Driancourt M A, Reynaud K, Smitz J
INRA-URA CNRS 1291, PRMD, 37380 Monnaie, France.
Reproduction. 2001 Mar;121(3):463-74.
After in vitro maturation, fertilization and development, the percentage of fertilized eggs developing to the blastocyst stage is usually lower in calves compared with cows. It is unknown whether this low ability to develop in vitro is inherent to calf oocytes or is caused by altered follicular maturation. The latter possibility was explored in the present study using two markers of follicle function: in vitro steroidogenesis by intact follicles and aromatase activity of follicular walls. Calf follicles > 9 mm in diameter had a low ability to produce oestradiol (ten times reduction compared with cows) despite a testosterone output by theca cells which was similar to that observed in cows. This finding is in agreement with the low aromatase activity of granulosa cells of calf follicles measured by tritiated water release assay. Qualitative and quantitative differences between calf and cow follicular fluids were assessed using western blotting (inhibin and activin, heat shock protein 90, Müllerian inhibiting substance) and assays (inhibin and activin) to determine whether this defective aromatase could be produced by alterations in the amounts of follicular proteins modulating aromatase (inhibin and activin, heat shock protein 90, Müllerian inhibiting substance). Western blotting of follicular fluid proteins demonstrated three main bands (59, 57 and < 30 kDa) and one minor band (34 kDa) with the anti-alpha inhibin antibody, whereas a single 18 kDa band was detected when an anti-beta inhibin antibody was used. Calf follicular fluid contained similar amounts of all main inhibin forms (alpha and beta) but a 34 kDa alpha inhibin form was missing. The amounts of dimeric inhibin were similar between cows and calves but small follicles from calves contained more activin. Single bands at 70 kDa (Müllerian inhibiting substance) and 90 kDa (heat shock protein 90) were detected by western blotting. Müllerian inhibiting substance was missing from calf follicular fluid and heat shock protein 90 was present in smaller amounts in calf versus cow follicular fluid. None of the above differences could explain the defective aromatase of calf follicles. Two-dimensional separation of the [35S]-labelled proteins secreted by follicular walls originating from calf or cow follicles matched for size and follicle health was performed and 151 spots were observed on the master gel, which summarized all the spots present at least once. Fifteen spots were present in calves and not in cows. Quantitative differences were also detected with three spots containing more proteins in cows than in calves. Whether some of these proteins can alter maturation of follicles or oocytes requires further investigation.
在体外成熟、受精和发育后，与母牛相比，犊牛受精卵发育到囊胚阶段的百分比通常较低。目前尚不清楚这种体外发育能力低下是犊牛卵母细胞所固有的，还是由卵泡成熟改变所致。本研究使用卵泡功能的两个标志物探讨了后一种可能性：完整卵泡的体外类固醇生成和卵泡壁的芳香化酶活性。尽管犊牛卵泡直径>9mm时，其膜细胞的睾酮分泌量与母牛相似，但产生雌二醇的能力较低（与母牛相比降低了10倍）。这一发现与通过氚化水释放试验测得的犊牛卵泡颗粒细胞低芳香化酶活性一致。使用蛋白质印迹法（抑制素和激活素、热休克蛋白90、苗勒管抑制物质）和检测方法（抑制素和激活素）评估犊牛和母牛卵泡液之间的定性和定量差异，以确定这种有缺陷的芳香化酶是否可能由调节芳香化酶的卵泡蛋白（抑制素和激活素、热休克蛋白90、苗勒管抑制物质）含量改变所致。卵泡液蛋白的蛋白质印迹法显示，抗α抑制素抗体出现三条主要条带（59、57和<30kDa）和一条次要条带（34kDa），而使用抗β抑制素抗体时检测到一条单一的18kDa条带。犊牛卵泡液中所有主要抑制素形式（α和β）的含量相似，但缺少34kDa的α抑制素形式。母牛和犊牛之间二聚体抑制素的含量相似，但犊牛的小卵泡中激活素含量更多。通过蛋白质印迹法检测到70kDa（苗勒管抑制物质）和90kDa（热休克蛋白90）的单一条带。犊牛卵泡液中缺少苗勒管抑制物质，与母牛卵泡液相比，犊牛卵泡液中热休克蛋白90的含量较少。上述差异均无法解释犊牛卵泡芳香化酶缺陷。对来自犊牛或母牛且大小和卵泡健康状况匹配的卵泡壁分泌的[35S]标记蛋白进行二维分离，在主凝胶上观察到151个斑点，这些斑点汇总了至少出现一次的所有斑点。有15个斑点只存在于犊牛中而不存在于母牛中。还检测到定量差异，有三个斑点中母牛的蛋白质含量比犊牛多。这些蛋白质中的一些是否会改变卵泡或卵母细胞的成熟，需要进一步研究。
