Regulation of the corpus luteum of early pregnancy in the marmoset monkey: local interactions of luteotrophic and luteolytic hormones in vivo and their effects on the secretion of progesterone

The interaction between luteotrophic and luteolytic agents in controlling progesterone production by the marmoset corpus luteum in the late luteal phase/early pregnancy was investigated at the local level in vivo using a perfusion cannula system. Perfusion of the prostaglandin F2 alpha (PGF2 alpha) analogue, cloprostenol (0.5 microgram/ml), resulted in an immediate fall in progesterone production. This response was not sustained in two out of five corpora lutea but pregnancy was terminated in all animals exposed to PGF2 alpha. Perfusion of human chorionic gonadotrophin (hCG) (4 micrograms/ml) alone significantly stimulated progesterone secretion but there was no response to hCG when the corpus luteum had previously been perfused with PGF2 alpha. Perfusion with hCG together with PGF2 alpha prevented a fall in progesterone secretion. The results suggest that the luteolytic action of PGF2 alpha in the marmoset may be to prevent luteotrophic support of the corpus luteum. Melatonin (860 pmol/l), perfused either with PGF2 alpha or after PGF2 alpha, stimulated progesterone production. The ability of melatonin to influence progesterone production by the primate corpus luteum may therefore be by both a direct luteotrophic action and the prevention of luteolysis. Application of the perfusion system in order to investigate the ability of deglycosylated hCG to antagonize the action of hCG at the corpus luteum showed the necessity of testing pure preparations of hormones.     

Prostaglandin F2 alpha stimulates cAMP phosphodiesterase via protein kinase C in cultured human granulosa cells.

To investigate the involvement of cyclic AMP phosphodiesterase in the antigonadotrophic actions of prostaglandin F2 alpha (PGF2 alpha), human granulosa cells were cultured in serum-supplemented medium for 72 h, treated for 30 min with cloprostenol or phorbol myristate acetate (PMA) and then exposed to human chorionic gonadotrophin (hCG) +/- isobutyl-methylxanthine (IBMX) for a further 4 h. In the absence of IBMX, cloprostenol and PMA inhibited hCG-stimulated progesterone production. However, in the presence of 0.5 mM IBMX, inhibition of phosphodiesterase prevented these antigonadotrophic effects. Phosphodiesterase activity was assessed by a novel direct assay performed on intact cells after 3 days of culture. PGF2 alpha, cloprostenol and 4 beta-PMA all enhanced cAMP degradation whilst an inactive phorbol ester (4 alpha-PMA) had no effect. Down-regulation of protein kinase C by 4 beta-PMA pre-treatment prevented the subsequent stimulation of phosphodiesterase activity by both cloprostenol and 4 beta-PMA. We conclude that the antigonadotrophic actions of PGF2 alpha in cultured human granulosa cells involve a stimulation of cAMP phosphodiesterase mediated via protein kinase C.     

Mechanism of action of prostaglandin F2 alpha-induced luteolysis: evidence for a rapid effect on the guanine nucleotide binding regulatory component of adenylate cyclase in rat luteal tissue

The initial events in prostaglandin F2 alpha-(PGF2 alpha)-induced luteolysis were studied in pregnant mare serum gonadotropin/human chorionic gonadotropin-(PMSG/hCG)-treated rats with luteinized ovaries. Injection with a potent PGF2 alpha analog (cloprostenol, 5 micrograms/ml) induced functional luteolysis, as assessed by plasma levels of progesterone and 20 alpha-dihydroprogesterone. At 0.5 and 3 h after cloprostenol administration the luteolytic effect was also evident as a reduced response of luteal adenylate cyclase to all stimulatory agents tested, LH, isoproterenol, fluoride, guanylylimidodiphosphate and forskolin. 24 h after cloprostenol the response to all agents, except to LH, had returned to normal. This general and transient block of the luteal adenylate cyclase system indicates that a common factor, possibly the stimulatory guanine nucleotide binding protein (Ns), is involved in the mechanism of action of PGF2 alpha. To test this hypothesis, we measured the functional coupling of the Ns protein to the beta-adrenergic receptor in luteal membranes. Binding competition curves showed a marked shift to the right in membranes prepared from rats injected with cloprostenol 0.5 and 3 h before membrane preparation, while at 24 h after cloprostenol the shift had disappeared. The total number of beta-adrenergic receptors was, however, not affected by the cloprostenol treatment. Computer analysis of the data indicates that, at 0.5 and 3 h after cloprostenol treatment, there was a reduced number of high affinity binding sites, 38 and 41%, respectively, compared to 53% for control membranes. The cellular mechanism for this action of PGF2 alpha on the Ns protein remains to be elucidated.     

