Assessment of the mechanism by which prolactin stimulates progesterone production by early corpora lutea of pigs.

Previously, we reported that administration of prolactin (PRL) during the early luteal phase in sows increases plasma progesterone concentrations. In the current study, we searched for the mechanisms by which PRL exerts this luteotrophic effect. The objectives of the study were (1) to examine the effect of PRL and/or low-density lipoproteins (LDL) on progesterone production by porcine luteal cells derived from early corpora lutea, and (2) to assess the ability of PRL to activate phosphoinositide-specific phospholipase C (PI-PLC) and protein kinase C (PKC) in these luteal cells. Ovaries with early corpora lutea (day 1-2 of the oestrous cycle) were obtained from the slaughterhouse. Progesterone production by dispersed luteal cells was measured after treatment with PRL, phorbol 12-myristate 13-acetate or inhibitors of PKC in the presence or absence of LDL. LDL increased progesterone concentration in the incubation medium (304.5 vs 178.6 ng/ml in control, P<0.05). PRL augmented LDL-stimulated progesterone secretion by luteal cells (to 416 ng/ml, P<0.05), but PRL alone did not affect progesterone production (209.6 ng/ml, P>0.05). Staurosporine, a PKC inhibitor, inhibited progesterone secretion stimulated by the combined action of LDL and PRL; however, such inhibition was not demonstrated when cells were treated with the PKC inhibitor, H-7. PKC activation was assessed by measuring the specific association of [H]phorbol dibutyrate (H-PDBu) with luteal cells after treatment with PRL or ionomycin (a positive control). PRL and ionomycin increased H-PDBu-specific binding in early luteal cells by 28+/-5.5% (within 5 min) and 70.2+/-19.3% (within 2 min) over control binding respectively (P<0.05). In addition, PRL did not augment the LDL-stimulated progesterone production in PKC-deficient cells. In contrast with PKC, total inositol phosphate accumulation, as well as intracellular free calcium concentrations, were not affected by PRL in the current study. We conclude that PRL, in the presence of LDL, stimulates progesterone production by early corpora lutea in vitro. Moreover, PRL appears to activate PKC, but not PI-PLC, in these cells. Thus intracellular transduction of the PRL signal may involve activation of PKC that is not dependent on PI-PLC.

Depletion of vitamin C from pig corpora lutea by prostaglandin F2 alpha-induced secretion of the vitamin.

The luteolytic effects of prostaglandin F2 alpha (PGF2 alpha) are thought to be mediated in part by the promotion of an increasingly oxidative cellular environment. Loss of antioxidants is one mechanism by which PGF2 alpha might induce or exacerbate oxidative damage within the corpus luteum. This study was performed to establish whether depletion of vitamin C is an acute effect of PGF2 alpha on the pig corpus luteum and to gain insight into the mechanism of luteal vitamin C loss at luteolysis. Gilts (n = 4) were anaesthetized and both utero-ovarian veins and an ear vein were catheterized. Each corpus luteum on the treated ovary received an intraluteal injection of PGF2 alpha (1 microgram) followed by a sustained release implant containing 100 micrograms of the prostaglandin. The other ovary served as the control and each corpus luteum received corresponding volumes of injection vehicle and blank implant. Blood was collected from the ear vein and both utero-ovarian veins every 15 min beginning 15 min before the onset of treatment. Collection of blood stopped when animals were ovariectomized and corpora lutea were collected at 2 h after treatment. Progesterone and vitamin C (ascorbate) concentrations were measured in tissue and plasma samples. PGF2 alpha-treated luteal tissue had similar progesterone, but significantly lower ascorbate, concentrations when compared with control corpora lutea. PGF2 alpha treatment resulted in a rapid and sustained increase in plasma ascorbate within the treatment-side utero-ovarian vein, while the control utero-ovarian vein and ear vein showed little change in plasma ascorbate during the experimental period. No effect of PGF2 alpha on plasma progesterone was evident. This finding suggests that PGF2 alpha depletes the pig corpus luteum of vitamin C by inducing secretion of the vitamin into the bloodstream. Further studies are necessary to determine whether the depletion of vitamin C that is induced by PGF2 alpha contributes to the demise of the pig corpus luteum.

Arachidonic acid inhibits hCG-stimulated progesterone production by corpora lutea of primates: potential mechanism of action.

