Multiple Classes of Prostaglandin F2α Binding Sites in Subpopulations of Ovine Luteal Cells1

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Abstract
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.degree. C, and dispersed cell preparations were frozen in a programmable cell freezer and stored at -196.degree. C. After recovery from cryopreservation, cell viability and prostaglandin F2.alpha.(PGF2.alpha.) 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 PGF2.alpha. binding characteristics. From saturation analyses (increasing amounts of radiolabeled PGF2.alpha.) of PGF2.alpha. binding to unfractionated cells, we detected a single class of high affinity binding sites (Kd = 17.4 .+-. 2.3 nM) in addition to the nonspecific binding component. Using displacement analyses (constant radiolabeled PGF2.alpha. and increasing amounts of unlabeled PGF2.alpha.) and unfractionated cells, we detected additional binding sites of lower affinity (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 PGF2.alpha. predominantly at high-affinity 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 PGF2.alpha. receptor occupancy in the presence of nanomolar PGF2.alpha. concentrations. Significant occupancy of the lower affinity sites on small luteal cells would require higher concentrations of PGF2.alpha. approaching the micromolar range. Differential binding of PGF2.alpha. by the two luteal cell types is suggestive of functional differences during events such as luteolysis that involve interaction of the corpus luteum and PGF2.alpha.