Inhibition of ovulation in the gonadotropin-primed immature rat by exogenous prostaglandin E2.

In the past two decades there have been innumerable reports that prostaglandins (PGs) are essential for mammalian ovulation. However, we have recently found that a relatively low dose of 0.03 mg indomethacin (INDO) sc to PMSG/hCG-primed immature Wistar rats can significantly reduce ovarian PG levels without inhibiting the control ovulation rate of 60+ ova/rat (1-3). In view of this information, the present study was an effort to duplicate the earlier reports that PGs can reverse the "inhibitory" effect of INDO on ovulation. In control animals, which received PMSG and hCG only, the ovulation rate was 63.8 +/- 4.5 ova/rat. This rate was reduced to 4.1 +/- 1.1 ova/rat when the animals were injected with 1.0 mg INDO at 3 h after hCG. In no instance was this inhibition reversed when the animals were treated with 1.0 mg of PGE2 or PGF2 alpha, or a combination of both prostanoids in either a single dose at 3 h after hCG, or in 4x doses at 2-h intervals beginning at 3 h after hCG. Furthermore, in animals that did not receive INDO, the ovulation rate in PGE2-treated animals was reduced to 20.0 +/- 6.7 ova/rat, and in animals treated with PGE2 and PGF2 alpha (combined) it was reduced to 19.4 +/- 6.5 ova/rat. In summary, not only did the PGs fail to reverse the anti-ovulatory effect of INDO, PGE2 actually suppressed the ovulation rate.     

Inhibition of ovulation in the gonadotropin-primed immature rat by exogenous prostaglandin E2

In the past two decades there have been innumerable reports that prostaglandins (PGs) are essential for mammalian ovulation. However, we have recently found that a relatively low dose of 0.03 mg indomethacin (INDO) sc to PMSG/hCG-primed immature Wistar rats can significantly reduce ovarian PG levels without inhibiting the control ovulation rate of 60+ ova/rat (1–3). In view of this information, the present study was an effort to duplicate the earlier reports that PGs can reverse the “inhibitory” effect of INDO on ovulation. In control animals, which received PMSG and hCG only, the ovulation rate was 63.8 ± 4.5 ova/rat. This rate was reduced to 4.1 ± 1.1 ova/rat when the animals were injected with 1.0 mg INDO at 3 h after hCG. In no instance was this inhibition reversed when the animals were treated with 1.0 mg of PGE₂ or PGF_2α, or a combination of both prostanoids in either a single dose at 3 h after hCG, or in 4× doses at 2-h intervals beginning at 3 h after hCG. Furthermore, in animals that did not receive INDO, the ovulation rate in PGE₂-treated animals was reduced to 20.0 ± 6.7 ova/rat, and in animals treated with PGE₂ and PGF_2α (combined) it was reduced to 19.4 ± 6.5 ova/rat. In summary, not only did the PGs fail to reverse the anti-ovulatory effect of INDO, PGE₂ actually suppressed the ovulation rate.     

Cross-Talk between cAMP and MAPK Pathways in HSD11B2 Induction by hCG in Placental Trophoblasts

Overexposure of the fetus to glucocorticoids in gestation is detrimental to fetal development. The passage of maternal glucocorticoids into the fetal circulation is governed by 11beta-Hydroxysteroid Dehydrogenase Type 2 (HSD11B2) in the placental syncytiotrophoblasts. Human chorionic gonadotropin (hCG) plays an important role in maintaining placental HSD11B2 expression via activation of the cAMP pathway. In this study, we investigated the relationship between the activation of the cAMP pathway by hCG and subsequent phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) or p38 mitogen-activated protein kinase (MAPK) pathways in the regulation of placental HSD11B2 expression in human placental syncytiotrophoblasts. We found that treatment of the placental syncytiotrophoblasts with either hCG or dibutyl cAMP (dbcAMP) could promote the phosphorylation of p38 and ERK1/2. Inhibition of p38 MAPK with SB203580 not only reduced the basal HSD11B2 mRNA and protein levels but also attenuated HSD11B2 levels induced by either hCG or dbcAMP. By contrast, inhibition of ERK1/2 with PD98059 increased the basal mRNA and protein levels of HSD11B2 and had no effect on HSD11B2 mRNA and protein levels induced by either hCG or dbcAMP. These data suggest that p38 MAPK is involved in both basal and hCG/cAMP-induced expression of HSD11B2, and ERK1/2 may play a role opposite to p38 MAPK at least in the basal expression of HSD11B2 in human placental syncytiotrophoblasts and that there is complicated cross-talk between hCG/cAMP and MAPK cascades in the regulation of placental HSD11B2 expression.     

