Effects of enclomiphene and zuclomiphene on basal and gonadotrophin-stimulated progesterone secretion by isolated subpopulations of small and large ovine luteal cells.

We examined the effects of enclomiphene and zuclomiphene, alone and in combination with oestradiol, on basal and gonadotrophin-stimulated progesterone secretion by isolated subpopulations of both large (granulosa-lutein) and small (theca-lutein) ovine luteal cells. Isolated large and small luteal cells derived from intact, enucleated ovine corpora lutea were incubated for 48-120 h with or without 22R-hydroxycholesterol or pregnenolone (2.5 microM) and a range of enclomiphene, zuclomiphene, and/or oestradiol concentrations (3-100 microM), both with and without ovine luteinizing hormone (100 ng/ml). Spent media were assayed in duplicate for progesterone content by radioimmunoassay. Enclomiphene, zuclomiphene, and oestradiol exhibited equivalent dose-dependent inhibitory effects on basal and gonadotrophin-stimulated small and large ovine luteal cell progesterone secretion under all substrate conditions. Both cell types became more sensitive to clomiphene inhibition with increasing time in culture. In combined treatments, the effects of oestradiol and either enclomiphene or zuclomiphene became additive in longer-term cultures and were never antagonistic. In this model system, (i) clomiphene, like oestradiol, appears to inhibit 3beta-hydroxysteroid dehydrogenase activity, (ii) both stereoisomers act as oestrogen agonists, (iii) neither demonstrates any anti-oestrogenic properties, and (iv) both large and small luteal cells become more sensitive to clomiphene inhibition with increasing duration of exposure.

Prostaglandin F2 alpha receptor concentrations in corpora lutea of cycling, pregnant, and pseudopregnant pigs.

The objective of this study was to measure and compare the concentrations of PGF 2 alpha receptors on luteal cells taken from cycling, pregnant, and pseudopregnant pigs. Corpora lutea were removed surgically from cycling, pregnant, and pseudopregnant (induced with 5 mg estradiol valerate/day i.m. beginning on Day 11) pigs on Days 12, 13, and 14 (postestrus) and were subjected to collagenase dissociation. Dissociated luteal cells (approximately 100,000 large viable cells per tube) were assayed for specific PGF 2 alpha binding by Scatchard analysis, using [3H]PGF 2 alpha and varying doses (0-5 microM) of unlabeled PGF 2 alpha. Luteal cells from all three types of pigs were shown to possess two specific PGF 2 alpha binding sites (high affinity, Kd = 9-47 nM; low affinity, Kd = 243-1359 nM). The concentrations of the high-affinity PGF 2 alpha binding site (PGF 2 alpha "receptor") on Days 12 and 13 were not significantly different (NS) between cycling (1.7 and 1.1 x 10(6) receptors per large luteal cell, respectively), pregnant (1.3 and 1.2 x 10(6)), and pseudopregnant (1.1 and 0.8 x 10(6)) pigs. However, on Day 14, luteal PGF 2 alpha receptor concentrations were significantly higher (p < 0.05) in cycling (4.2 x 10(6)) compared with pregnant (1.3 x 10(6)) and pseudopregnant (1.4 x 10(6)) pigs. We speculate that reduced luteal PGF 2 alpha receptor concentrations on Day 14 in pregnant and pseudopregnant compared with cycling pigs may lead to decreased luteal sensitivity to PGF 2 alpha in these animals, and that this mechanism may play a role in the maternal recognition of pregnancy in this species.

Effects of individual and combined treatment with prostaglandins E2 and F2 alpha on progesterone secretion by ovine luteal cells supplemented with homologous serum lipoproteins in vitro.

