Oussaid B, Lonergan P, Khatir H, Guler A, Monniaux D, Touze J L, Beckers J F, Cognie Y, Mermillod P
INRA, Unité Physiologie de la Reproduction des Mammiféres Domestiques, Nouzilly, France.
J Reprod Fertil. 2000 Jan;118(1):137-44.
A GnRH antagonist (Antarelix) was used to suppress endogenous pulsatile secretion of LH and delay the preovulatory LH surge in superovulated heifers to study the effect of a prolonged follicular phase on both follicle and oocyte quality. Oestrous cycles were synchronized in 12 heifers with progestagen (norgestomet) implants for 10 days. On day 4 (day 0 = day of oestrus), heifers were stimulated with 24 mg pFSH for 4 days and luteolysis was induced at day 6 with PGF2 alpha (2 ml Estrumate). Animals in the control group (n = 4) were killed 24 h after the last FSH injection. At this time, heifers in group A36h (n = 4) and group A60h (n = 4) were treated with 1.6 mg of Antarelix every 12 h for 36 and 60 h, respectively, and then killed. After dissection of ovarian follicles, oocytes were collected for individual in vitro maturation, fertilization and culture; follicular fluid was collected for determination of steroid concentrations, and granulosa cells were smeared, fixed and stained for evaluation of pycnosis rates. Granulosa cell smears showed that 90% of follicles were healthy in the control group. In contrast, 36 and 58% of the follicles in group A36h showed signs of early or advanced atresia, respectively, while 90% of the follicles in group A60h showed signs of late atresia. Intrafollicular concentrations of oestradiol decreased (P < 0.0001) from healthy follicles (799.14 +/- 40.65 ng ml-1) to late atretic follicles (3.96 +/- 0.59 ng ml-1). Progesterone concentrations were higher (P < 0.0001) in healthy follicles compared with atretic follicles, irrespective of degree of atresia. Oestradiol:progesterone ratios decreased (P < 0.0001) from healthy (4.58 +/- 0.25) to late atretic follicles (0.07 +/- 0.009). The intrafollicular concentrations of oestradiol and progesterone were significantly higher (P < 0.0001) in the control than in the treated groups. The oestradiol:progesterone ratio was higher (P < 0.0001) in the control (4.55 +/- 0.25) than in the A36h (0.40 +/- 0.05) and A60h (0.07 +/- 0.009) groups. Unexpectedly, the cleavage rate of fertilized oocytes, blastocyst rate and number of cells per blastocyst were not significantly different among control (85%, 41% and 95 +/- 8), A36h (86%, 56% and 93 +/- 5) and A60h (88%, 58% and 79 +/- 4) groups. In addition, there were no significant differences in the blastocyst rates from oocytes derived from healthy (45%), early atretic (54%), advanced atretic (57%) and late atretic follicles (53%). In conclusion, the maintenance of the preovulatory follicles in superovulated heifers with a GnRH antagonist induced more atresia and a decrease in oestradiol and progesterone concentrations. However, the developmental potential in vitro to day 8 of the oocytes recovered from these atretic follicles was not affected.
使用促性腺激素释放激素（GnRH）拮抗剂（安塔瑞克）抑制超排小母牛内源性促黄体生成素（LH）的脉冲式分泌，并延迟排卵前LH峰，以研究延长卵泡期对卵泡和卵母细胞质量的影响。12头小母牛用孕激素（诺孕美特）植入物同步发情周期10天。在第4天（第0天=发情日），用24毫克垂体促卵泡素（pFSH）刺激小母牛4天，并在第6天用前列腺素F2α（2毫升氯前列醇）诱导黄体溶解。对照组（n = 4）的动物在最后一次FSH注射后24小时处死。此时，A36h组（n = 4）和A60h组（n = 4）的小母牛分别每12小时用1.6毫克安塔瑞克处理36小时和60小时，然后处死。解剖卵巢卵泡后，收集卵母细胞进行个体体外成熟、受精和培养；收集卵泡液测定类固醇浓度，将颗粒细胞涂片、固定并染色以评估核固缩率。颗粒细胞涂片显示，对照组90%的卵泡健康。相比之下，A36h组分别有36%和58%的卵泡显示早期或晚期闭锁迹象，而A60h组90%的卵泡显示晚期闭锁迹象。卵泡内雌二醇浓度从健康卵泡（799.14±40.65纳克/毫升）降至晚期闭锁卵泡（3.96±0.59纳克/毫升）（P < 0.0001）。无论闭锁程度如何，健康卵泡中的孕酮浓度均高于闭锁卵泡（P < 0.0001）。雌二醇与孕酮的比值从健康卵泡（4.58±0.25）降至晚期闭锁卵泡（0.07±0.009）（P < 0.0001）。对照组卵泡内雌二醇和孕酮的浓度显著高于处理组（P < 0.0001）。对照组的雌二醇与孕酮比值（4.55±0.25）高于A36h组（0.40±0.05）和A60h组（0.07±0.009）（P < 0.0001）。出乎意料的是，对照组（85%、41%和95±8）、A36h组（86%、56%和93±5）和A60h组（88%、58%和79±4）中受精卵母细胞的分裂率、囊胚率和每个囊胚的细胞数没有显著差异。此外，来自健康卵泡（45%）、早期闭锁卵泡（54%）、晚期闭锁卵泡（57%）和晚期闭锁卵泡（53%）的卵母细胞的囊胚率也没有显著差异。总之，用GnRH拮抗剂维持超排小母牛的排卵前卵泡会诱导更多闭锁，并降低雌二醇和孕酮浓度。然而，从这些闭锁卵泡中回收的卵母细胞在体外发育到第8天的潜力并未受到影响。
