improving ovine capacitation in vitro system with 10 μg/mL heparin for 45 min,caffeine don ' t cooperate with heparin to improve the rate of capacitation.

Objective To investigate the distribution of progesterone receptors(PR),induced by estrogen or progesterone,on the endometrium in between mice treated by estrus synchronization and those in natural estrus.Methods Totally 45 mice were divided into five groups: Group A: pseudopregnant group in natural estrus,Group B: pseudopregnant group in estrus synchronization,Group C: ovariectomy group,Group D: group treated by progesterone,and Group E: group treated by estrogen.On day 4,6,and 8 after mating,the murine uteri were obtained to observe and analyze the distribution of PR in endometrium by immunohistochemistry.Results The progesterone receptors distributed in the three types of endometrium cells among all the groups.The positively stained cell number of endometrium in the group of natural estrus was significantly different from that in the groups by estrus synchronization(P0.05).On day 4 and day 6,the positively stained cell number of all three cellular types in the group treated by progesterone was much higher than that in ovariectomy group(P0.05).In addition,the positively stained cell number in glandular epithelium and endometrial stroma of the estrogen-treated group was much higher than that in the ovariectomy group on day 4,day 6 and day 8(P0.05),but the positively stained cell number in luminal epithelium was much lower than that in the ovariectomy group(P0.05).Conclusion The PR distribution in mouse endometrium by estrus synchronization and that in natural estrus,is mainly induced by their endogenetic estrogen and progesterone.

To discuss the effects of calcium ion concentration on ovine sperm capacitation.The sheep spermatozoa were incubated in the ovine oviduct synthesis fluid with different calcium ion concentration,then induced by calcium ionophore A23187.The results showed that the sperm capacitation was not significantly affected by adding with different concentrations of calcium ion,but the acrosome reaction rates induced by 1.7,2 mmol/L were significantly higher than the others.The appropriate sperm enduring period of 1.7,2 mmol/L were 16 h and 10 h respectively.It suggested that the suitable concentration of calcium ion for ovine sperm capacitation in vitro is 1.7 mmol/L,and this process should accompany with calcium ion.

This study was aimed to provide some experimental references for improving the in vitro fertilization system of sheep.The effect of different ovine serum concentrations during their different estrous stages on their sperm capacitation,enduring durations and fertilization were analyzed.After the basic sperm capacitation medium(Sheep Oviduct Fluid,SOF medium) was supplied respectively with 0%,2%,5%,10% and 20% ovine serum collected during their different estrous stages(the first day of estrus,the last day of estrus,and diestrus),the effects of their sperm capacitation,enduring durations were compared and analyzed reasonably by CTC(chlortetracycline)staining and appropriate fertilization in vitro.The results showed as following:①While the SOF medium was supplied with the ovine serum collected on the first day of their estrus,its best effect was initiated by treating for 45 min with 10% and 20% serum.Their sperm rates(45.28% and 44.52%) by capacitation but without acrosome reaction(AR) were significantly higher than those among the other groups treated with 0%,2% and 5% serum,respectively(30.14%,28.71% and 35.49%,P0.05);②While the SOF medium was supplied with the ovine serum collected on the last day of estrus and diestrus,there were no significant differences of the ovine sperm capacitation percentage among all the groups treated for 45 minutes(P0.05);③While the SOF medium was supplied with 20% ovine serum collected on the first day of their estrus,enduring duration of sperm(18 h)was much longer than that of the other two groups with 20% serum collected on their last day of estrus or diestrus respectively(11 h,11 h);④Following their appropriate fertilization in vitro,the cleavage and blastocyst rates(62.5% and 26.25%)in the group treated with the serum collected on the first day of their estrus,were significantly higher than those in the other two groups treated with the serum collected on the last day of estrus(42.22% and 11.11%,P 0.05)or diestrus(37.36% and 15.38%,P 0.05),respectively.It can be recommended that the serum be applicable for improving ovine IVF system,while the sheep sperm is treated for 45 minutes by SOF medium with 20% ovine estrous serum collected on the first day of their estrus.

<正>体外获能是哺乳动物体外受精体系中很重要的一步,获能处理的好坏直接关系着受精的成功与否。目前,绵羊体外受精效果与牛相比尚有较大差距,虽然原因是多方面的,但绵羊精子体外获能效果不十分理想是一个重要方面。因此,试验拟从精子获能的角度来探寻绵羊受精效果不理想的原因。1材料和方法1.1试剂CTC、BSA、乳酸钠、乳酸钙、丙酮酸钠、L-谷氨

了解发情期、妊娠前期、发情间期的生殖道对获能的影响,从而选取一个适当时期的生殖道作为实验的基础,希望能为体外获能的研究提供一些参考。将离心后的绵羊精液输入发情期、妊娠前期、发情间期的绵羊生殖道作用一定时间后,发现获能的比例均会增加,但不同时期的生殖道间差异不显著,且顶体反应的比例变化也不大;说明处于不同生殖周期的绵羊生殖道对绵羊精子获能的影响是相同的。