Arachidonic acid (AA) is a precursor of metabolites known to affect the corpus luteum (CL) in many species, including primates. We have shown that some of these products (prostaglandins F2 alpha and E2) inhibit pro-gesterone (P4) production and activate the phosphatidylinositol (PI) pathway in CL of rhesus monkeys. A direct role of AA in luteal function has also been suggested. The current experiments were designed to investigate the effect of AA on P4 synthesis and to examine the ability of AA to activate the PI pathway in CL of rhesus monkeys. Basal and hCG-stimulated P4 production by luteal cells collected during the midluteal phase was measured after treatment with AA (1, 5, and 10 microM) or linoleic acid (1, 5, and 10 microM). Dispersed cells (50,000/tube) were incubated at 37 degrees C for 2 h. AA elicited a dose-dependent decrease in hCG-stimulated, but not in basal, P4 production. hCG-stimulated P4 production was reduced (P < 0.01) at AA doses of 5 microM (12.1 +/- 1.5 ng/mL) and 10 microM (8.6 +/- 1.8 mg/mL) to hCG alone (18 +/- 1.6 ng/mL). There was no significant effect of 1 microM AA (15.2 +/- 1.6). Response to linoleic acid was dissimilar and was not dose-dependent. Viability of cells was not affected by any treatment. Indomethacin, a prostaglandin synthesis inhibitor, and nordihydroguaiaretic acid, an inhibitor of lipoxygenase, did not interfere with the inhibitory effect of AA. Activation of the PI pathway was assessed by monitoring the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol phosphates and by monitoring increases in intracellular free calcium concentrations ([Ca2+]i) in individual cells. Moreover, the ability of AA to activate protein kinase C (PKC) in luteal cells was measured using a [3H]phorbol dibutyrate (PDBu) binding assay. AA did not alter PIP2 hydrolysis or [Ca2+]i, however, AA (10 microM) increased specific binding of [3H]PDBu to luteal cells (P < 0.05). We conclude that AA inhibits hCG-stimulated P4 production by primate luteal cells. AA exerts this action without being converted to prostaglandins or leukotrienes. This inhibition may be mediated through the activation of PKC. These results suggest a possible role for AA in the regulation of luteal function in primates, and that PKC-activation by AA may promote its effects.

Acute versus chronic effects of human chorionic gonadotrophin on relaxin secretion in rhesus monkeys.

Corpora lutea (CL) were removed from rhesus monkeys (N = 26) at 0 h, 9 h, 3 days, 6 days and 10 days during treatment with hCG to simulate blood concentrations of CG during normal pregnancy. Dispersed luteal cells were incubated in vitro at 37 degrees C for 8 h. Immunoreactive relaxin was measured in incubation medium and in cell extract by radioimmunoassay (RIA). Cellular content and release of relaxin into medium increased as simulated early pregnancy progressed. By 3 days, relaxin content had significantly increased (P less than 0.05) and continued to rise throughout simulated early pregnancy. Significant increases in cellular content and release were observed before the time when relaxin has been detected in the peripheral circulation during this treatment regimen. Within group, total relaxin (cells plus medium) was similar before and after incubation (P greater than 0.05). As such, production of relaxin during the 8-h incubation was not evident. In-vitro exposure of the luteal cells to hCG or dbcAMP had no acute effect on cell content or medium concentration of relaxin at any stage of simulated early pregnancy. Since acute effects of hCG and dbcAMP were not evident in vitro, a sustained gonadotrophic influence may be necessary to augment relaxin production/secretion in the primate CL.

Luteal production of steroids and prostaglandins during simulated early pregnancy in the primate: differential regulation of steroid production by chorionic gonadotropin.

