King W, Ghosh S, Thomas P, Sullivan C V
Department of Zoology, North Carolina State University, Raleigh 27695, USA.
Biol Reprod. 1997 Jan;56(1):266-71. doi: 10.1095/biolreprod56.1.266.
Previous studies have shown that blood plasma levels of 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP) and 17alpha, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S) increase in striped bass (Morone saxatilis) undergoing final oocyte maturation (FOM). Both hormones are produced by ovarian fragments undergoing hCG-induced germinal vesicle breakdown (GVBD) in vitro. In the present study, we investigated binding of DHP and 20beta-S to ovarian membranes from striped bass undergoing FOM. Saturable binding sites for DHP were not detected. Saturation of 20beta-S binding sites with 5 nM [3H]20beta-S occurred within 40 min at 0 degrees C (at 3 min, half of the maximum specific binding of steroid was calculated to have occurred), and the binding was pH-dependent. Scatchard analyses revealed the presence of a single class of high-affinity (dissociation constant [Kd] = 1.4 +/- 0.2 nM), limited-capacity (estimated concentration [Bmax] = 2.7 +/- 0.3 pmol/g ovary) 20beta-S binding sites on membranes from striped bass ovaries undergoing FOM. In contrast, only low levels of specific binding (Bmax < 0.04 pmol/g tissue) were detected on membranes from testes, liver, brain, and muscle. Ovarian membranes prepared from vitellogenic females also had low levels (Bmax < 0.1 pmol/g ovary) of specific 20beta-S binding, less than 5% of that found during FOM. Results of competition assays showed that DHP was approximately 250 times less effective than 20beta-S for displacing 20beta-S from ovarian membranes. In contrast, 20beta, 21-dihydroxy-4-pregnen-3-one was a very effective competitor, although it is only a weak inducer of oocyte GVBD in vitro. Of several other steroids tested, only progesterone showed affinity for the 20beta-S binding site within a physiological range of concentrations. Taken together with previous studies of striped bass FOM, these findings indicate that 20beta-S is the oocyte maturation-inducing steroid hormone in striped bass.
先前的研究表明，在经历最终卵母细胞成熟（FOM）的条纹鲈（Morone saxatilis）中，血浆中17α,20β-二羟基-4-孕烯-3-酮（DHP）和17α,20β,21-三羟基-4-孕烯-3-酮（20β-S）的水平会升高。这两种激素都是由体外经人绒毛膜促性腺激素（hCG）诱导发生生发泡破裂（GVBD）的卵巢片段产生的。在本研究中，我们研究了DHP和20β-S与经历FOM的条纹鲈卵巢膜的结合情况。未检测到DHP的可饱和结合位点。在0℃下，用5 nM [3H]20β-S可在40分钟内使20β-S结合位点饱和（在3分钟时，计算得出类固醇最大特异性结合的一半已经发生），并且这种结合是pH依赖性的。Scatchard分析显示，在经历FOM的条纹鲈卵巢膜上存在一类单一的高亲和力（解离常数[Kd]=1.4±0.2 nM）、有限容量（估计浓度[Bmax]=2.7±0.3 pmol/g卵巢）的20β-S结合位点。相比之下，在睾丸、肝脏、大脑和肌肉的膜上仅检测到低水平的特异性结合（Bmax<0.04 pmol/g组织）。从卵黄生成期雌性制备的卵巢膜也具有低水平（Bmax<0.1 pmol/g卵巢）的特异性20β-S结合，不到FOM期间发现水平的5%。竞争试验结果表明，DHP从卵巢膜上置换20β-S的效力约为20β-S的1/250。相比之下，20β,21-二羟基-4-孕烯-3-酮是一种非常有效的竞争者，尽管它在体外只是卵母细胞GVBD的弱诱导剂。在测试的其他几种类固醇中，只有孕酮在生理浓度范围内对20β-S结合位点表现出亲和力。结合先前对条纹鲈FOM的研究，这些发现表明20β-S是条纹鲈中诱导卵母细胞成熟的类固醇激素。
