Genome-wide gene expression analysis reveals a dynamic interplay between luteotropic and luteolytic factors in the regulation of corpus luteum function in the bonnet monkey (Macaca radiata).

Although LH is essential for survival and function of the corpus luteum (CL) in higher primates, luteolysis occurs during nonfertile cycles without a discernible decrease in circulating LH levels. Using genome-wide expression analysis, several experiments were performed to examine the processes of luteolysis and rescue of luteal function in monkeys. Induced luteolysis with GnRH receptor antagonist (Cetrorelix) resulted in differential regulation of 3949 genes, whereas replacement with exogenous LH (Cetrorelix plus LH) led to regulation of 4434 genes (1563 down-regulation and 2871 up-regulation). A model system for prostaglandin (PG) F(2alpha)-induced luteolysis in the monkey was standardized and demonstrated that PGF(2alpha) regulated expression of 2290 genes in the CL. Analysis of the LH-regulated luteal transcriptome revealed that 120 genes were regulated in an antagonistic fashion by PGF(2alpha). Based on the microarray data, 25 genes were selected for validation by real-time RT-PCR analysis, and expression of these genes was also examined in the CL throughout the luteal phase and from monkeys treated with human chorionic gonadotropin (hCG) to mimic early pregnancy. The results indicated changes in expression of genes favorable to PGF(2alpha) action during the late to very late luteal phase, and expressions of many of these genes were regulated in an opposite manner by exogenous hCG treatment. Collectively, the findings suggest that curtailment of expression of downstream LH-target genes possibly through PGF(2alpha) action on the CL is among the mechanisms underlying cross talk between the luteotropic and luteolytic signaling pathways that result in the cessation of luteal function, but hCG is likely to abrogate the PGF(2alpha)-responsive gene expression changes resulting in luteal rescue crucial for the maintenance of early pregnancy.

Simulated conditions of microgravity suppress progesterone production by luteal cells of the pregnant rat.

The purpose of this study was to assess whether simulated conditions of microgravity induce changes in the production of progesterone by luteal cells of the pregnant rat ovary using an in vitro model system. The microgravity environment was simulated using either a high aspect ratio vessel (HARV) bioreactor with free fall or a clinostat without free fall of cells. A mixed population of luteal cells isolated from the corpora lutea of day 8 pregnant rats was attached to cytodex microcarrier beads (cytodex 3). These anchorage dependent cells were placed in equal numbers in the HARV or a spinner flask control vessel in culture conditions. It was found that HARV significantly reduced the daily production of progesterone from day 1 through day 8 compared to controls. Scanning electron microscopy showed that cells attached to the microcarrier beads throughout the duration of the experiment in both types of culture vessels. Cells cultured in chamber slide flasks and placed in a clinostat yielded similar results when compared to those in the HARV. Also, when they were stained by Oil Red-O for lipid droplets, the clinostat flasks showed a larger number of stained cells compared to control flasks at 48 h. Further, the relative amount of Oil Red-O staining per milligram of protein was found to be higher in the clinostat than in the control cells at 48 h. It is speculated that the increase in the level of lipid content in cells subjected to simulated conditions of microgravity may be due to a disruption in cholesterol transport and/or lesions in the steroidogenic pathway leading to a fall in the synthesis of progesterone. Additionally, the fall in progesterone in simulated conditions of microgravity could be due to apoptosis of luteal cells.

Mitochondrial peripheral-type benzodiazepine receptor expression. Correlation with gonadotropin-releasing hormone (GnRH) agonist-induced apoptosis in the corpus luteum.

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) decreases the expression of the mitochondrial peripheral-type benzodiazepine receptor (PBR) and increases the rate of DNA degradation in a time-dependent manner in the corpora lutea of pregnant rats. In the present study, we show in situ the GnRH-Ag-induced DNA fragmentation and correlate the increase of the rate of DNA degradation with the decrease in mitochondrial PBR ligand binding (r = 0.89). The GnRH-Ag-induced decrease in the 18-kDa PBR protein also correlated with the reduction in the Bcl-X(L), but not Bcl-2 (cell survival), gene product levels and the increase in the Bax (cell death) gene product expression in the luteal mitochondrial preparations. Considering the function of PBR in cholesterol uptake and intramitochondrial movement, we propose that decreased PBR expression may lead to reduced levels of mitochondrial membrane cholesterol, which, together with the ability of Bcl-X(L) and Bax to form ion channels, produces breaks in the outer membranes allowing the exit of cytochrome c, thus triggering apoptosis. Alternatively, PBR may exert an as yet unidentified anti-apoptotic function.