Stimulation of the primate corpus luteum (CL) by endogenous chorionic gonadotropin (CG) in early pregnancy, or by exogenous human (h)CG in simulated early pregnancy, results in a transient elevation of serum progesterone (P) and a persistent elevation of serum 17 beta-estradiol (E). Luteal prostaglandins (PG) may play a role in these responses. The objective of the current study was to correlate luteal PG production and steroidogenic response of CL in vitro with patterns of serum steroids during simulated early pregnancy. CL were removed from rhesus monkeys (n = 26) at 0 h, 9 h, 3 days, 6 days, and 10 days, during prolonged CG exposure of simulated early pregnancy. Dispersed cells were incubated in vitro at 37 degrees C for 8 h. Changes in basal production of P were not significantly correlated with patterns of serum steroids. Maximal stimulation of P production by hCG in vitro (stimulated minus basal) continuously declined (p less than 0.01) from 0 h (means +/- SE, 59.6 +/- 17.9 ng/ml) to 10 days (4.7 +/- 1.8 ng/ml) of simulated early pregnancy. In contrast to patterns of response to hCG, the level of enhancement in P production in response to a maximally stimulatory dose of dibutyryl (db) cyclic adenosine 3',5'-monophosphate (cAMP) declined (p less than 0.05) from 0 h (52.4 +/- 17.6 ng/ml) to 3 days (20.3 +/- 8.4 ng/ml), but was maintained through 10 days (23.7 +/- 11.6 ng/ml) of simulated early pregnancy. As such, desensitization to gonadotropin, which occurred in terms of P production, appears to involve an event subsequent to stimulation of adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin production by corpora lutea of rhesus monkeys: characterization of incubation conditions and examination of putative regulators.

Prostaglandins (PGs) are produced by the corpus luteum (CL) of many domestic and laboratory species and may play a role in CL regulation. The production of PGs by luteal tissue of the rhesus monkey has yet to be clearly elucidated. The production of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha by CL from rhesus monkeys and the incubation conditions (time and cell number) that permit assessment of their synthesis were examined. CL (n = 3 per characterization) were surgically removed from nonpregnant monkeys during the mid-luteal phase of the menstrual cycle (approximately 8-10 days after ovulation). Luteal tissue was dissociated and the cells were incubated at varying concentrations for increasing periods of time at 37 degrees C. Subsequent to defining incubation conditions, various exogenous factors were examined for their potential to modify PG production. Indomethacin, calcium ionophore, human chorionic gonadotropin (hCG), estradiol-17 beta (E2), progesterone (P), testosterone (T), dihydrotestosterone (DHT), and 1-4-6 androstatriene-3, 17-dione (ATD) were incubated with luteal cells in increasing doses. PG and P concentrations in the medium were determined by radioimmunoassay. PGs in the medium after 6 h incubation were detectable at all cell concentrations tested (50,000, 100,000, 200,000 cells/tube). Concentrations of PGs and P increased with cell number (p less than 0.05). Luteal cells (50,000 cells/tube) were incubated for times of 0-24 h. Concentrations of P, PGE2, and PGF2 alpha in the medium were relatively low prior to incubation (0 h), increased (p less than 0.05) linearly within the first 6-12 h, and plateaued through the remaining 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of subpopulations of luteal cells obtained from cyclic and superovulated ewes.

Peripheral blood samples were collected daily (Days 1-10 after ovulation) and analysed for progesterone content. Luteal tissue was collected on Day 10 after the LH surge, or Day 10 after hCG injection from cyclic and superovulated ewes, respectively. The tissue was enzymically dispersed and an aliquant was utilized for measurement of cell diameters, and staining for 3 beta-hydroxy-delta 5-steroid dehydrogenase-delta 5, delta 4-isomerase activity (3 beta-HSD). The remaining cell preparation was separated into small (10-22 micron) and large (greater than 22 micron) cell fractions by elutriation. Small and large cell suspensions were incubated (37 degrees C, 2 h) in the presence or absence or ovine LH (100 ng/ml) or dbcAMP (2 mM) and progesterone content of the medium was measured. Superovulation did not affect circulating progesterone concentrations, when expressed per mg luteal tissue recorded; basal progesterone production by small or large luteal cells; the unresponsiveness of large luteal cells to ovine LH or dbcAMP; the ratio of small:large cells recovered by dissociation the mean diameter of total cells; or the mean diameter of large cells. However, the mean cell diameter and LH stimulation of progesterone production by small cells were greater (P less than 0.05) in luteal tissue collected from superovulated than in that from cyclic ewes. These differences appear to be an amplification of basic function. Therefore, we conclude that corpora lutea obtained from superovulated ewes can be used to study functional aspects of small and large cells.

Changes in available gonadotropin receptors in the corpus luteum of the rhesus monkey during simulated early pregnancy.