Phorbol ester causes desensitization of gonadotropin-responsive adenylate cyclase in a murine Leydig tumor cell line

The murine Leydig tumor cell line, MLTC-1, contains gonadotropin receptors and a gonadotropin-responsive adenylate cyclase system that became refractory (desensitized) when exposed to human chorionic gonadotropin (hCG). MLTC-1 cells also contain phorbol ester receptors with a Kd of 53 nM for [3H]phorbol dibutyrate. Exposing cells to 12-O-tetradecanoyl phorbol 13-acetate (TPA) also causes desensitization of the hCG response. TPA-induced desensitization was similar to hCG-induced desensitization by every criteria tested. Both TPA- and hCG-induced desensitization caused approximately 50% loss of the hormone response within 30 min. Neither TPA or hCG altered receptor affinity for hCG. The dose response of adenylate cyclase to hCG or GTP in isolated membranes was not affected by either hCG- or TPA-induced desensitization. Similarly the dose response to hCG of cAMP accumulation in intact cells was not altered by desensitization with hCG or TPA. It was determined that MLTC-1 cells have Ca2+/phospholipid-dependent protein kinase activity that displayed a dose-dependent response to TPA. The concentration of TPA required to activate the protein kinase was similar to that required for desensitization. Phorbol esters that were unable to activate protein kinase C were also unable to desensitize MLTC-1 cells. The protein kinase from MLTC-1 cells was also activated by diacylglycerol. In addition, diacylglycerols caused desensitization of the hCG response. TPA- and diacylglycerol-induced desensitization is probably mediated by protein kinase C, and the similarities between hCG- and TPA-induced refractoriness suggests a convergence of mechanisms at some point of MLTC-1 cell desensitization.     

Reversal of indomethacin blockade of ovulation in gilts by prostaglandins

Prepubertal gilts given 750 IU pregnant mares′ serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation fail to ovulate when 10 mg/kg indomethacin (INDO) is injected 24 h after hCG administration. This study examines the effects of administration of exogenous prostaglandins F_2α and E₂ (PGF_2α and PGE₂) alone or in combination, and at various times prior to the expected time of ovulation, on the INDO blockade of ovulation in PMSG/hCG-treated gilts. Occurrence of ovulation was determined by visual observation at laparotomy 48 h after hCG. When 5 mg or 10 mg PGF_2α was injected at each of 38, 40 and 42 h after hCG injection, 63% and 79%, respectively, of preovulatory follicles ovulated. In contrast, injection of 5 mg PGE₂ or 5 mg PGE₂ plus 5 mg PGF_2α induced ovulation in 0% and 24% of preovulatory follicles, respectively. In control groups, 100% of folicles in PMSG/hCG-treated gilts ovulated whereas none did so in PMSG/hCG/INDO-treated animals. These results indicate that administration of PGF_2α can induce ovulation in the PMSG/hCG/INDO-treated prepubertal gilt and suggest that PGE₂ is ineffective and may be antagonistic to PGF_2α in overcoming the ovulation blocking effect of INDO.     

Mitogen-activated protein kinase activation and regulation of cyclooxygenase 2 expression by platelet-activating factor and hCG in human endometrial adenocarcinoma cell line HEC-1B

The activation of mitogen-activated protein kinase (MAP kinase) and the regulation of cyclooxygenase 2 (COX-2) were investigated in the human endometrial adenocarcinoma cell line HEC-1B by treatment with platelet-activating factor (PAF) and hCG. Pre-treatment of the cells with both oestradiol and medroxyprogesterone acetate was required for MAP kinase activation and COX-2 expression to respond to PAF and hCG. PAF-induced MAP kinase activation was sensitive to MAP kinase kinase (MEK) inhibitor, PD098059, and phosphatidylinositol-3-OH kinase (PI3K) inhibitor, wortmannin. In contrast, hCG-induced MAP kinase activation was sensitive to PD098059 and protein kinase A inhibitor, H-89, but not to wortmannin. PAF-induced COX-2 expression was insensitive to PD098059 but sensitive to wortmannin, whereas hCG-induced COX-2 expression was sensitive to PD098059 and H-89 but insensitive to wortmannin. 8-(4-chlorophenylthio)-cAMP, a potent cAMP analogue, induced activation of MAP kinase and expression of COX-2. These results indicate that MAP kinase is activated with PAF and hCG in HEC-1B cells. In addition, COX-2 expression is stimulated through the MAP kinase activation pathway with hCG and the wortmannin sensitive pathway with PAF in HEC-1B cells. These results also imply that protein kinase A remains upstream of hCG-induced activation of MAP kinase in HEC-1B cells.     