GnRH action on luteal steroidogenesis during pregnancy.

The results of our study presented here establishes that gonadotropin-releasing hormone (GnRH) acts directly on the corpus luteum, leading to suppressed production and release of progesterone and thus disrupting pregnancy. A GnRH-agonist (GnRH-Ag) treatment suppressed the luteal and serum progesterone levels. This suppression is neither mediated by a fall in ovarian testosterone production nor its conversion to estradiol. Although the treatment suppressed the nuclear estradiol-receptor content and binding sites for LH in the corpus luteum, it had no effect on the luteal binding sites for GnRH and prolactin within 24 h. GnRH-Ag augmented the plasma levels of luteinizing hormone, decreased the magnitude of nocturnal surges of prolactin, and had no effect on luteal cyclic adenosine 5'-monotriphosphate levels. Yet, the treatment had no effect on the luteal content of free cholesterol. We have also demonstrated, for the first time, the presence of steroidogenic acute regulatory protein and peripheral benzodiazepine receptor in the rat corpus luteum, and the suppression of these proteins by GnRH-Ag leads to reduced steroidogenesis by the corpus luteum. Concomitantly, P450 side-chain cleavage enzyme, its activity, and its mRNA content and 3beta-hydroxy-steroid dehydrogenase content in the corpus luteum decreased. The treatment suppressed the plasma levels of pregnenolone and 20alpha-dihydroprogesterone. These data suggest that the suppression of luteal steroidogenesis by GnRH-Ag may be due to its inhibitory effect on the cholesterol transport and/or on the enzymes involved in the steroidogenic pathway. Furthermore, based on other observations made in our laboratory, we propose a hypothesis that an endogenous GnRH is present in the corpus luteum/ovary during pregnancy in the rat and that this GnRH may play a physiological role in the regulation, maintenance, and/or termination of pregnancy.

Immunolocalization and expression of prohibitin, a mitochondrial associated protein within the rat ovaries.

This study was designed to determine the cellular distribution and pattern of expression for the mitochondria-associated protein, prohibitin, during the transitional stages of follicular differentiation within the rat ovary. Immunohistochemical staining techniques were used on frozen sections to examine the localization of prohibitin to preantral, antral, preovulatory, and atretic follicles. Prohibitin localization was also determined in corpus luteum from adult rats, in addition to those from infant and juvenile ovaries, before and after gonadotropin stimulation. Western and Northern blotting techniques were used for qualitative and quantitative assessment of prohibitin expression levels within the ovary. Prohibitin was localized within granulosa cells of infant and juvenile ovaries in a relatively heterogeneous staining pattern. The oocyte also exhibited robust prohibitin expression at all stages of follicular development. In addition, strong prohibitin expression was evident in the corpus luteum as well as in follicles undergoing atresia. Additional data derived from studies involving a GnRH-agonist indicate that increases in prohibitin protein expression correlate with the initial events of apoptosis. Collectively, these results support a growth regulatory role for prohibitin within the rat ovary. Therefore, we propose that prohibitin may serve as an important regulator of granulosa cell fate during follicular development.

GnRH agonist treatment decreases progesterone synthesis, luteal peripheral benzodiazepine receptor mRNA, ligand binding and steroidogenic acute regulatory protein expression during pregnancy.

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) suppresses luteal steroidogenesis in the pregnant rat. We further demonstrated that the peripheral-type benzodiazepine receptor (PBR) and the steroidogenic acute regulatory protein (StAR) play key roles in cholesterol transport leading to steroidogenesis. The purpose of this study was to understand the cellular and molecular mechanisms involved in the suppression of luteal steroidogenesis leading to a fall in serum progesterone levels in GnRH-Ag-treated rats during early pregnancy. Pregnant rats were treated individually starting on day 8 of pregnancy with 5 microgram/day GnRH-Ag using an osmotic minipump. Sham-operated control rats received no treatment. At 0, 4, 8 and 24 h after initiation of the treatment, rats were killed and corpora lutea (CL) were removed for PBR mRNA, protein and radioligand binding analyses, immunoblot 1-D gel analysis of StAR, P450 scc and 3beta-hydroxysteroid dehydrogenase as well as 2-D gel analysis of StAR. The treatment decreased the luteal PBR mRNA expression at all time periods starting at 4 h compared with that in corresponding sham controls. GnRH-Ag also reduced, in the CL, the PBR protein/ligand binding, the StAR protein and P450 scc protein and its activity as early as 8 h after the treatment and they remained low compared with those in corresponding sham controls. The data from 2-D gel studies suggest that the majority of the decrease in StAR protein appears to be in the phosphorylated forms of StAR. Thus, we have demonstrated, for the first time, the presence of PBR and StAR in the pregnant rat CL and that the coordinated suppression of these proteins involved in the mitochondrial cholesterol transport along with P450 scc by GnRH-Ag leads to reduced ovarian steroidogenesis.

