牦牛输卵管上皮细胞和卵泡颗粒细胞的培养

从牦牛输卵管壶腹部获取上皮细胞,用抽吸法从牦牛卵泡中获取颗粒细胞,对牦牛输卵管上皮细胞和卵泡颗粒细胞进行体外培养和传代的培养,观察培养过程中2种细胞原代和传代培养的生长方式及形态特点。两者培养144~216 h可形成融合的单层细胞,其传代密度保持105~106/mL为宜。     

牦牛输卵管上皮细胞和卵泡颗粒细胞的培养

分离培养牦牛输卵管上皮细胞和卵泡颗粒细胞的目的是克服体外受精中早期胚胎发育阻断,建立有利于牦牛早期胚胎体外发育的共培养体系.采集牦牛输卵管上皮细胞进行原代及传代培养,同时从卵泡液中获取颗粒细胞进行原代培养,培养过程中观察2种细胞的生长方式和形态特点.输卵管上皮细胞贴壁生长时,呈多边形,且呈单层成簇生长.培养144 h～216 h,可形成细胞单层.颗粒细胞贴壁生长时,呈现聚集生长特性,贴壁细胞形状不规则,呈放射状.培养72 h～96 h,形成颗粒细胞单层.     

卵泡大小和颗粒细胞对山羊卵泡卵母细胞体外成熟和体外受精的影响

用切割法采集卵泡液,收集卵丘一卵母细胞复合体（Cumulus oocytes comlexs,COCs）和自然裸卵,将部分COCs去除卵丘细胞获得机械裸卵,COCs放入体外成熟培养液中培养为成熟卵母细胞,加入获能的精子液,进行体外受精。结果表明：卵母细胞的体外成熟率和卵裂率与卵泡直径密切相关,大卵泡（80．95％,P〈0．01）和中等卵泡（75．50％,P〈0．05）的卵母细胞成熟率高于小卵泡（50．27％）;犬卯泡（53．53％）和中等卵泡（47．13％）的卵裂率显著高于小卵泡的32．26％（P〈0．05）。COCs、机械裸卵和自然裸卵的体外成熟率分别为75．0％、54．2％和10．5％,差异极显著（P〈0．01）,卵裂率分别为53．8％、10．8％和0％,差异极显著（P〈0．01）。对照组和1×10^5、1×10^6个／mL颗粒细胞组卵母细胞体外成熟率分别为68．6％、69．6％和67．8％,无显著差异（P〉0．05）,但均显著高于1×10^7个／mL（51．5％,P〈0．05）和1×10^10个／mL（35．5％,P〈0．05）颗粒细胞组,但各组间的体外受精率无显著差异（P〉0．05）。结果提示,大卵泡和中卵泡的卵母细胞的体外成熟率和卵裂率显著高于小卵泡,体外成熟培养液中添加高浓度的颗粒细胞能显著抑制卵母细胞的体外成熟。     

输卵管和颗粒细胞单层对牛体外受精胚胎发育的影响

以屠宰场牛卵巢为试验材料,研究输卵管细胞单层(OCM)和颗粒细胞单层(GCM)对牛卵母细胞体外成熟(IVM)、体外受精(IVF)和体外培养(IVC)后胚胎发育能力的影响。(1)从卵泡抽取卵丘卵母细胞复合体(COCs),并根据卵母细胞外面卵丘细胞的层数将其分为3类:1级(≥4层);2级(2～3层);3级(0～1层)。作分别在IVM和IVC培养液中添加GCM(1×106个/mL)与不添加的对比试验。结果显示:添加GCM对1级卵母细胞的卵裂率、6～8细胞发育率和囊胚率无明显影响(P>0.05);但添加GCM的2级、3级卵母细胞,受精后的卵裂率、6～8细胞发育率和囊胚率分别高于未添加组(P<0.05)。(2)所有卵母细胞(包括COCs和裸卵)被随机分为3个组,在其IVM和IVC培养液中分别添加OCM、GCM或不添加体细胞(对照组)。结果显示:OCM和GCM组的卵裂率、6～8细胞发育率和囊胚率均高于对照组(P<0.05),而两试验组之间差异不显著。     

BOEC＋CCs、TAURIN对水牛卵母细胞体外受精及早期胚胎发育的影响

利用屠宰场废弃的水牛卵巢、观察在受精液和早期胚胎培养液中添加BOEC＋CCs(输卵管上皮细胞＋颗粒细胞）、Taurin(牛磺酸）对体外受精率及早期胚胎发育的影响。分别在受精液和早期胚胎培养液中添加BOEC＋CCs或70μM Taurin，获得的受精率分别为39．5％、40．9％，囊胚率分别为28．1％、20％，均极显著地高于对照组（P＜0．01）；同时比较发现，两实验组的受精率无明显差异，但囊胚率差异显著（P＜0．05）。     