Role of mitogen-activated protein kinase in prostaglandin f(2alpha) action in human granulosa-luteal cells

In the ovary it has been demonstrated that PGF(2alpha) activates the phospholipase C (PLC)/diacylglycerol/protein kinase C pathway. However, little is known about the downstream signaling events that mediate subsequent cellular responses such as steroidogenesis. The present study was designed to examine the effect of PGF(2alpha) on activation of the mitogen-activated protein kinase (MAPK) signaling pathway and its physiological role in human granulosa-luteal cells (hGLCs). Human GLCs, obtained from women undergoing in vitro fertilization-embryo transfer, were treated with increasing concentrations of PGF(2alpha) (10 nmol/L to 10 micromol/L) for 5 min. For time-course experiments, hGLCs were treated with 1 micromol/L PGF(2alpha) for 1, 5, 10, or 20 min. Western blot analysis, using a monoclonal antibody that detected the phosphorylated forms of extracellular signal-regulated kinases 1 and 2 (p42(mapk) and p44(mapk), respectively), demonstrated that PGF(2alpha) activated MAPK in hGLCs in a dose- and time-dependent manner. Treatment of the cells with neomycin (10 mmol/L; a PLC inhibitor), bisindolylmaleimide I (5 micromol/L; a PKC inhibitor), or PD98059 (50 micromol/L; a MEK inhibitor and a MAPK kinase inhibitor) significantly attenuated the PGF(2alpha)-induced activation of MAPK. In contrast, MAPK activation was not significantly affected by pertussis toxin (200 ng/mL; a G(i) inhibitor) pretreatment. To determine the role of MAPK in steroidogenesis, hGLCs were treated with PGF(2alpha) (1 micromol/L), hCG (1 IU/mL), or PGF(2alpha) plus hCG in the presence or absence of PD98059. Progesterone levels in the culture medium were examined by RIA. Treatment of hGLCs with PGF(2alpha) significantly inhibited hCG-induced progesterone production. The presence of the MEK inhibitor, PD98059, reversed the inhibitory effect of PGF(2alpha) on hCG-induced progesterone production. To our knowledge, it is the first demonstration of PGF(2alpha)-induced activation of the MAPK signaling pathway in the human ovary. These results indicated that PGF(2alpha) activated MAPK subsequent to PLC and PKC activation through pertussis toxin-insensitive G protein in hGLCs. Further, we demonstrated that PGF(2alpha)-induced MAPK activation is associated with modulation of progesterone production. These results support the idea that the MAPK signaling pathway is involved in mediating PGF(2alpha) actions in the human ovary.     

Steroidogenesis by the immature rhesus monkey ovary in vitro

Primates are believed to have a low level of ovarian steroidogenic activity during prepubertal development. In order to study the rate limiting factors associated with the low level of steroidogenesis, ovaries from prepubertal rhesus monkeys were quartered and incubated for 48 h at 37 C in minimum essential medium. These ovaries secreted 687 +/- 347 pg estradiol/mg ovary and 299 +/- 35 pg progesterone/mg ovary during 48 h of incubation. The addition of 100 ng luteinizing hormone (LH) or 1 mM dibutyryl (Bu)2 cAMP failed to increase significantly estradiol or progesterone secretion. Furthermore, the addition of either progesterone or androstenedione failed to augment estradiol secretion. The presence of either LH or (Bu)2 cAMP with the steroidal substrates also failed to augment estradiol secretion. In contrast, the addition of (Bu)2 cAMP with lipoprotein-derived cholesterol significantly stimulated a two-fold increase in progesterone secretion. The presence of LH in the lipoprotein-supplemented medium failed to augment progesterone secretion. These results suggest that prepubertal monkey ovaries lack the ability to respond to LH, probably due to a lack of gonadotropin receptors or failure of the receptor to stimulate cAMP synthesis. Furthermore, the failure of progesterone and androstenedione to augment estradiol secretion suggests that some cellular components needed to induce aromatase activity are not functional in the prepubertal primate ovary.     