Expression of mRNA and proteins for testicular steroidogenic enzymes and brain and pituitary mRNA for glutamate receptors in rats exposed to immobilization stress.

The objectives of this study were to determine whether stress attenuates the pituitary LH response to excitatory amino acids by altering expression of glutamate receptor 1 (GluR1) and N-methyl-D-aspartic acid (NMDA) receptor mRNA levels in the hypothalamus or pituitary, and assess whether stress influences testicular levels of mRNA or protein for steroidogenic enzymes. Three hours (h) of immobilization stress was associated with a greater than 7-fold increase in serum corticosterone, and a marked reduction in serum testosterone (T) concentrations. Stress did not significantly alter hypothalamic or pituitary GluR1 and NMDA receptor mRNA levels. Although transcript levels for P450SCC and P45017alpha mRNA in the testis were unchanged in stressed rats, western blotting of testicular fractions revealed reduced amounts of P450SCC and 3beta-HSD, but not P45017alpha. The data suggest that immobilization stress reduces T production by suppressing the translation of transcripts for P450SCC and 3beta-HSD, but the attenuated LH response of stressed animals to NMDA is not mediated by altered hypothalamic or pituitary expression of GluR1 and NMDA receptor levels.

In vivo studies on the role of the peripheral benzodiazepine receptor (PBR) in steroidogenesis.

In various steroidogenic cell models, mitochondrial preparations and submitochondrial fractions, the expression of the mitochondrial 18 kDa peripheral-type benzodiazepine receptor (PBR) protein confers the ability to take up and release, upon ligand activation, cholesterol. Thus, cholesterol becomes available to P450scc on the inner mitochondrial membrane. These in vitro studies were validated by in vivo experiments. Treatment of rats with ginkgolide B (GKB), specifically reduced the ligand binding capacity, protein, and mRNA expression of the adrenocortical PBR and circulating glucocorticoid levels. Treatment with GKB also resulted in inhibition of PBR protein synthesis and corticosterone production by isolated adrenocortical cells in response to ACTH. The ontogeny of both PBR binding capacity and protein directly paralleled that of ACTH-inducible steroidogenesis in rat adrenal cells and in rats injected with ACTH. In addition, the previously described suppression of luteal progesterone synthesis in the pregnant rat by continuous in vivo administration of a gonadotropin-releasing hormone agonist may be due to decreased luteal PBR ligand binding and mRNA. These results suggest that (i) PBR is an absolute prerequisite for adrenocortical and luteal steroidogenesis, (ii) regulation of adrenal PBR expression may be used as a tool to control circulating glucocorticoid levels and (iii) the stress hypo-responsive period of neonatal rats may result from decreased adrenal cortical PBR expression.

Induction of apoptosis by a gonadotropin-releasing hormone agonist during early pregnancy in the rat.

We have demonstrated that continuous administration of a GnRH-agonist (GnRH-Ag) in the rat during early pregnancy suppressed plasma progesterone levels within 8 h after the commencement of treatment. The purpose of the present study is to evaluate the hypothesis that in vivo GnRH-Ag treatment induces apoptotic cell death during early pregnancy. Rats were treated individually on day 8 of pregnancy with 5 microg/day of GnRH-Ag via an osmotic minipump. Sham-controlled rats received no treatment. Rats were killed at 4, 8 or 24 h after the treatment. At autopsy, blood samples were obtained and ovaries were removed. The corpora lutea (CL) from one ovary were removed for DNA laddering and RNA studies. As early as 8 h after the commencement of treatment, GnRH-Ag suppressed serum progesterone levels as expected, and increased the degree of DNA degradation in the CL that was time-dependent. At 24 h after the treatment, GnRH-Ag increased the Bax, a cell death gene, expression in the CL. Collectively, these data suggest that GnRH-Ag treatment induces apoptosis during early pregnancy in the rat.

