Webb Molly A H, Feist Grant W, Trant John M, Van Eenennaam Joel P, Fitzpatrick Martin S, Schreck Carl B, Doroshov Serge I
Department of Animal Science, One Shields Avenue, University of California, Davis, CA 95616, USA.
Gen Comp Endocrinol. 2002 Oct 15;129(1):27-38. doi: 10.1016/s0016-6480(02)00508-7.
Ovarian follicles and plasma were collected from two female white sturgeon, Acipenser transmontanus, injected with sturgeon pituitary homogenate followed 12h later with GnRHa to induce ovulation. The oocytes of one female underwent germinal vesicle breakdown (GVBD) but ovulation did not occur in response to hormonal stimulation (Female 1), while the oocytes of the other female underwent GVBD and ovulation (Female 2). Follicles collected 12h after the first injection to induce ovulation were incubated with radioinert pregnenolone (P5) or tritiated-P5 ([3H]P5) plus radioinert P5. Steroids were extracted from media and intact follicles, and the extracts were fractionated by high performance liquid chromatography (HPLC). Fractions from the incubation with radioinert precursor were used in a bioassay to determine the potency of the steroid products to induce GVBD. Plasma levels of testosterone (T), estradiol, and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) were measured by radioimmunoassay during induced ovulation, and plasma collected at the time of ovulation (actual or expected) was analyzed by HPLC. A peak in plasma 17,20beta-P was detected at the time of the second injection to induce ovulation in Female 2 (the time at which follicles were collected for incubation with [3H]P5). The HPLC analysis revealed several progestins in the plasma of Female 2 at ovulation that were not present in Female 1 at the time of expected ovulation. A variety of C19 and C21 steroids were produced in vitro by ovarian follicles from both females. The "suggestive" identities of the major metabolites were 11-deoxycortisol, androstenedione, 17-hydroxyprogesterone (17OHP), and 17,20beta-P in Female 1 and cortisol, 17,20beta, 21-trihydroxyprogesterone (20beta-S), 11-deoxycortisol, T, 17OHP, and 17,20beta-P in Female 2. Several of the steroids were active in a GVBD bioassay, but the fractions that contained the steroid coeluting the authentic 11-deoxycortisol on the HPLC and 17,20beta-P (positively identified by gas chromatography-mass spectrometry) were found to be the most potent. The results from this study combined with the results of Webb et al. (2001b) suggest the potential roles of 11-deoxycortisol, 17,20beta-P, 20beta-S, and P4 as maturation-inducing steroids in sturgeon.
从两条雌性白鲟（Acipenser transmontanus）中采集卵巢卵泡和血浆，先给它们注射鲟鱼垂体匀浆，12小时后再注射促性腺激素释放激素类似物（GnRHa）以诱导排卵。其中一条雌性的卵母细胞经历了生发泡破裂（GVBD），但对激素刺激没有发生排卵（雌性1），而另一条雌性的卵母细胞经历了GVBD并排卵（雌性2）。在首次注射诱导排卵12小时后收集的卵泡与放射性惰性孕烯醇酮（P5）或氚标记的P5（[3H]P5）加放射性惰性P5一起孵育。从培养基和完整卵泡中提取类固醇，提取物通过高效液相色谱（HPLC）进行分离。与放射性惰性前体孵育的馏分用于生物测定，以确定类固醇产物诱导GVBD的效力。在诱导排卵期间通过放射免疫测定法测量血浆中睾酮（T）、雌二醇和17,20β-二羟基-4-孕烯-3-酮（17,20β-P）的水平，并对排卵时（实际或预期）采集的血浆进行HPLC分析。在给雌性2注射第二次诱导排卵时（即收集卵泡与[3H]P5孵育的时间）检测到血浆17,20β-P出现峰值。HPLC分析显示，雌性2排卵时血浆中有几种孕激素，而在预期排卵时雌性1的血浆中不存在这些孕激素。两条雌性的卵巢卵泡在体外都产生了多种C19和C21类固醇。在雌性1中，主要代谢产物的“推测”身份为11-脱氧皮质醇、雄烯二酮、17-羟孕酮（17OHP）和17,20β-P；在雌性2中为皮质醇、17,20β,21-三羟基孕酮（20β-S）、11-脱氧皮质醇、T、17OHP和17,20β-P。几种类固醇在GVBD生物测定中具有活性，但发现在HPLC上与 authentic 11-脱氧皮质醇和17,20β-P（通过气相色谱-质谱法确证）共洗脱的类固醇馏分效力最强。本研究结果与Webb等人（2001b）的结果表明，11-脱氧皮质醇、17,20β-P、20β-S和P4在鲟鱼中作为成熟诱导类固醇的潜在作用。