Prostaglandin F2 alpha (PGF2 alpha) is a potent luteolysin, whereas prostaglandin E2 (PGE2) is generally luteotropic in vivo. To establish a model system for investigations of the mechanisms involved in these actions, we examined the effects of individual and combined treatment with PGE2 and PGF2 alpha on basal and ovine LH-stimulated progesterone secretion during long-term incubations conducted with and without supplemental homologous low-density lipoprotein (LDL) as substrate. Effects of both PGF2 and PGF2 alpha were concentration- and time-dependent and were further influenced by the presence of LDL and/or LH in medium. Neither of the prostaglandins exerted any significant effect before 48 h in culture, but distinctly different patterns of response to PGE2 and PGF2 alpha emerged thereafter. Low, but not high, concentrations of PGE2 increased progesterone secretion in the absence of LH, whereas PGF2 alpha (alone and in combination with PGE2) inhibited progesterone production in all medium formulations. The transcriptional inhibitor actinomycin effectively blocked the actions of PGF2 alpha, but had no effect on response to LH or PGE2. These data demonstrate that both the putative luteotropic actions of PGE2 and the potent, luteolytic effects of PGF2 alpha in vivo can be reproduced in long-term cultures of ovine luteal cells in vitro, and they suggest that the mechanism of PGF2 alpha-induced luteolysis may involve new protein synthesis.

Effects of substrate supplementation with hydroxycholesterol analogues and serum lipoproteins on ovine luteal cell progesterone secretion in vitro: demonstration of prostaglandin F2 alpha luteolytic actions in a defined model system.

In an attempt to establish a defined model system for studies aimed at elucidating the mechanism of PGF2 alpha action, we examined the effects of medium supplementation with soluble hydroxycholesterol analogues, alone and in combination with ovine luteinizing hormone (oLH) in the presence and absence of PGF2 alpha, on progesterone secretion by mixed ovine luteal cells in vitro. In short-term cultures (2-6 h), supplementary 22R-hydroxycholesterol (22R-OHC; 0.16-20 micrograms ml-1) increased (P < 0.05) progesterone production in a dose-dependent manner, whereas similar concentrations of 22S-hydroxycholesterol (22S-OHC) and 25-hydroxycholesterol (25-OHC) had little effect. In incubations of < or = 24 h duration, 22R-OHC (1 micrograms ml-1) dramatically increased progesterone secretion, whereas oLH (100 ng ml-1) in the presence or absence of PGF2 alpha (250 ng ml-1) had no consistent effects, alone or in combination with 22R-OHC. In contrast, 22R-OHC (1 micrograms ml-1) alone had no effect in long-term incubations (72-192 h), nor did treatment with oLH (100 ng ml-1) in the presence or absence of PGF2 alpha (250 ng ml-1) in the absence of 22R-OHC. Together, however, 22R-OHC and oLH stimulated (P < 0.05) progesterone secretion, a synergistic effect consistently inhibited (P < 0.05) by PGF2 alpha. Equimolar (2.5 mumol l-1) concentrations of 22R-OHC and homologous serum low- or high-density lipoprotein cholesterol exhibited comparable capacities to maintain progesterone secretion in long-term cultures (24-168 h), with and without gonadotrophin (oLH or human chorionic gonadotrophin, 100 ng ml-1) stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin photoaffinity probes: synthesis and binding affinity of C-18 substituted PGF2 alpha prostanoids bearing a perfluorinated aryl azide.

C-18 Phenoxy analogs of prostaglandin F2 alpha (PGF2 alpha) that possessed a perfluorinated aryl azide and an aryl iodide substituent were synthesized and evaluated as potential photoaffinity probes for PGF2 alpha. Prior studies indicated that only hydrophobic modifications in the omega-side chain of PGF2 alpha were compatible with high binding affinity, and this finding excluded the use of a hydroxyl-substituted C-18 phenoxy group as an activated aryl ring capable of radioiodination. Consequently, an alternate means of introducing the iodine substituent using an ipsosubstitution of a trimethylsilyl arene was developed. Although this strategy was successful from a synthetic perspective, the potential PGF2 alpha photoaffinity probe, (15S)-18-[3'-((4''-azido-2'',3'',5'',6''-tetrafluorophenyl)- methoxy) methyl-5'-iodophenoxy]-19,20-bisnorprostaglandin F2 alpha, exhibited only marginal competitive binding with [3H]-PGF2 alpha to ovine luteal cells and to plasma membranes of bovine corpora lutea. The hydrophobic but bulky C-18 substituent was presumably incompatible with effective receptor binding.

Prostaglandin photoaffinity probes: synthesis and binding affinity of aryl azide-substituted C-1 esters of prostaglandin F2 alpha.