Differential actions of gangliosides on gonadotropin and cholera enterotoxin stimulated adenosine3':5' cyclic monophosphate dependent protein kinase in isolated rat ovarian cells.

Rat ovarian cells were exposed to cholera enterotoxin, and the effect on progesterone synthesis as well as on protein kinase stimulation was examined. Cholera enterotoxin stimulated ovarian steroidogenesis in a dose dependent manner similar to that of hCG. The stimulation of protein kinase by cholera toxin was followed by a lag period, whereas hCG effect was immediate. Mixed gangliosides, when added to the incubation medium, blocked the cholera enterotoxin-stimulated protein kinase activity and abolished the decrease in exogenous [³H] cyclic AMP receptor activity brought about by the toxin. In contrast, under similar experimental conditions ganglioside addition elicited no effect on protein kinase activation produced by hCG or LH. The data suggest that gangliosides do not appear to be directly involved in gonadotropin binding to ovarian cell membrane and subsequent mediation of physiological response.     

Human chorionic villous macrophages as a fetal biological shield from maternal chorionic gonadotropin

In addition to its most well characterized biological role in the rescue and maintenance of corpus luteum function, human chorionic gonadotropin (hCG) also stimulates the onset of fetal gonadal steroidogenesis. However, excess hCG is teratogenic to fetal gonadal tissues, and therefore hCG must be tightly regulated. Although there is an anatomical barrier between the fetal vessels and maternal blood, other mechanisms may regulate hCG levels. In the present study, we investigated whether human chorionic villous macrophages degraded maternal hCG. Isolated human macrophages incorporated and degraded hCG in a time-dependent manner. Human placental villous macrophages and phorbol myristate acetate (PMA)-treated THP-1 cells expressed the gene encoding an exon 9-deleted form of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor; expression of the full-length receptor was not determined. While both PMA-treated or untreated THP-1 cells could uptake hCG into their cytoplasms, hCG degradation and excretion of its byproducts only progressed in PMA-treated THP-1 cells. In conclusion, hCG internalization and degradation are different processes in macrophages that protect fetal gonadogenesis from excess hCG. The exon 9-deleted LH/CG receptor, but not the full-length receptor, is involved in the degradation of cytoplasmic hCG by organ-specific, dominant–negative interactions.     

Modulatory function of protein kinase C in the activation of ornithine decarboxylase and in cAMP production in rat osteoblasts

The effect of activation of protein kinase C on stimulation of ornithine decarboxylase (ODC) activity and cAMP production was studied in fetal rat osteoblasts. Both phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, and 4 alpha-phorbol, ineffective in activating protein kinase C, failed to stimulate ODC activity and cAMP production. We tested the effect of protein kinase C on stimulation of ODC activity by parathyroid hormone (PTH) and forskolin. In contrast to PTH-stimulated ODC activity, which was not affected by PMA, forskolin-stimulated (1 and 10 microM) ODC activity was dose dependently reduced. PMA (400 nM) reduced both 1 and 10 microM forskolin-stimulated ODC activity to the same level, approximately 3 nmol CO2/mg protein, which suggests a controlling role of protein kinase C in forskolin-stimulated ODC activity. The study of the effect of protein kinase C on PTH- and forskolin-stimulated cAMP production also revealed differences between PTH and forskolin. When PMA was added simultaneously with PTH (4 and 20 nM) or forskolin (1 and 10 microM) the PTH-stimulated cAMP production was dose-dependently potentiated by PMA, whereas forskolin-stimulated cAMP production was not affected. However, both PTH- and forskolin-stimulated cAMP production was dose-dependently augmented when PMA was added 3 min prior to PTH or forskolin. With increasing preincubation periods (up to 24 h) with PMA instead of a potentiation an inhibition was observed. This inhibition is not due to PTH receptor desensitization, although, on basis of the present results desensitization can not completely be excluded. In all cases 4 alpha-phorbol was without effect. The present results show that protein kinase C modulates stimulation of ODC activity and cAMP production in fetal rat osteoblasts. The modulation of both ODC activity and cAMP production appears to be dependent on the nature of the stimulator. The present data suggest a role for protein kinase C in limiting the cAMP-mediated stimulation of ODC activity in these cells. Furthermore, it is suggested that protein kinase C can interfere at more than one site in the cAMP-generating system.     