山羊卵泡母细胞体外受精及受精卵的体外发育

对山羊卵泡卵母细胞的体外成熟,体外及受精卵体外培养的系列研究表明、在成熟培养液中添加１５μｇ／Ｌ,重组牛生长激素,与ＦＳ睡ＬＨ配合使用,卵泡卵母细胞的成熟率（６７．９％）显著高于不含ｒｂＧＨ的培养组;３８．５℃和３９．０℃的培养温度以 卵母细胞体外成熟培养的影响无显著差异;ＢＯ液上浮法对山羊冷冻精子的活精子分离效果优于Ｐｅｒｃｏｌｌ分层液法,并得到较高的受精率;颗粒细胞单层和输卵管上皮细胞单层扔共     

山羊卵泡卵母细胞体外受精及受精卵的体外发育

对山羊卵泡卵母细胞的体外成熟、体外受精及受精卵体外培养的系列研究表明,在成熟培养液中添加１５μｇ／Ｌ重组牛生长激素（ｒｂＧＨ）,与ＦＳＨ和ＬＨ配合使用,卵泡卵母细胞的成熟率（６７．９％）显著高于不含ｒｂＧＨ的培养组（５３．１％,Ｐ＜０．０５）;３８．５℃和３９．０℃的培养温度对卵泡卵母细胞体外成熟培养的影响无显著差异（Ｐ＞０．０５）;ＢＯ液上浮法对山羊冷冻精子的活精子分离效果优于Ｐｅｒｃｏｌ分层液法,并得到较高的受精率（６４．３％∶４９．０％,Ｐ＜０．０５）;颗粒细胞单层和输卵管上皮细胞单层的共同培养体系均有益于受精卵的体外发育。     

颗粒细胞对卵泡发育的影响

卵泡发育是一个复杂的生理过程,通过间隙连接,颗粒细胞在卵母细胞的生长发育过程中起营养作用,并促进卵母细胞的成熟;颗粒细胞和膜细胞的相互作用是卵泡发育和维持正常功能的重要条件。作为卵泡发育的标志,颗粒细胞的生长分化是原始卵泡启动和生长的关键,并通过受体介导途径调控生长期卵泡的发育及卵泡闭锁,从而在卵泡发育过程中起重要的调控作用。     

牛输卵管上皮细胞培养的研究

用０．７６％的ＥＤＴＡ分离剥取输卵管上皮细胞是一种好的试剂,１０％胎牛血清（ＦＣＳ）ＴＣＭ－１９９培养其上皮细胞,５－６ｄ即可生长成单层,寿命可延至３－４周,共同培养从第７天开始,其囊胚发育率显著高于与颗粒细胞共同培养。     

高繁殖力黄淮山羊大卵泡颗粒细胞上调表达基因的研究

旨在对山羊卵巢有腔卵泡发育中基因表达特点进行研究。本研究应用抑制消减杂交技术(SSH)对黄淮山羊卵泡期卵巢非闭锁大卵泡(6 mm)和小卵泡(4 mm)颗粒细胞内差异表达基因进行了研究,建立了消减cDNA文库,通过斑点杂交分析,从正向文库中随机挑取96个克隆进行差异表达的筛选,结果发现,其中有8个与已知功能基因高度相似,12个全新的表达序列标签(EST)。结果显示,这些基因可能影响着山羊卵泡的发育成熟和排卵。     

Establishment of an advanced chemically defined medium for early embryos derived from in vitro matured and fertilized bovine oocytes

A chemically defined medium would be useful for analyzing promoters or inhibitors in in vitro culture (IVC) of bovine embryos. However, an IVC system for bovine embryos in a chemically defined medium has not been fully established. The present study was carried out to establish an advanced chemically defined medium for bovine embryos that supports a high rate of embryo development to the blastocyst stage. In the first experiment, we examined the effects of addition of Medium RD (RPMI1640 and Dulbecco's MEM, 1:1 v/v) to mKSOM/aa on developmental competence. The addition of 10% RD to mKSOM/aa with BSA improved the rate of development to the blastocyst stage; however, 10% RD-mKSOM/aa with PVP, which is a chemically defined medium, caused a reduction in the percentage of hatching blastocysts. In the second experiment, embryos were cultured in the chemically defined medium of 10% RD-mKSOM/aa containing 11.7, 23.4, 46.8, 70.2 or 96.8 μM inositol. Inositol at the concentration of 70.2 μM improved the rate of development to the hatching blastocyst stage. In the third experiment, the optimal RD concentration in the IVC medium was evaluated. Embryos were cultured in the chemically defined medium supplemented with 10, 20 or 30% (v/v) RD. The rate of development to the blastocyst stage was highest with 20% RD. In the fourth experiment, the effects of N-acetylglucosamine (GlcNAc) as an IVC medium supplement on developmental competence were examined. The rate of development to the blastocyst stage with 1.0 mM GlcNAc was significantly higher than that without GlcNAc, but the rate of development with 1.2 mM GlcNAc was not different from that without GlcNAc. We also evaluated the ability of blastocysts produced in RD-mKSOM/aa to develop to normal calves after being transferred into recipients. Ten of the 16 recipients became pregnant, with 9 delivering normal calves. These results indicate that 20% RD-mKSOM/aa containing 70.2 μM myo-inositol and 1 mM GlcNAc is useful as a chemically defined medium for IVC of bovine embryos.     