In seeking prostaglandin F2 alpha (PGF2 alpha) photoaffinity probes possessing both an efficient, photoactive cross-linking substituent and a radiolabel of high specific activity, the synthesis and binding affinity of PGF2 alpha C-1 esters in which the alcohol component possessed either an aryl azide or a perfluorinated aryl azide was investigated. These derivatives showed great promise due to their ability to compete for the binding of [3H]-PGF2 alpha in both a luteal membrane binding assay and in a whole luteal cell binding assay. Identification of the C-1 site in PGF2 alpha as a site for modification of the PGF2 alpha molecule with photoactive alcohol derivatives represented a logical step toward the goal of developing a useful PGF2 alpha photoaffinity probe.

Relationship of oestrus synchronization method, circulating hormones, luteinizing hormone and prostaglandin F-2 alpha receptors and luteal progesterone concentration to premature luteal regression in superovulated sheep.

Ewes were treated with exogenous follicle-stimulating hormone (FSH) and oestrus was synchronized using either a dual prostaglandin F-2 alpha (PGF-2 alpha) injection regimen or pessaries impregnated with medroxy progesterone acetate (MAP). Natural cycling ewes served as controls. After oestrus or AI (Day 0), corpora lutea (CL) were enucleated surgically from the left and right ovaries on Days 3 and 6, respectively. The incidence of premature luteolysis was related (P less than 0.05) to PGF-2 alpha treatment and occurred in 7 of 8 ewes compared with 0 of 4 controls and 1 of 8 MAP-exposed females. Sheep with regressing CL had lower circulating and intraluteal progesterone concentrations and fewer total and small dissociated luteal cells on Day 3 than gonadotrophin-treated counterparts with normal CL. Progesterone concentration in the serum and luteal tissue was higher (P less than 0.05) in gonadotrophin-treated ewes with normal CL than in the controls; but luteinizing hormone (LH) receptors/cell were not different on Days 3 and 6. There were no apparent differences in the temporal patterns of circulating oestradiol-17 beta, FSH and LH. High progesterone in gonadotrophin-treated ewes with normal CL coincided with an increase in total luteal mass and numbers of cells, which were primarily reflected in more small luteal cells than in control ewes. Gonadotrophin-treated ewes with regressing CL on Day 3 tended (P less than 0.10) to have fewer small luteal cells and fewer (P less than 0.05) low-affinity PGF-2 alpha binding sites than sheep with normal CL. By Day 6, luteal integrity and cell viability was absent in ewes with prematurely regressed CL. These data demonstrate that (i) the incidence of premature luteal regression is highly correlated with the use of PGF-2 alpha; (ii) this abnormal luteal tissue is functionally competent for 2-3 days after ovulation, but deteriorates rapidly thereafter and (iii) luteal-dysfunctioning ewes experience a reduction in numbers of small luteal cells without a significant change in luteal mass by Day 3 and, overall, have fewer low-affinity PGF-2 alpha binding sites.

cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation.

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.

Interaction of leukotriene C4 and Chinese hamster lung fibroblasts (V79A03 cells). 1. Characterization of binding.

A novel, specific, and potent biological action of leukotriene C4 (LTC4) was demonstrated in the Chinese hamster lung fibroblast cell line V79A03 (V79 cells), namely the confirment of protection against subsequent gamma-irradiation. Consequently, studies were conducted to determine whether LTC4-conferred radioprotection could be attributed to a receptor-mediated phenomenon. Specific binding sites for leukotriene C4 (LTC4) were identified and characterized using intact V79 cells incubated at 4 degrees C in the presence of serine-borate, during which time conversion of LTC4 to LTD4 or LTE4 was undetectable. Binding was maximal in a broad region between pH 6.2 and 8.8. Ca2+, Mg2+, and Na+ were not required for binding, and binding was not altered by GTP, ATP, or cAMP, by leukotrienes B4, D4, or E4, or by the leukotriene end point antagonists LY 171883, FPL 55712, or Revlon 5901-5. Scatchard analyses and kinetic experiments indicated the presence of high-affinity [Kd = 2.5 +/- 0.63 nM, approximately 9.9 x 10(5) sites/cell] and low-affinity [Kd = 350 +/- 211 nM, approximately 2.7 x 10(6) sites/cell] binding sites. The observed binding characteristics of LTC4 to V79 cells are consistent with a receptor-mediated phenomenon. In a companion communication which follows this report, we report the subcellular distribution of LTC4 binding to V79 cells and demonstrate that this binding is unlikely to be attributed principally to interaction with glutathione-S-transferase.