Interaction of mouse placental lactogens and androgens in regulating progesterone release in cultured mouse luteal cells.

Pituitary hormones are essential for the maintenance of the corpus luteum in the pregnant mouse during the first half of gestation. Thereafter, hormones from the placenta take over the luteotropic role of the pituitary hormones. Mouse placental lactogen-I (mPL-I) and mPL-II, two PRL-like hormones produced in the placenta, are probably necessary for the maintenance of the corpus luteum in the latter half of pregnancy. A culture system of luteal cells from pregnant mice was developed to investigate the role of hormones from the placenta that may be important for the function of the corpus luteum. Mice were killed on days 10, 14, and 18 of pregnancy, and the corpora lutea were excised from the ovaries and digested in 0.1% collagenase, 0.002% DNase for 1 h. The resulting luteal cell suspension was plated onto 96-well plates coated with fibronectin (1 x 10(5) cells/well) and cultured for 1-3 days. Medium was changed daily. The cells were treated with various concentrations and combinations of mPL-I, mPL-II, mouse PRL, androstenedione, dihydrotestosterone, 17beta-estradiol (E2), testosterone, hydroxyflutamide, cycloheximide, actinomycin D, and fadrozole to study the effects of these different treatments on progesterone (P4) production. The three lactogens (mPL-I, mPL-II, and mouse PRL) all stimulated the release of P4 from the luteal cells. The potency of the lactogens was similar and did not depend on the stage of pregnancy at which the luteal tissue was obtained. However, the responsiveness of the cells to all hormone-stimulated P4 release was gradually reduced the later in pregnancy the tissue was collected. Androgens also stimulated the release of P4 from the luteal cells, and when administered together, the lactogens and the androgens acted synergistically to stimulate P4 release. The androgens acted directly but not through conversion to E2, as determined by the findings that 1) the effects of the androgens could not be reproduced by E2 administration, 2) nonaromatizable androgen dihydrotestosterone was as effective as aromatizable androgens, and 3) aromatase inhibitor did not prevent the action of the androgens to stimulate the P4 release. The effect of the androgens on the P4 release was rapid, occurring within 15 min of hormone administration. It was not prevented by inhibitors of protein and RNA synthesis, and the intracellular androgen receptor antagonist hydroxyflutamide did not affect the androgen action. Therefore, the androgen effects were not mediated through the intracellular androgen receptor and de novo protein synthesis was not needed for androgen-stimulated P4 release.

Effects of short-term treatment of a gonadotropin-releasing hormone agonist on the follicular development and gonadotropin secretion in the rat.

The gonadotropin-releasing hormone (GnRH) controls pituitary gonadotropin biosynthesis and secretion and therefore it is indispensable in regulating reproductive function. We have studied the effects of a short term treatment (7-days) of a GnRH-agonist (GnRH-Ag) in vivo (a) on reproductive cyclicity and follicular development and (b) on peripheral gonadotropin secretion of normal cycling rat. GnRH-Ag (0.2, 1 or 5 micrograms/day), administered continuously through an osmotic minipump for 7-days, had a varied effect on ovarian cyclicity as is evident by vaginal cytology and it also decreased ovarian weight. A dose of 1 as well as 5 micrograms/day for 7-days of GnRH-Ag caused the complete demise of early and late antral follicles, whereas a dose of 0.2 microgram/day of GnRH-Ag for 7-days caused a significant decrease in the number of late antral follicles. There was a remarkable increase in the number of atretic follicles in the ovary of rats that were treated with 5 micrograms/day of GnRH-Ag for 7-days. Ovarian histology showed the predominance of corpora lutea in rats treated with 1 or 5 micrograms/day of GnRH-Ag and the interstitial cells in general appeared pycnotic. GnRH-Ag treatment significantly enhanced the serum LH secretion but FSH secretion remained unaffected. Serum PRL concentration diminished in rats that were treated with the highest dose (5 micrograms/day) of GnRH-Ag. Results from this study clearly demonstrate that GnRH-Ag treatment interferes with normal cyclicity of rats and it profoundly affects the follicular development. Therefore, it acts as an atretognic factor in the ovary.