Influence of luteinizing hormone and prostaglandin F2 alpha on progesterone secretion in superfused bovine luteal tissue slices

Tissue slices from bovine corpus luteum from Days 12 or 13 of the oestrous cycle were superfused for 8 h, and the progesterone secretion under the influence of prostaglandin F2 alpha (PGF2 alpha) and/or LH was measured. PGF2 alpha at concentrations of 0.28 to 2800 nmol/l medium did not affect the basal progesterone secretion, whereas higher doses (7000 to 28,000 nmol/l) induced a slight increase in hormone secretion. LH, 3.4 nmol/l, caused an increase in the progesterone concentration in superfusates which exceeded the control levels (P less than 0.01). This luteotropic effect of LH was not influenced by simultaneous addition of 28 to 2800 nmol/l PGF2 alpha. PGF2 alpha, 2800 nmol/l, did not inhibit progesterone secretion, when administered together with 0.034 to 34 nmol LH/l. Pre-superfusion with 2800 nmol/l PGF2 alpha had no effect on the LH-stimulated increase in progesterone secretion. It is concluded that in cattle, a direct cellular effect of PGF2 alpha, antagonizing the luteotropic function of LH, may be of less importance than other possible direct and indirect PGF2 alpha actions.     

Prostaglandin F2 alpha-induced calcium transient in ovine large luteal cells: II. Modulation of the transient and resting cytosolic free calcium alters progesterone secretion

A previous study demonstrated that prostaglandin F2 alpha (PGF2 alpha) stimulates a transient increase in cytosolic free Ca2+ levels [( Ca2+]i) in ovine large luteal cells. In the present study, the magnitude of the PGF2 alpha (0.5 microM)-induced calcium transient in Hanks' medium (87 +/- 2 nM increase above resting levels) was reduced (P less than 0.05) but not completely eliminated in fura-2 loaded large luteal cells incubated in Ca2(+)-free or phosphate- and carbonate-free medium (10 +/- 1 nM, 32 +/- 6 nM, above resting levels; respectively). Preincubation for 2 min with 1 mM LaCl3 (calcium antagonist) eliminated the PGF2 alpha-induced calcium transient. The inhibitory effect of PGF2 alpha on secretion of progesterone was reduced in Ca2(+)-free medium or medium plus LaCl3. Resting [Ca2+]i levels and basal secretion of progesterone were both reduced (P less than 0.05) in large cells incubated in Ca2(+)-free medium (27 +/- 4 nM; 70 +/- 6% control, respectively) or with 5 microM 5,5'-dimethyl bis-(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (40 +/- 2 nM; 49 +/- 1% control; respectively). In addition, secretion of progesterone was inhibited (P less than 0.05) by conditions that increased (P less than 0.05) [Ca2+]i; that is LaCl3 ([Ca2+]i, 120 +/- 17 nM; progesterone, 82 +/- 8% control) and PGF2 alpha ([Ca2+]i, 102 +/- 10 nM; progesterone, 82 +/- 3% control). In small luteal cells, resting [Ca2+]i levels and secretion of progesterone were reduced by incubation in Ca2(+)-free Hanks ([Ca2+]i, 28 +/- 2 nM; progesterone, 71 +/- 6% control), however, neither LaCl3 nor PGF2 alpha increased [Ca2+]i levels or inhibited secretion of progesterone. The findings presented here provide evidence that extracellular as well as intracellular calcium contribute to the PGF2 alpha-induced [Ca2+]i transient in large cells. Furthermore, whereas an adequate level of [Ca2+]i is required to support progesterone production in both small and large cells, optimal progesterone production in large cells depends upon an appropriate window of [Ca2+]i.     