Interaction of leukotriene C4 and Chinese hamster lung fibroblasts (V79A03 cells). 2. Subcellular distribution of binding and unlikely role of glutathione-S-transferase.

It was reported previously that radiation-induced cytotoxicity in V79A03 (V79) cells was attenuated by pretreatment of cells with leukotriene C4 (LTC4), leading us to determine that V79 cells possessed specific binding sites, with characteristics of receptors, for LTC4 (see the preceding, companion communication). Additional studies were conducted to determine the subcellular distribution and the chemical nature of the LTC4 binding site in V79 cells. Trypsin treatment of cells before LTC4 binding assays resulted in a 74% reduction in high-affinity binding. In tests to examine the subcellular location of LTC4 binding, plasma membrane and nuclear fractions were obtained from V79 cells. In contrast to Scatchard analyses of LTC4 binding to intact cells which were curvilinear, Scatchard analyses of nuclear and plasma membrane fractions were linear, indicative of the presence in these cellular substituents of low and high-affinity binding, respectively. To examine the nature of the high-affinity LTC4 binding sites, intact V79 cells were photolyzed with [3H]-LTC4 rendered photoactive by preincubation with N-hydroxysuccinimidyl-4-azidobenzoate. The cell-bound radioactivity migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of approximately 40 kdal. Five different commercial preparations of glutathione-S-transferase (GST), which has been implicated as a source of LTC4 "specific binding" in other cells, migrated in the same SDS-PAGE system with an apparent molecular weight of 20-24 kdal. Furthermore, preincubations of V79 cells with three antisera generated against GST had minimal effects upon subsequent LTC4 binding to intact cells. These data, taken together with the data from the preceding companion communication, suggest that the radioprotective effect of LTC4 upon V79 cells may be attributable to a receptor-mediated phenomenon which appears distinct from leukotriene binding to GST.

Prostaglandin F2 alpha receptors on enzyme-dissociated pig luteal cells throughout the estrous cycle.

A deficiency of luteal cell prostaglandin F2 alpha (PGF2 alpha) receptors might help explain the well documented refractoriness of pig corpora lutea to the luteolytic effects of PGF2 alpha administered in vivo before day 12 of the estrous cycle. Accordingly, experiments were conducted to measure the levels of [3H] PGF2 alpha-binding sites/receptors on collagenase-dispersed pig luteal cells taken at different stages of the estrous cycle. Pig corpora lutea were obtained surgically at various stages of the estrous cycle and dissociated with collagenase in medium 199. Dissociated mixed luteal cells (approximately 5-15 x 10(4) large luteal cells/tube) were assayed for [3H]PGF2 alpha-binding activity by saturation (Scatchard) analysis. In preliminary experiments it was determined that PGF2 alpha binding was maximal after incubation for 45 min at 30 C in assay buffer of pH 5.75. Additionally, it was determined that [3H]PGF2 alpha binding was displaceable by PGF2 alpha = PGD2 greater than PGE2 greater than 13,14-dihydro-15-keto-PGF2 alpha. Other eicosanoids did not inhibit [3H]PGF2 alpha binding. Two distinct classes of binding sites (high affinity Kd = 19-64 nM; low affinity Kd = 262-3103 nM) were observed at all stages of the estrous cycle. From studies using enriched (by elutriation) large (greater than 30 microns) and small (10-20 microns) luteal cells it appeared that the high affinity binding site was largely confined to large luteal cells, whereas the low affinity binding site was found on both large and small luteal cells. The concentrations (number per large luteal cell) of high affinity PGF2 alpha-binding sites of mixed (unelutriated) luteal cell preparations was low on days 6-8 (0.6 x 10(6) sites/cell) and increased gradually up to 1.4 x 10(6) sites/cell on day 12. The concentrations of binding sites were increased approximately 3-fold on day 13 (4.6 x 10(6) sites/cell; P less than 0.05 vs days 6-12) and remained elevated on days 14 and 16-17 (approximately 3 x 10(6) sites/cell). In summary, these results indicate the existence of a high affinity PGF2 alpha-binding site in pig (large) luteal cells, which is probably the luteal PGF2 alpha receptor. The numbers of these putative PGF2 alpha receptors are low during the early luteal phase (before day 12), but increase thereafter (days 13, 14, and 16-17). This may provide one explanation for the observed refractoriness in vivo of pig corpora lutea to PGF2 alpha before, and increased sensitivity to PGF2 alpha after, day 12 of the estrous cycle.