Follicular development, steroidogenesis and gonadotrophin secretion in response to long-term treatment with a gonadotrophin-releasing hormone agonist in the rat.

Gonadotrophin-releasing hormone (GnRH) and its agonists are implicated in the local control of rat ovarian function. We have evaluated the effects of long-term administration of different doses of GnRH agonist (GnRH-Ag) in vivo (a) on reproductive cyclicity and follicular development, (b) on peripheral gonadotrophin and steroid concentrations and (c) on in vitro cAMP and progesterone production by the follicles in response to stimulatory doses of FSH or LH (1 microgram/ml). GnRH-Ag (0.2, 1 or 5 micrograms/day) administration for 28 days had a profound impact on the oestrous cycle of rats as revealed by vaginal cytology. GnRH-Ag treatment caused a decrease in ovarian and uterine weights, which correlated very well with the decrease in the number of follicles present in the ovary. GnRH-Ag (5 micrograms/day) reduced the number of early preantral follicles and there was complete disappearance of early as well as late antral follicles. However, a dose of 1 microgram GnRH-Ag/day was effective in the complete demise of only late antral follicles with a significant attenuation in the number of early antral follicles. There was an enhancement in serum LH concentrations in response to the highest dose of GnRH-Ag administration with serum FSH concentrations declining in rats treated with the two higher doses. However, serum prolactin concentrations were attenuated only in rats treated with the highest dose of GnRH-Ag. GnRH-Ag treatment decreased serum progesterone and oestradiol concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of luteal steroidogenesis by two LHRH antagonists (Nal-Glu and Nal-Arg antagonists) in the pregnant rat.

We have examined the effect of two LHRH antagonists (Nal-Glu and Nal-Arg antagonists) on the basal progesterone (P4), pregnenolone (P5) and 20 alpha-dihydroprogesterone (20 alpha-DHP) production by luteal cells obtained from day 8 pregnant rats. A dose of 0.1 mmol/l of Nal-Glu or Nal-Arg attenuated basal P4 production by luteal cells after 12, 24 or 48 h of incubation. P5, a precursor for P4 synthesis was also reduced by both doses of Nal-Glu or Nal-Arg (0.1 mmol or 0.1 mumol/l) after 24 h of incubation. A period of 12 h was not sufficient to inhibit P5 production by luteal cells incubated with both doses of Nal-Glu or with the lower dose of Nal-Arg. The higher dose of Nal-Glu and Nal-Arg remained effective in attenuating P5 production by luteal cells after 48 h of incubation. We measured the production of a metabolite of P4, i.e., 20 alpha-DHP to assess whether this suppression in P4 production is due to an enhancement in the P4 metabolism by increasing the activity of 20 alpha-hydroxydehydrogenase. However, instead, we observed (i) a decrease in the production of 20 alpha-DHP by the higher dose of Nal-Glu and Nal-Arg after 12, 24 or 48 h of incubation and (ii) a decrease or no change in the production of 20 alpha-DHP by the lower dose of Nal-Glu or Nal-Arg at all time periods of incubation. Based on these observations we conclude that LHRH antagonists exert a direct effect on the corpus luteum and suppress luteal steroidogenesis. This suppression in luteal steroidogenesis could be due to a decrease in the activity of any one of these enzymes of the steroidogenic pathway, viz., cholesterol side chain cleavage (P450scc), a rate limiting enzyme involved in the synthesis of P5 from cholesterol, or 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), which catalyzes the oxidation of P5 to P4 or due to a decrease in activity of both enzymes.

Suppression of luteal steroidogenesis by an LHRH antagonist (Nal-Lys antagonist: antide) in vitro during early pregnancy in the rat.

