Izadyar F, Colenbrander B, Bevers M M
Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
Mol Reprod Dev. 1996 Nov;45(3):372-7. doi: 10.1002/(SICI)1098-2795(199611)45:3<372::AID-MRD15>3.0.CO;2-0.
Regulatory effect of GH on follicular growth and development in the cow is well documented. The aim of this study was to investigate the role of GH on in vitro bovine oocyte maturation. Therefore bovine cumulus oocyte complexes (COCs) were cultured in M199 without FCS and gonadotropins and in the presence of 10, 100, or 1,000 ng/ml bovine GH (NIH-GH-B18). The COCs were incubated at 39 degrees C in a humidified atmosphere with 5% CO2 in air and nuclear stage was assessed after 2, 4, 8, 16, 22, and 24 hr of incubation using DAPI staining. To assess the effect of GH on developmental capacity of the oocytes, COCs were incubated in the presence of GH for 22 hr, followed by IVF and in vitro embryo culture. Cultures without GH served as controls. For subsequent development, the embryos were cultured in M199 supplemented with 10% FCS on a monolayer of BRL cells. Embryos were scored morphologically and the efficiency of the culture system was evaluated as (1) the percentage of cleaved embryos 4 days after IVF, (2) the percentage of blastocysts on day 9 expressed on the basis of the number of oocytes at the onset of culture, and (3) the percentage of hatched blastocysts on day 11 expressed on the basis of the total number of blastocysts present at day 9. GH (100 and 1,000 ng/ml) significantly accelerated nuclear maturation (P < 0.001). At 4 and 8 h the percentage of oocytes in GV stage after GH treatment (54% and 19%) was significantly lower than the control (64% and 41%). Similarly at 16 and 22 h the percentage of oocytes in MII stage was significantly higher in the GH-treated group; (58% and 77%) and (46% and 62%) for GH and control respectively. The number of oocytes in MII beyond 22 hr of culture did not differ; 100 and 1,000 ng/ml GH induced significant cumulus expansion (P < 0.05), which was not observed in the absence of GH. Addition of 100 and 1,000 ng/ml GH during maturation significantly (P < 0.01) enhanced subsequent cleavage rate from (64% and 67%) in control to (75% and 81%) in GH-treated group; embryonic development in terms of day 9 blastocyst formation was also significantly increased in the presence of GH (29% and 34%) compared to the control (18% and 24%). The hatchability of the blastocysts was not influenced by GH. From the present data, it can be concluded that GH present during IVM has a beneficial effect on subsequent development.
生长激素（GH）对奶牛卵泡生长和发育的调节作用已有充分记录。本研究的目的是调查GH在体外牛卵母细胞成熟过程中的作用。因此，将牛卵丘卵母细胞复合体（COCs）培养于不含胎牛血清（FCS）和促性腺激素的M199培养基中，并添加10、100或1000 ng/ml的牛生长激素（NIH-GH-B18）。将COCs在39℃、含5%二氧化碳的湿润空气中孵育，使用4',6-二脒基-2-苯基吲哚（DAPI）染色在孵育2、4、8、16、22和24小时后评估核阶段。为了评估GH对卵母细胞发育能力的影响，将COCs在GH存在下孵育22小时，然后进行体外受精（IVF）和体外胚胎培养。不添加GH的培养物作为对照。为了后续发育，将胚胎培养于添加10% FCS的M199培养基中，置于单层水牛视网膜成纤维细胞（BRL细胞）上。对胚胎进行形态学评分，并将培养系统的效率评估为：（1）IVF后4天的胚胎分裂率；（2）基于培养开始时卵母细胞数量计算的第9天囊胚率；（3）基于第9天存在的囊胚总数计算的第11天孵化囊胚率。100和1000 ng/ml的GH显著加速了核成熟（P < 0.001）。在4和8小时时，GH处理后处于生发泡（GV）期的卵母细胞百分比（54%和19%）显著低于对照组（64%和41%）。同样，在16和22小时时，GH处理组处于第二次减数分裂中期（MII）的卵母细胞百分比显著更高；GH组和对照组分别为（58%和77%）以及（46%和62%）。培养超过22小时后，MII期的卵母细胞数量没有差异；100和1000 ng/ml的GH诱导了显著的卵丘扩展（P < 0.05），在不添加GH时未观察到这种情况。在成熟过程中添加100和1000 ng/ml的GH显著（P < 0.01）提高了后续的分裂率，从对照组的（64%和67%）提高到GH处理组的（75%和81%）；与对照组（18%和24%）相比，在GH存在下第9天囊胚形成方面的胚胎发育也显著增加（29%和34%）。囊胚的孵化率不受GH影响。从目前的数据可以得出结论，体外成熟（IVM）过程中存在的GH对后续发育具有有益作用。