Multiple classes of prostaglandin F2 alpha binding sites in subpopulations of ovine luteal cells.

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 degrees C, and dispersed cell preparations were frozen in a programmable cell freezer and stored at -196 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural requirements for prostaglandin analog interaction with the ovine corpus luteum prostaglandin F2 alpha receptor. Implications for development of a photoaffinity probe.

The capacity of structurally modified analogs of prostaglandin F2 alpha (PGF2 alpha) to inhibit binding of [3H]PGF2 alpha to receptors on ovine luteal cells was evaluated by radioreceptor assay using dispersed, viable, ovine luteal cells. Binding assays were conducted at pH 5.75, since binding to both high (Kd 17.4 +/- 2.3 nM) and low (Kd 409 +/- 166 nM) affinity sites was enhanced markedly at reduced pH. The capability to compete with [3H]PGF2 alpha for binding was evaluated for different prostaglandin analogs having modifications in the C-8 "upper" side-chain, in the cyclopentane ring, or in the C-12 "lower" side-chain. Prostaglandin J2 was a surprisingly potent competitor for binding to the PGF2 alpha receptor. Several phenyl-substituted analogs exhibited receptor-binding potency greater than or equal to native PGF2 alpha, while most other analogs had reduced capacity to compete with native PGF2 alpha for binding. Several 17-azidophenol PGF2 alpha analogs were synthesized and tested, but analogs having hydroxyl groups on the aryl ring had low affinity for receptors. However, 17-(4-azidophenyl)-18,19,20-trinor-PGF2 alpha as well as 17-(3-iodo-4-azidophenyl)-18,19,20-trinor-PGF2 alpha exhibited binding affinities that were approximately 10% of native PGF2 alpha, and the radioiodinated analogs of PGF2 alpha may be useful as probes of the PGF2 alpha receptor.

Physiologic characterization of transformed and cloned rat granulosa cells.

Properties of a clonal line of SV40-transformed rat granulosa cells (DC3 cells) were elucidated. DC3 cells were maintained in vitro in Iscove Modified Dulbecco Medium that contained 20% fetal bovine serum. The cells had a logarithmic growth phase doubling time of approximately 18 h and produced detectable quantities of estrone, estradiol, and progesterone. Steroidogenesis was increased by supplementation with either steroidogenic substrates or agents that stimulated activity of adenylate cyclase. Production of progesterone and estrogens was enhanced when medium was supplemented with 25-hydroxycholesterol, and production of estradiol was enhanced by medium supplementation with androstenedione. Treatments with forskolin and cholera toxin resulted in marked increases of cyclic adenosine 3',5'-monophosphate (cAMP) in medium and cells and enhanced steroidogenesis. Isoproterenol and vasoactive intestinal peptide, but not follicle-stimulating hormone (FSH), luteinizing hormone (LH), insulin or prolactin, stimulated cAMP secretion by suspended cells. DC3 cells had small but detectable levels of binding to FSH, but binding of LH and epidermal growth factor could not be detected. DC3 cells possess characteristics expected of granulosa cells arrested in an early stage of differentiation and may provide a useful model for studies of "immature" granulosa cell functions.

Prostaglandin photoaffinity probes: synthesis and biological activity of azide-substituted 16-phenoxy- and 17-phenyl-PGF2 alpha prostaglandins.

