Multiple classes of prostaglandin F2 alpha binding sites in subpopulations of ovine luteal cells.
Multiple classes of prostaglandin F2 alpha binding sites in subpopulations of ovine luteal cells.
复制标题
绵羊黄体细胞亚群中多类前列腺素 F2 α 结合位点。
DOI：
10.1095/biolreprod41.3.385
复制
发表时间：
1989
影响因子：
3.6
通讯作者：
Fitz,TA
中科院分区：
文献类型：
--
作者：
Balapure,AK;Caicedo,IC;Kawada,K;Watt,DS;RexroadJr,CE;Fitz,TA
一种冷冻保存程序的开发，以提供绵羊黄体细胞在整个季节性乏情期。从黄体中期获得的黄体，超数排卵的母羊酶促分散。一些分散的细胞通过淘洗分成亚群。在4 °C下，将Hanks缓冲盐水中的二甲亚砜（7.5%终浓度）加入细胞中。并将分散的细胞制备物在可编程细胞冷冻器中冷冻并储存在-196 °C下。冻存复苏后，细胞活力和前列腺素F2α（PGF 2 α）结合特性与新鲜细胞无明显差异。此外，解冻的细胞保留的能力，附着到培养皿和保留的反应性孕酮分泌前列腺素E2（PGE 2）和绵羊促黄体生成素（LH），虽然孕酮分泌率在解冻相比，新鲜细胞减弱。本研究通过研究绵羊黄体细胞与前列腺素F2 α的结合特性，对绵羊黄体细胞的冻存效果进行了评价。通过对PGF 2 α与未分级细胞结合的饱和度分析（放射性标记的PGF 2 α的量增加），我们检测到除非特异性结合组分外的一类高亲和力结合位点（Kd= 17.4 ± 23 nM）。使用置换分析（恒定的放射性标记PGF 2 α和增加量的未标记PGF 2 α）和未分级分离的细胞，我们检测到了低亲和力（Kd= 409 ± 166 nM）的额外结合位点以及非特异性结合组分。通过淘洗获得的小黄体细胞基本上没有大细胞污染，只有低亲和力结合位点。大黄体细胞主要在高亲和力位点结合PGF 2 α，但具有与制备物中存在的小细胞比例相称的低亲和力组分。在体内，在存在纳摩尔PGF 2 α浓度的情况下，绵羊大黄体细胞可实现可观的PGF 2 α受体占用。小黄体细胞上低亲和力位点的显著占据需要接近微摩尔范围的较高浓度的PGF 2 α。两种黄体细胞对PGF 2 α的不同结合提示了在黄体溶解等事件中的功能差异，这些事件涉及黄体和PGF 2 α的相互作用
A cryostorage procedure was developed to provide ovine luteal cells throughout the period of seasonal anestrus. Corpora lutea obtained from midluteal phase, superovulated ewes were dispersed enzymatically. Some dispersed cells were fractionated into subpopulations by elutriation. Dimethylsulfoxide (7.5% final concentration) in Hanks’ buffered saline was added to cells at 4 °C. and dispersed cell preparations were frozen in a programmable cell freezer and stored at −196 °C . After recovery from cryopreservation, cell viability and prostaglandin F2α(PGF2α) binding characteristics of thawed cells were not different from those of corresponding fresh cells. Additionally, thawed cells retained the capacity to attach to culture dishes and retained responsiveness of progesterone secretion to prostaglandin E2(PGE2) and ovine luteinizing hormone (LH), although rates of progesterone secretion were attenuated in thawed compared with fresh cells. The cryopreservation procedure will prove useful to relieve constraints in utilization of ovine luteal cells arising from reproductive seasonality in sheep.Cells retrieved from cryostorage were evaluated by studying PGF2αbinding characteristics. From saturation analyses (increasing amounts of radiolabeled PGF2α) of PGF2αbinding to urtfractionated cells, we detected a single class of high affinity binding sites (Kd= 17.4 ± 23 nM) in addition to the nonspecific binding component. Using displacement analyses (constant radiolabeled PGF2αand increasing amounts of unlabeled PGF2α) and unfractionated cells, we detected additional binding sites of lower affmity (Kd= 409 ± 166 nM) as well as the nonspecific binding component. Small luteal cells obtained by elutriation, which were essentially devoid of large cell contamination, had only low affinity binding sites. Large luteal cells bound PGF2αpredominantly at highaffinity sites but had a low affinity component that was commensurate with the proportion of small cells present in the preparation. In vivo, ovine large luteal cells may achieve appreciable PGF2αreceptor occupancy in the presence of nanomolar PGF2αconcentrations. Significant occupancy of the lower affinity sites on small luteal cells would require higher concentrations of PGF2αapproaching the micromolar range. Differential binding of PGF2αby the two luteal cell types is suggestive of functional differences during events such as luteolysis that involve interaction of the corpus luteum and PGF2α