Pregnancies from bovine oocytes matured and fertilized in vitro

Production of transgenic animals and embryo cloning are only a few examples of new biotechnological methods applied to animal embryos. All these techniques require large amounts of oocytes and early embryos. In many laboratory animals, embryos matured and fertilized in vivo are easily obtained, but with larger domestic species it requires laborious surgical procedures and the number of embryos obtained remains relatively small (Bracken et al. 1982). The in vitro maturation of follicular oocytes derived from slaughterhouse ovaries and their in vitro fertilization provides large numbers of oocytes and embryos with considerably less effort. The final proof of the success in the in vitro maturation and fertilization procedure is the birth of healthy progeny. Also the normal preimplantation development of the embryos gives useful information about the efficiency of the method employed.     

不同激活条件及半胱氨酸处理对猪ICSI胚胎发育影响研究

本试验探讨了不同辅助激活方法(Calciumionophore A23187激活、Calciumionophore A23187+6-DMAP联合激活和电激活)、不同精子预处理方法(液氮冻融处理和0.1%Triton X-100处理)和在添加半胱氨酸的胚胎培养液中培养不同时间(0 h、4 h、12 h和168 h)对猪卵母细胞内单精子注射(ICSI)胚胎体外发育的影响。结果显示:与无辅助激活相比,A23187+6-DMAP联合激活和电激活均能显著提高ICSI卵母细胞的激活率、卵裂率和囊胚率(P0.05),A23187+6-DMAP联合激活能显著提高ICSI卵母细胞的受精率(P0.05)。液氮冻融精子组ICSI卵母细胞的雄原核形成率显著高于活精子组(P0.05)。在添加半胱氨酸的胚胎培养液中培养4 h的ICSI卵母细胞受精率、雄原核形成率和囊胚率显著高于0 h组(P0.05)。以上结果表明,猪卵母细胞在ICSI后需要辅助激活来启动胚胎顺利发育,A23187+6-DMAP激活效果较好。液氮冻融精子可以促进ICSI后雄原核的形成。半胱氨酸处理4 h对猪ICSI卵母细胞受精和发育均有促进作用。     

Effects of sera and steroid hormones on development of bovine oocytes matured and fertilized in vitro and co-cultured with bovine oviduct epithelial cells

The present experiment was designed to identify possible effects of sera and steroid hormones added to a co-culture with bovine oviduct epithelial cells on embryonic development in vitro. Bovine oocytes were matured in vitro for 24 h and then fertilized in vitro using swim-up and heparin-treated, frozen-thawed spermatozoa. At 18 and 20 h after insemination, oocytes were cultured for 3 or 7 d in a co-culture system with bovine oviduct epithelial cells containing either fetal calf serum (FCS) or estrous cow serum (ECS) and one of six hormonal additions (none, 1 or 10 micrograms/ml estradiol [E]; 1 microgram/ml progesterone [P]; 1 microgram/ml E + P; and 10 micrograms/ml E + P). A total of 2,666 oocytes were cultured for 3 d and examined for cleavage. Of those, 2,280 oocytes were cultured up to 7 d for development to the late morula or blastocyst stage. Greatest cleavage rates for 2- to 8-cell and 8-cell stages were observed in FCS (71 and 24%) and ECS (66 and 23%) without steroid addition. For development into blastocysts, no serum effect was observed. Greatest rates for development into blastocysts were observed in FCS (14%) and ECS (16%) without steroid addition. These results indicate that addition of E and P at the doses and combinations tested did not enhance developmental capacity of in vitro fertilized bovine oocytes. Compared with FCS, ECS tended to increase cleavage rates and development into blastocysts.     

