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 F(2α) (PGF(2α)) 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 PGF(2α) binding characteristics. From saturation analyses (increasing amounts of radiolabeled PGF(2α) of PGF(2α) binding to unfractionated cells, we detected a single class of high affinity binding sites (K(d) = 17.4 ± 2.3 nM) in addition to the nonspecific binding component. Using displacement analyses (constant radiolabeled PGF(2α) and increasing amounts of unlabeled PGF(2α)) and unfractionated cells, we detected additional binding sites of lower affinity (K(d) = 409 ± 166 nM) as well as the nonspecific binding component. Small luteal cells obtained by elutriation, which were essentialy devoid of large cell contamination, had only low affinity binding sites. Large luteal cells bound PGF(2α) pedominantly 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 PGF(2α) receptor occupancy in the presence of nanomolar PGF(2α) concentrations. Significant occupancy of the lower affinity sites on small luteal cells would require higher concentrations of PGF(2α) approaching the micromolar range. Differential binding of PGF(2α) by the two luteal cell types is suggestive of functional differences during events such as luteolysis that involve interaction of the corpus luteum and PGF(2α).
|Number of pages||8|
|Journal||Biology of Reproduction|
|State||Published - 1989|
ASJC Scopus subject areas
- Reproductive Medicine