LHRH and its analogues are known to exert direct effects on the ovary. Herein we have described a direct inhibitory effect of an LHRH antagonist (Nal-Lys antagonist: antide) on the basal progesterone (P4) and pregnenolone (P5) production by luteal cells obtained from the day-8 pregnant rat. Luteal cells incubated with two doses of antide (10(-4) and 10(-7) M) for 24 or 48 h showed suppression of P4 production. P5 production was suppressed by both doses of antide within 12 h of incubation. Neither dose of antide interfered with P5 production when the duration of incubation was extended beyond 12 h. The 20 alpha-dihydroprogesterone yield from the luteal cells treated with these doses of antide remained unaffected. We estimated the activities of the cholesterol side-chain cleavage (P450scc) enzyme (which is a key enzyme involved in the conversion of cholesterol to P5) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) (which catalyses the conversion of P5 to P4) in the luteal cells treated with different doses of antide. Both doses of antide suppressed the activity of the P450scc enzyme after 12 h of incubation and the 3 beta-HSD content of the luteal cells after 48 h of incubation. These observations indicate that antide exerts a direct inhibitory effect at the level of the corpus luteum, that differential suppression of P5 and P4 during different periods of incubation with antide is due to a defect in either the P450scc or the 3 beta-HSD enzyme system, or both.

Inhibition of steroidogenesis by luteal cells of early pregnancy in the rat in response to in vitro administration of a gonadotropin-releasing hormone agonist.

Previous studies from this laboratory have demonstrated that the administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo in early or mid-pregnancy to rats induces antifertility effects by suppressing the luteal production of progesterone (P4) within 24h with a concomitant increase in luteal lipid droplets and decreases in the luteal cytochrome P450 side chain cleavage (P450scc) enzyme and its mRNA content. These observations suggest a direct inhibitory effect of GnRH-Ag on the corpus luteum. Here we demonstrate a suppressive effect of GnRH-Ag in vitro on the basal P4, pregnenolone (P5) and 20 alpha-dihydroprogesterone (20 alpha-DHP) production by luteal cells obtained during early pregnancy in rats. We further studied its effect on two key enzymes, namely P450scc and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), which participate in the conversion of cholesterol to P5 and conversion of P5 to P4, respectively. We observed that two doses of GnRH-Ag, 10(-4) and 10-7 M, suppress the basal P4 production in vitro after 12 h of incubation by luteal cells; P4 remained suppressed after 48 h of incubation. Basal P5 production was also suppressed after luteal cells were incubated for 12 h with 10(-4) M and 10(-7) M GnRH-Ag, but incubation for 48 h with GnRH-Ag failed to alter P5 production by these cells. 20 alpha-DHP production was suppressed after incubating the luteal cells with both doses of GnRH-Ag for 12 h. GnRH-Ag inhibited P450scc activity after 12 h of incubation and 3 beta-HSD protein content at all time periods measured. These results suggest that GnRH exerts a direct inhibitory effect on luteal steroidogenesis. This inhibition is due to its suppressive effect on P450scc and/or 3 beta-HSD and not due to an increase in P4 metabolites.

Antireproductive effect of a potent LHRH antagonist (Nal-Lys antagonist:antide) during early pregnancy in the rat.

Effects of a potent third generation LHRH antagonist [Nal-Lys antagonist:antide] have been observed during the first half of pregnancy in rats. A daily dose of 40 micrograms or above, when administered from day 8 of pregnancy, suppressed serum progesterone levels within 48 h and by day 12 there was complete absence of live fetuses. Lower doses of antide (10-20 micrograms) reduced progesterone levels in circulation but were unable to induce abortion consistently in all treated rats. Administration of 150 or 300 micrograms of antide once on day 8 suppressed the progesterone levels within 24 h. Only a dose of 300 micrograms was effective in completely interfering with gestation by day 12 with no observable live fetuses.

Effects of in vivo dihydrotestosterone treatment on changes in nocturnal surge of prolactin, luteal ultrastructure and P-450scc mRNA and protein content in pregnant rats.

We have demonstrated previously that the administration of dihydrotestosterone (DHT) decreases plasma progesterone levels within 24 h and thus, results in abortion during the first half of pregnancy (Am. J. Physiol. 241 (1981) E444-E448). The purpose of this study was to determine (a) if the administration of DHT suppresses plasma prolactin levels or its nocturnal surge within 24 h after the treatment, (b) how soon after the commencement of treatment do the concentrations of DHT increase and progesterone levels decrease in the circulation, (c) the ultrastructural changes that occur in corpora lutea, and (d) the changes in luteal P-450 side-chain cleavage (P-450scc) enzyme and mRNA content upon DHT treatment. Within 24 h after the commencement of DHT treatment, the nocturnal surge of prolactin, detected in both groups on day 10 at 03.30 h, was inhibited in DHT-treated rats as compared to controls. The non-surge levels of prolactin at 05.00 and 06.00 h were not different between groups. The intraovarian DHT pellet increased plasma levels of the steroid 3-fold within 2 h (blood samples were taken at 2-hourly intervals) when compared to controls. By 24 h DHT levels were decreased but were still higher than controls. Plasma progesterone levels began to fall 6 h after the commencement of treatment. Luteal tissue from animals treated with DHT appeared steroidogenic, and contained more lipid droplets than controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in rat luteal ultrastructure and P450scc mRNA and protein content after in vivo treatment with a gonadotropin-releasing hormone agonist.