Epidermal growth factor inhibits follicular response to human chorionic gonadotropin: possible role of cell to cell communication in the response to gonadotropin.

Epidermal growth factor (EGF) affects follicular steroidogenesis and expression of gonadotropin receptors. The effects of EGF on hCG-induced estradiol and progesterone secretion and ovulation were examined in the in vitro perfused rabbit ovary. We also examined the effects of EGF on hCG-induced progesterone secretion by isolated granulosa cells. In addition, distribution of hCG within the follicle was probed by immunohistochemical means 30 min after its administration to the in vitro perfused ovary. EGF significantly (P less than 0.05) reduced hCG-induced secretion of estradiol (control, 117 +/- 12 pg/min.follicle; 10 ng/ml EGF, 55 +/- 10) and progesterone (control, 18.2 +/- 1.2 ng/min.follicle; 10 ng/ml EGF, 11.9 +/- 0.8) by the perfused ovary. In contrast, EGF did not inhibit hCG-induced progesterone secretion by isolated granulosa cells. Ovulatory efficiency (number of ovulated ova per number of mature follicles x 100) when EGF was given 30 min before hCG was reduced dose-dependently from 58.2% with no EGF to 8.3% with 10 ng/ml EGF (P less than 0.001). Ovulation was not inhibited by EGF when it was given 30 min after hCG. Distribution of hCG in the preovulatory follicle was confined to the basement membrane, thecal cell layer, and a small fraction of the outer granulosa cell layer. These observations suggest that gonadotropin stimulates the follicle through the release of a secondary signal(s) from ligand-bound granulosa cells near the follicle wall to unexposed cells of the inner avascular area. EGF may inhibit the follicular response to hCG by attenuation of this cell to cell communication.     

Prostaglandins and progesterone secretion in the in vitro perfused pseudopregnant rabbit ovary

The relationship between progesterone and prostaglandin (PG) secretion in the pseudopregnant rabbit corpus luteum was investigated using isolated in vitro perfused ovaries. Progesterone and PG secretion were measured on days 1, 11, and 18 of hCG-induced pseudopregnancy. The mean progesterone secretion increased significantly from days 1 to 11, and then decreased significantly by day 18. PG secretion was inversely correlated with progesterone secretion, suggesting that PG might inhibit progesterone secretion. To test this hypothesis, indomethacin, an inhibitor of PG secretion, was administered to intact rabbits from days 11-18 of pseudopregnancy and/or on day 18 ovaries were perfused in vitro with indomethacin. Indomethacin administered in vivo, in vitro, or both in vivo and in vitro significantly reduced PG secretion compared to that in controls, but did not affect progesterone secretion. In addition, perfusion of ovaries in vitro with PGF2 alpha did not alter progesterone secretion on either day 11 or day 18. Thus, although there is an inverse relationship between progesterone and PG secretion during pseudopregnancy, PGF2 alpha alone had no effect on progesterone secretion. These results question the hypothesis that PGF2 alpha alone is the luteolytic factor.     

Prostaglandin-induced secretion of oxytocin and prolactin in red (Cervus elaphus) and Pere David's (Elaphurus davidianus) deer hinds: evidence for oxytocin of luteal origin

Peripheral plasma concentrations of oxytocin in female red deer during the luteal phase of the oestrous cycle (9.3 +/- 2.1 fmol/ml) exceeded those in the follicular phase (3.1 +/- 1.4) or during seasonal anoestrus (3.2 +/- 1.3). In both red and Père David's deer hinds during the mid-luteal phase of the cycle, systemic administration of a luteolytic dose of the prostaglandin F2 alpha analogue, cloprostenol, caused the concentration of oxytocin in the peripheral circulation to rise. Mean (+/- SEM) concentrations increased from 8.1 +/- 0.7 to 97 +/- 8 fmol/ml in red and from 6.2 +/- 0.7 to 153 +/- 30 fmol/ml in Père David's hinds within 5 min of treatment. During seasonal anoestrus oxytocin secretion in response to cloprostenol was reduced to less than 10% of that during the breeding season, in both species. Cloprostenol treatment raised circulating concentrations of prolactin in both species during the breeding season, and during anoestrus in red deer only. The concentration of oxytocin in a single corpus luteum removed at laparotomy from one red deer hind at the mid-luteal phase of the cycle was 66 nmol/g wet wt; identification was authenticated by HPLC. These results suggest that the corpus luteum secretes oxytocin in the Cervidae, as established previously in the Bovidae, and that luteal oxytocin secretion is stimulated by prostaglandin.     