<正>颗粒细胞在卵母细胞体外成熟和体外受精进程中具有重要的作用,能显著提高卵母细胞体外成熟和体外受精率;但也有研究发现,颗粒细胞存在与否并不影响卵母细胞体外成熟和体外受精率,甚至还可显著降低其体外成熟率和体外受精率。鉴于以上现状,试验在卵母细胞体外成熟的基础上,研究了成熟时间、血清和颗粒细胞条件液对绵羊卵母细胞体外成熟及孤雌胚发育的影响,旨在探索为体外受精等技术提供更高质量卯母细胞的方法。

In order to investigate nuclear maturation , ovine(ovis arise) oocytes were in vitro cultured by inhibiting with butyrolactoneⅠ(BL-Ⅰ). They were moved, after 8 h, and in vitro matured (IVM) randomly in appropriate medium for different durations to analyze the synchronization of both their nuclear and cytoplasmic maturation. The results showed that only the rate of ovine oocytes in MⅡ stage, in IVM group for 8 h, was significantly lower than that of the others (P 0.01), but there were no significant differences of both oocyte maturation and blastocyst rates among all the groups (P 0.05). It suggests that such kind of inhibition by BL-I should be reversible although, it may not improve the cleavage and developmental competence of ovine embryos.

After matured ovine oocytes were activated by 5 μmol/L A23187 for 5 min and then 2 mmol/L 6-DMAP for 4 h, it was cultured in vitro in five types of cell monolayers ( including pre-matured granulosa cells, matured granulosa cells, oviduct ampulla epithelium cells, oviduct isthmus epithelium cells and endometrium cells) respectively. The cleavage rates and blastocyst rates of embryos treated between pre-matured granulosa cells and matured granulosa cells, were not significantly different; and the blastocyst rates of oviduct ampulla epithelium cell and endometrium cell groups(27.5%,27.7%), were significantily higher than the others of oviduct isthmus epithelium cell and granulosa cell groups(17.7%,20.3%)(P0.05). These results indicated that the pre-matured granulosa and matured granulosa cells may play the same role on the development of ovine parthenogenetic embryos. The oviduct ampulla epithelium cells and endometrium cells are suitable for the development of ovine parthenogenetic embryos cultured in vitro, and their blastocyst rates reach to 27.5% and 27.7% respectively.

研究并建立适合小鼠囊胚的双重荧光染色方法。用建立的双重荧光染色法对小鼠体内外囊胚细胞计数,分别计数囊胚不同时期的内细胞团和滋养层的细胞数,比较不同发育时期囊胚的内细胞团和滋养层细胞的比例,评定体内外囊胚质量。

Matured ovine oocytes were activated by 5 μmol/L A23187 for 5 min and then 2 mmol/L 6-DMAP for 4 h,afterwards cultured in SOFaa and M-199 medium supplemented with BSA and serum of different concentrations,respectively.The cleavage rate of group M-199+FCS(29.9%) was significantly lower than SOFaa+BSA(52.7%) and SOFaa+FCS(52.8%) groups(P0.05).And the blastocyst rate of SOFaa+BSA(0.8%) group was significantily lower than M-199+BSA(7.6%),SOFaa+FCS(11.8%),M-199+FCS(10.2%) groups(P0.05).The cleavage rate and blastocyst rate of oocyte treated with SOFaa+10% FCS(50.7%,11.3%)and SOFaa+20% FCS(59.2%,9.9%)were not significantly different,and those data of M-199+10% FCS(29.6%,5.6%) and M-199+20% FCS(32.4%,9.9%)were also same.These results showed that SOFaa medium is suitable for the development of ovine parthenogenetic embryos cultured in vitro,and SOFaa added with10% FCS can improve its function on those IVC embryos.

In this experiment,the discrepancy was compared among the Swim-up,Induction by A23187,Percoll and Incubation after Percoll methods for ovine sperm capacitation in vitro with chlortetracycline fluorescence(CTC) staining and in vitro fertilization.The results showed that the rates of sperm capacitation and the cleavage treated by Swim-up method were higher than the others.It might be suggested that the Swim-up method is exactly suitable for the application of in vitro fertilization of sheep.

In order to investigate the potential effects of progesterone on ovine sperm capacitation in vitro,ovine sperms were added in the SOF medium with different concentrations of progesterones(1、5、10 μmol/L),and some sperm samples were fetched respectively and treated with chlortetracycline(CTC) pigmentation during different times.Analysis of ovine sperm capacitation was assessed by different sperm staining types with CTC.The results showed that the percentage of capacitated sheep sperms in each treatment group for 240 min and 360 min was markedly higher than that in of the control one(P0.05).It presented that the progesterone may enhance the ovine sperm capacitation in vitro.