Stimulation of the primate corpus luteum (CL) by endogenous CG in early pregnancy or by exogenous human CG (hCG) in simulated early pregnancy, is transient, despite continued exposure to rising concentrations of CG. The objective of this study was to determine if the transitory response of the CL to CG is related to changes in gonadotropin receptors. Numbers and affinities of available LH/CG binding sites were characterized in the CL of rhesus monkeys (n = 27) during prolonged CG exposure in simulated early pregnancy, and the temporal relationship between changes in receptor parameters and in luteal function was examined. Administration of hCG increased progesterone concentrations above pretreatment levels within 9 h (2.2 +/- 0.8 vs. 7.6 +/- 1.1 ng/ml, mean +/- SE, P less than 0.05); the relative increase (345% of control) in serum progesterone was more profound than that of available hCG binding sites (135%, P greater than 0.05) or luteal weight (128%, P greater than 0.05) over this interval. Receptor affinities for hCG remained comparable to pretreatment values (Kd = 1.1 +/- 0.2 X 10(-10) M) throughout this 9-h period. A significant diminution in available hCG receptors occurred between 9 h (12.7 +/- 2.1 fmol/mg tissue) and 3 days (7.4 +/- 1.5 fmol/mg tissue) of hCG treatment (P less than 0.05). The loss of available CG receptors preceded a significant decline in serum progesterone concentrations. Serum progesterone decreased by 6 days (4.0 +/- 0.6 ng/ml, P less than 0.05) of hCG treatment, as did receptor affinity for hCG (Kd = 4.7 +/- 0.9 X 10(-10) M, P less than 0.05). Numbers and affinities of available receptors for hCG and serum progesterone concentrations fell before any decrease in luteal weight. Binding capacities and receptor affinities for human LH were comparable to those for hCG throughout simulated early pregnancy. In conclusion, the population of available LH/CG receptors in the macaque CL is maintained, or perhaps modestly increased, amidst dramatic stimulation of luteal function during early CG exposure. The subsequent diminution of number and affinity of available LH/CG receptors during prolonged exposure to CG in early pregnancy may compromise CL function and thus participate in the establishment of the transient nature of the luteal response to CG.

Follicular fluid treatment during the follicular versus luteal phase of the menstrual cycle: effects on corpus luteum function.

Administration of charcoal-extracted porcine follicular fluid (pFF) to rhesus monkeys at the time of menses impairs the subsequent function of the corpus luteum of the menstrual cycle. The following studies were performed: 1) to characterize the luteal phase defect induced by pFF treatment at menses, and 2) to determine whether pFF treatment in the luteal phase alters corpus luteum function. Adult, female rhesus monkeys were injected sc for 3 days with pFF (10, 5, and 5 ml) beginning on day 1 (n = 5) or day 18 (n = 4) of the menstrual cycle. Femoral venous blood was collected daily throughout the treatment cycle and during the posttreatment cycle of day 18 to 20-treated monkeys. Serum LH, FSH, 17 beta-estradiol (E2), and progesterone (P) were measured by RIA. After pFF treatment on days 1-3, FSH and E2 levels in the early follicular phase were less (P less than 0.05) than those of control cycles (n = 7). Serum LH was not suppressed by pFF treatment. Moreover, the preovulatory rise in circulating E2 and the amplitude of the LH/FSH surge were similar in control and pFF-treated monkeys. Although timely midcycle gonadotropin surges occurred in four of five pFF-treated monkeys, serum P was markedly reduced (P less than 0.05) during the first half of the luteal phase. Circulating P increased to control levels during the late luteal phase before normal onset of menses 16.3 +/- 1.0 (SE) days after the LH surge. Treatment with pFF on days 18-20 of the cycle reduced the levels of circulating FSH, but serum LH, E2, P, and the length of the luteal phase remained comparable to control cycles. Moreover, the hormonal patterns and the length of the follicular and luteal phases in the posttreatment cycle indicated normal ovarian function. Thus, pFF treatment at menses results in an aberrant ovarian cycle characterized by an insufficient, rather than short, luteal phase. Whereas pFF treatment in the early follicular phase vitiates development of the dominant follicle and the related corpus luteum, similar treatment at midluteal phase does not suppress concurrent luteal function or subsequent folliculogenesis.