Prior exposure to gonadotrophins prevents the subsequent antigonadotrophic actions of cloprostenol by a cyclic AMP-dependent mechanism in cultured human granulosa cells.

The antigonadotrophic action of a prostaglandin F2 alpha analogue, cloprostenol, has been investigated in human granulosa cells obtained from cycles stimulated for in-vitro fertilization and induced to secrete luteal quantities of progesterone by culture in serum-supplemented medium. Cells were exposed to conditions which may mimic those occurring in early pregnancy to establish the roles of human chorionic gonadotrophin (hCG) versus LH and that of cyclic AMP (cAMP) in the anti-gonadotrophic action of cloprostenol. When human granulosa cells were cultured in the absence of treatment for 3 days, exposure to cloprostenol had no effect on basal progesterone production but inhibited hCG-stimulated progesterone (60% decrease; P less than 0.01), hCG-stimulated cAMP (40% decrease; P less than 0.05) and the progesterone response to dibutyryl cAMP (dbcAMP; 70% decrease; P less than 0.01), suggesting pre- and post-cAMP sites of cloprostenol action. The inhibitory actions of cloprostenol were prevented when the granulosa cells were either continuously exposed to treatment from the start of culture or pre-exposed for 3 days to maximum concentrations of LH, hCG, dbcAMP or 8-bromo-cAMP. We conclude that prior exposure either in vivo or in vitro to LH or hCG prevents the subsequent antigonadotrophic action of cloprostenol via a cAMP-dependent mechanism. Prevention of the antigonadotrophic action of cloprostenol after exposure to hCG may be a mechanism through which CG prevents regression of the corpus luteum in early pregnancy, while the suppressive effect of LH pretreatment may account for the refractory response of the early corpus luteum to cloprostenol following the midcycle LH surge.     

Prostaglandin F2 alpha- and phorbol 12-myristate-13-acetate-stimulated progesterone production by cultured human luteal cells in the mid-luteal phase: prostaglandin F2 alpha increases cytosolic Ca2+ and inositol phosphates.

While prostaglandin F2 alpha (PGF2 alpha) has been thought to be a natural luteolysin in non-primates, a luteolytic effect in the human corpus luteum is less evident. We therefore investigated the action of PGF2 alpha on monolayer cultures of human luteal cells obtained from mid-luteal phase corpora lutea. PGF2 alpha increased basal and human chorionic gonadotrophin (hCG)-stimulated progesterone production by human cultured luteal cells. A potent tumour-promoting phorbol ester, phorbol 12-myristate-13-acetate (PMA), also stimulated progesterone production by cultured human luteal cells. Although human luteal cells were incubated for 24 h with PMA, hCG was still able to stimulate the production of progesterone by PMA-pretreated cells. However, PMA pretreatment blocked the ability of PGF2 alpha to stimulate progesterone production. It is possible that the luteotrophic effect of PGF2 alpha may be mediated, in part, by the activation of protein kinase C. Addition of PGF2 alpha to suspensions of human luteal cells preincubated with myo-[2-3H]inositol promoted an increase in labelled inositol phosphates. PGF2 alpha also rapidly increased intracellular free Ca2+ in human luteal cells loaded with the fluorescent Ca2+ probe, fura-2. We conclude that PGF2 alpha and PMA stimulate progesterone production and that PGF2 alpha increases the intracellular free calcium and inositol phosphates of human cultured luteal cells in the mid-luteal phase.     