Selective activation of rabbit ovarian protein kinase isozymes in rabbit ovarian follicles and corpora lutea

The magnitude of activation of the type I and type II forms of cAMP-dependent protein kinase was investigated in estrous follicles and corpora lutea (CL) obtained from ovaries of control rabbits and rabbits injected acutely with human chorionic gonadotropin (hCG). To this end, a chromatographic technique which permitted quantitative evaluation of the in vivo activational state of the two forms of cAmP-dependent protein kinase was developed and verified. Results revealed that in follicles obtained from ovaries of untreated estrous rabbits, 15% of the soluble cAMP-dependent protein kinase, all of which exists as the type II isozyme, is activated. Intravenous administration of a single bolus of hCG promoted a concentration-dependent activation (in 10 min) of this protein kinase isozyme. In CL obtained from ovaries of control, 4-day pseudopregnant rabbits, 32% of the total soluble cAMP-dependent protein kinase exists as the type I form and 68% exists as the type II form. Both types of protein kinase are approximately 10% dissociated in CL from ovaries of untreated rabbits. Upon intravenous administration of hCG, only the type I form of cAMP-dependent protein kinase is further activated (in 10 min). Dissociation of this protein kinase is dependent upon the time and concentration of hCG. Preferential activation of the type I form of cAMP-dependent protein kinase in CL is also demonstrable in in vitro studies using exogenous cAMP. These data suggest that the physiological intracellular mediator of acute cAMP-regulated, hCG-triggered functions in rabbit ovarian follicles is the type II isozyme of cAMP-dependent protein kinase while in CL of 4-day pseudopregnant rabbits, it is the type I enzyme form.     

Effect of indomethacin on alcohol-induced morphological anomalies in mice

The purpose of the present study was 1) to examine the effect of indomethacin (INDO), a prostaglandin synthesis inhibitor, on alcohol-induced growth and morphological impairment in C57BL/6J mice (Study 1) and 2) to determine if INDO crosses the placenta (Study 2). On day 10 of gestation, mice were injected (s.c.) acutely with either 0, 5, 10, or 20 mg/kg INDO, followed one hour later by alcohol (5.8 g/kg orally) or isocaloric sucrose. Fetuses were removed on day 19 of pregnancy, weighed, and examined for anomalous development. As expected, Study 1 demonstrated that maternal alcohol treatment decreased fetal weight and increased the number of fetuses with birth defects. INDO alone decreased fetal weight but did not affect morphologic development. More importantly, INDo antagonized alcohol-induced birth defects, but only at the highest dose. The results of Study 2 suggest that the relative ineffectiveness of INDO may be related to its inability to readily cross the placenta. Since high doses of INDO also caused maternal toxicity, the usefulness of this compound in future studies of this type was questioned.     

Gonadotropin stimulation of cyclic AMP levels in Chinese hamster ovary cells in culture.

When Chinese Hamster Ovary (CHO) cells, incubated in serum-free medium, are exposed to gonadotropins a transient increase in the intracellular concentration of cyclic AMP is observed. Maximum accumulation of cyclic AMP is noted 30 minutes after addition of either human chorionic gonadotropin (hCG) or follicle stimulating hormone (FSH). Within one to two hours after hormone addition, the intracellular concentrations of cyclic AMP have returned to basal levels. The enhancement of intracellular cyclic AMP levels by hCG is hormone concentration dependent, with maximal stimulation observed at 10 micrograms/ml hCG. The exogenous addition of gonadotropins also slows the growth rate of CHO cells. This effect on growth seems to be mediated through cyclic AMP since the growth rate of a mutant of CHO cells defective in the catalytic subunit of cyclic AMP dependent protein kinase is only slightly decreased.