The development of a prostaglandin PGF2 alpha photoaffinity probe led to the synthesis and biological evaluation of azide-substituted 17-phenyl-18,19,20-trinorprostaglandin F2 alpha and 16-phenoxy-17,18,19,20-tetranorprostaglandin F2 alpha derivatives. Two approaches for the preparation of iodinated versions of these prostaglandins were evaluated: (1) iodination of a phenyl azide bearing an activating hydroxyl group and (2) iodination of an aniline precursor to the phenyl azide group and subsequent conversion of the aniline to the phenyl azide. In the first approach, 17-(4-azido-2-hydroxyphenyl)-18,19,20-trinorprostaglandin F2 alpha, 16-(5-azido-3-hydroxyphenoxy)-17,18,19,20-tetranorprostaglandin F2 alpha, and 16-(4-azido-2-hydroxyphenoxy)-17,18,19,20-tetranorprostaglandin F2 alpha were prepared by using the Corey synthesis, but were biologically inactive presumably as a result of the hydrophilic phenolic hydroxyl group. In the second approach, the iodination of a 17-(4-aminophenyl)-18,19,20-trinorprostaglandin F2 alpha derivative delivered 17-(4-azido-3-iodophenyl)-18,19,20-trinorprostaglandin F2 alpha, which exhibited competitive binding with natural [3H]PGF2 alpha to ovine luteal cells and to plasma membranes of bovine corpora lutea. [125I]-17-(4-Azido-3-iodophenyl)-18,19,20-trinorprostaglandin F2 alpha was utilized in a preliminary photoaffinity cross-linking experiment.

Cytosolic receptor for estradiol in the corpus luteum of the ewe: variation throughout the estrous cycle and distribution between large and small steroidogenic cell types.

This study was designed to: 1) determine the concentration of the cytosolic receptor for estradiol-17 beta (E2 receptor) in the ovine corpus luteum throughout the estrous cycle and 2) determine the contents of E2 receptor in large and small steroidogenic and nonsteroidogenic luteal cells. Our data confirm the existence of a saturable, high-affinity receptor specific for estrogen in the cytosol fraction of ovine corpus luteum. The concentration of E2 receptor was assessed in corpora lutea collected at Days 4, 6, 8, 12 and 16 postestrus; it was lowest at Day 4 (6.9 +/- 0.5 fmol/mg protein) and maximal (19.0 +/- 2.8 fmol/mg protein) at Day 8 (P less than 0.05). The concentration was decreased somewhat at Day 12 (11.8 +/- 3.1 fmol/mg protein), but was higher again at Day 16 (18.0 +/- 4.6 fmol/mg protein). The affinity constants were not different between Days 5 and 15 (P less than 0.3). The large steroidogenic cells contained a 3.5X higher concentration of cytosolic E2 receptor than did the small steroidogenic cells at Day 10 (27.7 +/- 5.8 vs. 7.9 +/- 4.0 fmol/mg protein; P less than 0.05). There was no apparent difference between the affinity constants of the E2 receptor in large and small cells. The differences in concentration of cytosolic E2 receptor throughout the estrous cycle and the higher concentrations of the E2 receptor in the large steroidogenic cells suggests that estradiol may play a role in regulating luteal function.

Interactions of prostaglandins with subpopulations of ovine luteal cells. I. Stimulatory effects of prostaglandins E1, E2 and I2.

Preparations of small and large steroidogenic cells from enzymatically dispersed ovine corpora lutea were utilized to study the in vitro effects of luteinizing hormone (LH) and prostaglandins (PG) E1, E2 and I2. Cells were allowed to attach to culture dishes overnight and were incubated with either LH (100 ng/ml), PGE1, PGE2, or PGI2 (250 ng/ml each). The secretion of progesterone by large cells was stimulated by all prostaglandins tested (P less than 0.05) while the moderate stimulation observed after LH treatment was attributable to contamination of the large cell population with small cells. Prostaglandins E1 and E2 had no effect on progesterone secretion by small cells, while LH was stimulatory at all times (0.5 to 4 hr) and PGI2 was stimulatory by 4 hr. Additional studies were conducted to determine if the effects of PGE2 upon steroidogenesis in large cells were correlated with stimulated activity of adenylate cyclase. In both plated and suspended cells PGE2 caused an increase (P less than 0.05) in the rate of progesterone secretion but had no effect upon the activity of adenylate cyclase or cAMP concentrations within cells or in the incubation media. Exposure of luteal cells to forskolin, a nonhormonal stimulator of adenylate cyclase, resulted in marked increases in all parameters of cyclase activity but had no effect on progesterone secretion. These data suggest that the actions of prostaglandins E1, E2 and I2 are directed primarily toward the large cells of the ovine corpus luteum and cast doubt upon the role of adenylate cyclase as the sole intermediary in regulation of progesterone secretion in this cell type.