Activation of the phosphatidylinositol pathway in the primate corpus luteum by prostaglandin F2 alpha.

The current study was designed to investigate the ability of prostaglandin F2 alpha (PGF2 alpha) to activate a second messenger system (phosphatidylinositol pathway) in corpora lutea (CL) of rhesus monkeys. Activation of this pathway was assessed by monitoring the hydrolysis of phosphatidylinositol to inositol phosphates. Since inositol triphosphate mobilizes intracellular Ca2+, intracellular free calcium concentrations ([Ca2+]i) were also assessed in individual cells by fura-2 fluorescence photometry. These responses to PGF2 alpha were measured in luteal cells collected from nonpregnant rhesus monkeys. CL were collected during the early (days 4-5 after estimated LH surge; n = 4), mid (days 8-9; n = 4), and late (days 13-14; n = 5) luteal phase and 1 day after in vivo hCG treatment (15 IU/dose, morning and evening), which began during the midluteal phase (n = 5). PGF2 alpha significantly increased the accumulation of inositol phosphates in all groups (P less than 0.05), except the midluteal phase (P = 0.07). The luteal sensitivity to PGF2 alpha, judged by phosphatidylinositol hydrolysis, was low in the early to midluteal phase compared to that in the late luteal phase and after in vivo hCG treatment. PGF2 alpha also caused a rapid, yet transient, increase in [Ca2+]i in a large proportion of primate luteal cells. The proportion of luteal cells that responded to PGF2 alpha with an increase in [Ca2+]i was smaller (P less than 0.05) in CL collected during the early luteal phase than in the other groups. Luteal progesterone production was inhibited by PGF2 alpha in CL collected after in vivo hCG. CL treated in vivo with hCG also displayed in vitro the largest increases in phosphatidylinositol hydrolysis and [Ca2+]i in response to PGF2 alpha. Therefore, this study demonstrates that PGF2 alpha is a potent activator of the phosphatidylinositol pathway in the primate CL. This activation is augmented as the luteal phase progresses and is influenced by in vivo hCG treatment. This study also provides evidence that the inhibitory effects of PGF2 alpha on progesterone production are associated with the activation of the phosphatidylinositol pathway.     

Direct effects of the prostaglandins E2 and f2alpha on progesterone release by the corpus luteum of the marmoset monkey (Callithrix jacchus) studied by in vitro microdialysis.

The effects of the prostaglandins (PG) E2 and F2alpha on progesterone secretion in luteal tissue (32 corpora lutea) explanted from the mid-luteal ovary of the marmoset monkey (n=13) were investigated using an in vitro microdialysis system. Consecutive applications of 1, 10 and 100 microg/ml PGE2 resulted in a significant increase in secretion of progesterone at the maximum dose of 100 microg/ml, which was shown to be the stimulatory dose in both long-period and 20-min pulse (time to collect one fraction) applications. The response varied individually between 1.4- and 3. 4-fold above the baseline concentrations. Application of 500 microg/ml PGF2alpha led to similar hormone responses. In contrast, lower doses of PGF2alpha (0.5, 5 and 50 microg/ml) resulted in significantly increased levels of secretion of progesterone, to approximately 1.4-fold baseline values, only after the application was terminated (echo effect). Responses were less variable when a short pulse of 20 min duration was applied, instead of long applications of 1-2 h. On the basis of the passage rates measured for tritiated PGF2alpha, transfer through the dialysis membrane was assumed to be in the range of 1% for both PGs. Ultrastructurally, luteal cells lying in a sheath of five to seven cell layers around the dialysis tubing appeared intact and were interconnected by gap junctions. Vesiculation of the smooth endoplasmic reticulum was more prominent after PG treatment, indicating a stimulation of cellular synthesis/secretory activities that was in accordance with the stimulatory action of both PGs on progesterone release under these in vitro conditions.     