The effect of co-culture on the development of in vitro matured equine oocytes after intracytoplastic sperm injection

It is clear that, in the horse, there are many weak links in the process of in vitro embryo production; an optimal culture system for equine oocytes does not exist, and related data are conflicting. Therefore, the ability of 3 different culture systems to support embryonic development of ICSI horse oocytes was examined. Oocytes (n = 261) suitable for culture were collected from 55 ovaries and divided, according to cumulus morphology, into 2 categories: expanded cumulus and compacted cumulus. Oocytes with expanded and compacted cumulus were cultured for in vitro maturation in TCM 199 + 10% FCS + 0.1 iu/ml FSH/LH at 38.5 degrees C under 5% CO2 in air for 24 and 40 h, respectively. Oocytes (n = 149) reached metaphase II and were subjected to ICSI with frozen semen and then incubated in 3 different culture systems: A) TCM 199 + 10% FCS alone or B) on granulosa cell monolayer, C) SOF + MEM amino acids + 0.8% BSA. Cultural conditions were 39 degrees C and 5% CO2 in air for A and B, while a gas mixture (5% CO2, 5% O2, 90% N2) was used for C. The fertilisation rate was 32%. The cleavage rate in Group A was 74.4% (32/43); 18 embryos reached 2-6 cell stage, eight 8-16 cell, four 16-32 cell and two >32 cell. In Group B, the cleavage rate was 73.5% (36/49) with better results in embryonic development; 14 reached 2-6 cell stage, eighteen 8-16 cell, twelve 16-32 cell and five >32 cell. In Group C, the cleavage rate was significantly lower then in A and B; only 15 of 47 ICSI oocytes (39.1%) cleaved with maximum development to 2-6 cell stage. The remaining oocytes (68.1%) degenerated during culture. In conclusion, IVM horse oocytes can be fertilised in vitro with high efficiency with ICSI and co-culture systems showed to be superior in supporting in vitro embryo culture compared to simple ones. The identification of the factors beneficial to in vitro embryo development provided by the somatic cells could be important to optimise the embryo culture systems for equine embryos.     

Predictive value of the area of expanded cumulus mass on development of porcine oocytes matured and fertilized in vitro

The objective of the present study was to investigate the correlation between the degree of cumulus expansion and in vitro development of porcine cumulus-oocytes complexes (COCs) matured and fertilized in vitro. The COCs were matured in the maturation medium (IVMM) supplemented with 15% or 5% of porcine follicular fluid (PFF) from small, medium and large follicles (<2 mm, 2-5 mm and >5 mm, respectively). COCs cultured in IVMM with PFF for 48 h displayed less expansion than those cultured in IVMM alone (P<0.05), irrespective of follicle size. After culture for 24 h in IVMM with PFF and for another 24 h in IVMM alone, the degree of cumulus expansion was more prominent than culture in the presence of PFF for the entire 48 h period (P<0.05), but the percentages of oocytes with PB I showed no significant difference between the control and experimental groups (P>0.05). After in vitro fertilization, the oocytes failed to develop to the morula/blastocyst stages except for those matured in IVMM supplemented with 15% or 5% PFF obtained from >5 mm follicles for the first 24 h and followed by in IVMM alone for the second 24 h (12.5% and 11.1% of the embryos developed to morulae and blastocysts, respectively). The expanded cumulus areas of COCs were significantly positively correlated with their in vitro development (p=0.0058, 0.0001 and 0.0348 for the percentages of embryos developed to 2-4 cell, beyond 4 cell and morula and blastocyst stages, respectively). In conclusion, PFF had an inhibiting effect on cumulus expansion, and the inhibitory effect decreased progressively with the increase in size of follicles from which PFF was obtained, and the action of PFF on cumulus expansion was affected by the PFF culture time. The areas of the expanded cumulus mass may be used as a parameter to predict development of porcine oocytes matured and fertilized in vitro.     

Activin‐A receptor expression patterns in prepubertal goat oocytes and derived embryos

This study examines the presence of activin IIA and IIB receptors (ActR‐IIA and ActR‐IIB) by Western blotting and immunocytochemistry in immature and IVM‐oocytes, 2 to 8‐cells embryos and blastocysts from prepubertal goats. Western blotting revealed that activin receptors are synthesized during oocyte maturation and embryo development. In the immunocytochemistry experiments, no immunostaining for either receptor was detected in oocytes while both receptors were immunolabelled in all the cells of cleaved embryos. In blastocysts, while ActR‐IIA expression appeared evenly distributed in the two cell lineages, inner cell mass and trophectoderm, the ActR‐IIB immunosignal was restricted mainly to the inner cell mass. Our findings reveal the presence of activin type II receptors (ActR‐IIA and ActR‐IIB) in in vitro matured prepubertal goat oocytes and blastocyst‐stage embryos. The expression of these receptors could be a key factor in understanding differences between competent and incompetent oocytes.