Relaxin and prostaglandin on oxytocin secretion from bovine luteal cells during different stages of gestation

The objective of this study was to determine whether bovine luteal cells from different stages of gestation secrete oxytocin and whether relaxin, cloprostenol (a potent analogue of prostaglandin F2 alpha), estradiol-17 beta, and LH can acutely alter oxytocin secretion. Bovine luteal cells (10(5)) were cultured for 24 h without treatment and with medium-hormone replacement every 24 h. Oxytocin was quantified by radioimmunoassay of the culture media. Basal oxytocin secretion was similar (22-31 pmol/l, p less than 0.05) for all stages of gestation (days 100, 145, 160, 185, 200, 210, and 240). Relaxin induced a dose-dependent suppression of oxytocin release. After 24 h of incubation, addition of 0, 16.7, 83.5, and 167 nmol/l porcine relaxin (3000 U/mg) induced 54 +/- 4, 105 +/- 16, 47 +/- 4, and 38 +/- 4 pmol/l of oxytocin in cells from 160-day-old corpora lutea and 138 +/- 12, 21 +/- 2, 19 +/- 3, and 15 +/- 2 pmol/l oxytocin in cells from 240-day-old corpora lutea. From luteal cells of 160- and 240-day-old corpora lutea, 2 micromol/l cloprostenol induced a marked increase (p less than 0.01) of 208 +/- 39 and 371 +/- 34 pmol/l oxytocin, respectively. Addition of 167 nmol/l relaxin did not prevent cloprostenol-induced oxytocin secretion during the first 48 h, but a decrease (p less than 0.05) in oxytocin occurred in day 3 cell cultures. These results indicate that cultured luteal cells obtained from different stages of gestation in cattle can secrete oxytocin and suggest a role for relaxin in the regulation of oxytocin release.     

Prostaglandin F2 alpha administered in vivo induces Ca2+-dependent protein phosphorylation in rat luteal tissue

The present study was performed in order to further elucidate the mechanism of action of PGF2 alpha in luteolysis in the rat ovary. Seven days after priming with superovulatory doses of pregnant mare serum gonadotropin and human chorionic gonadotropin to induce luteal tissue formation, the rats were injected with a luteolytic dose of the prostaglandin F2 alpha analogue cloprostenol. The ovaries were then homogenized, a 30,000 x g supernatant and pellet were prepared, whereafter aliquots of the preparations were incubated in the presence of [gamma-32P]ATP with or without Ca2+. The phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and localized by autoradiography. The presence of Ca2+ caused an increased phosphorylation of a 45 kDa protein band in the particulate, but not in the cytosol, fraction. Furthermore, PGF2 alpha rapidly increased the 32P incorporation into the same protein band of 45 kDa. Thus, the PGF2 alpha-stimulated 32P incorporation was Ca2+-dependent and seen only in the particulate fraction. These results suggest that PGF2 alpha in its role as a luteolytic agent stimulates a Ca2+-dependent phosphorylation of a specific protein in luteal membranes of the rat ovary.     

Are ovarian steroids required for ovum maturation and fertilization? Effects of cyanoketone on the in vitro perfused rabbit ovary

An isolated perfused rabbit ovary preparation was used to determine the effects of cyanoketone, a potent inhibitor of 3 beta-hydroxysteroid dehydrogenase, on ovulation, ovum maturation and fertilizability, and steroid production. In the first experiment, cyanoketone (10(-4) M) was added to the perfusate of one ovary. The contralateral control ovary was perfused with medium alone. Thirty minutes after the onset of perfusion, hCG (50 IU) was added to the perfusate of both ovaries. The ovulatory efficiency of ovaries treated with cyanoketone plus hCG (82.3 +/- 4.6%) was similar to that of ovaries treated with hCG alone (84.8 +/- 4.4%). No difference was observed in the degree of ovum maturity or degeneration between control and cyanoketone-treated ovaries. Progesterone and estradiol production were significantly reduced by cyanoketone treatment; concentrations in the perfusate of ovaries treated with cyanoketone were 9.7% and 8.0% of the control values, respectively, 2 h after exposure to hCG. The concentration of 17-hydroxypregnenolone was not affected by cyanoketone treatment. Exposure to cyanoketone resulted in a significant (P less than 0.005) reduction in the fertilizability of ova ovulated and fertilized in vitro. In the second experiment, the percentage of ova that showed evidence of normal fertilization was significantly (P less than 0.025) increased in ovaries perfused with cyanoketone plus estradiol (64.5%) compared to that in ovaries perfused with cyanoketone alone (32.4%). In the third experiment, the addition of progesterone to the perfusate did not affect fertilizability of ovulated ova in ovaries perfused with cyanoketone plus estradiol. These results suggest that the presence of estradiol in the ovarian steroid environment may be essential for fertilizability of ova, but not for the processes of ovulation or meiotic maturation.     

Source of ovarian inhibin secretion during the oestrous cycle of the sheep

The source of inhibin secretion by the ovary in the sheep at different stages of the oestrous cycle was investigated by in-vivo cannulation of the ovarian veins. Twenty-four Scottish Blackface ewes were allocated to four groups of six ewes, i.e. those operated on during the luteal phase (day 10), and those operated on during the follicular phase 24-30, 36 and 60 h following an injection of 125 micrograms cloprostenol on day 10 of the luteal phase. Samples of jugular and timed ovarian venous blood were collected under anaesthesia before and after enucleation of the corpus luteum. Ovaries were then removed and follicles dissected out. Following injection of cloprostenol, luteal regression occurred as indicated by a fall in the secretion of progesterone. The concentration of inhibin in jugular venous plasma and its ovarian secretion rate were similar at all stages of the follicular phase and during the luteal phase. In contrast, the secretion rate of oestradiol rose from 2.86 +/- 0.73 pmol/min during the luteal phase to 8.70 +/- 2.24 pmol/min 24 h after injection of cloprostenol (P less than 0.05). Following enucleation of the corpus luteum the secretion rate of progesterone fell from 809 +/- 270 pmol/min to 86 +/- 30 pmol/min (P less than 0.001). There was also a smaller, artifactual fall in the secretion rate of oestradiol following enucleation of the corpus luteum, which was of similar size to a fall seen in the secretion rate of inhibin. This resulted in a significant (P less than 0.001) fall in the ratio of progesterone to inhibin, while the oestradiol to inhibin ratio remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct inhibitory effect of gonadotropin-releasing hormone upon luteal luteinizing hormone receptor and steroidogenesis in hypophysectomized rats

The effects of gonadotropin-releasing hormone (GnRH) and its potent agonist [des-Gly10, D-Leu6-N alpha Me) Leu7, Pro9,NHEt-GnRH (GnRH-A)] on ovarian luteal functions maintained by PRL were studied in vivo and in vitro. Hypophysectomized, diethylstilbestrol-treated female rats were primed with FSH for 2 days, followed by an ovulating dose of LH or hCG. Two days later, ovarian luteal functions were maintained by daily injections of 250 microgram PRL for 3 days. PRL treatment increased the serum progesterone level from 13.0 +/- 0.5 to 298 +/- 24 ng/ml and increased the ovarian hCG-binding capacity from 5.8 +/- 1.3 to 584 +/- 86 ng bound hCG/ovary. In contrast, concomitant treatment with GnRH or GnRH-A resulted in dose-dependent decreases in the PRL-induced increase of serum progesterone and ovarian LH/hCG receptor content. GnRH at 100 microgram/day caused a 60% decrease in serum progesterone and an 80% decrease in ovarian LH receptor content, whereas GnRH-A was effective at a 1-microgram dose level. Neither GnRH nor GnRH-A affected the binding affinity (Kd) of ovarian LH receptor. The direct inhibitory effects of GnRH and GnRH-A upon granulosa-luteal cell function were also tested in vitro. FSH treatment for 2 days induced functional LH and PRL receptors in cultured PRL, increased (by approximately 3-fold) progesterone production by these granulosa-luteal cells, whereas concomitant treatment with GnRH-A inhibited progesterone production in a dose-dependent manner. Thus, these studes demonstrated that GnRH and GnRH-A exert direct inhibition on ovarian luteal functions by decreasing LH receptor and progesterone production in vivo as well as inhibiting progesterone production by cultured granulosa-luteal cells in vitro.