Previous studies have demonstrated that plasma progesterone levels decrease in pregnant rats treated in vivo with a gonadotropin-releasing hormone agonist (GnRH-Ag), without changes in testosterone or estradiol levels in ovarian vein plasma. The objective of this study was to determine the loci of GnRH-Ag disruption of progesterone synthesis by examining luteal mitochondria, lipid droplets, cellular composition, and P450 side-chain cleavage (P450scc) enzyme and mRNA content in the pregnant rat. On Day 7 or 11 of pregnancy, osmotic minipumps containing GnRH-Ag were implanted into 5-7 rats. Sham operations were performed on 5-6 controls at each time period. Five micrograms per day of GnRH-Ag were released for about 24 h, after which corpora lutea and jugular vein plasma were collected. The corpora lutea were prepared for microscopy or analyzed for P450scc enzyme and mRNA content. Plasma progesterone levels were measured by RIA. In those rats treated with GnRH-Ag, progesterone levels had decreased, and within the luteal cells, there was an increase in the number of lipid droplets and a decrease in the number of tubular cristae within the mitochondria. Concomitantly, P450scc enzyme and mRNA content decreased on both Day 8 and Day 12 of pregnancy. Also, GnRH-Ag treatment decreased the ratio of large to small steroidogenic luteal cells on Day 8 of pregnancy, but did not alter cellular ratios on Day 12 of pregnancy. These observations suggest that treatment with GnRH-Ag inhibits progesterone synthesis by decreasing the amount of P450scc mRNA and enzyme content, which may alter the mitochondrial cristae structure on Day 8 and Day 12 of pregnancy. The reduction in tubular cristae and P450scc enzyme in the mitochondria may account for the increase in lipid droplets, as less cholesterol is converted to pregnenolone. An additional mechanism of inhibition may be the reduction in the number of large steroidogenic luteal cells, which appear to be the major source of progesterone in the rat corpus luteum on Day 8 of pregnancy.

Time-dependent biphasic effect of cytochalasin D on luteal progesterone release in the pregnant rat.

Numerous studies have examined the effects of cytoskeletal disruption on steroidogenesis; while some report an inhibition, other studies show a stimulation of steroid hormone production. In the present study, the possibility of a biphasic effect of cytoskeletal inhibitors on steroidogenesis was examined. Luteal tissue from day 12 pregnant rats was incubated for either 3.5 h (short-term) or 12.5 h (long-term) with cytochalasin D or colchicine at 10(-4) M in Medium 199 (medium). Controls were incubated in medium alone. After the incubation, the tissues were separated from the medium, and either processed for electron microscopy, or weighed and snap-frozen for subsequent homogenization and steroid hormone measurements. Progesterone, testosterone, and 17 beta-estradiol levels in the medium were measured by radioimmunoassay. After the short-term incubation, progesterone release decreased with cytochalasin D treatment, while cells became more rounded in shape with a loss of microfilaments. Upon long-term incubation, progesterone release increased and cell contact lessened. Colchicine had no effect at either incubation time, and estradiol and testosterone production remained unchanged throughout the experiments. These results demonstrate that cytochalasin D has a biphasic effect on luteal progesterone release in the rat and provides an explanation for the dichotomy of results thus far reported. In addition, the effects of cytochalasin D on rat luteal progesterone production appear to be the result of changes in cell shape or cell-to-cell contact.

Replacement of progesterone restores the nocturnal surge of prolactin following suppression with a gonadotropin-releasing hormone agonist during early pregnancy in the rat.

Continuous administration of a GnRH agonist (GnRH-Ag) at a dose of 5 micrograms/day, commencing on day 7 of pregnancy resulted in the suppression of daily nocturnal surges of prolactin (PRL) on day 8, and serum progesterone (P4) levels with subsequent termination of pregnancy. Replacement with dydrogesterone, a synthetic analog of P4 at a dose of 4 mg/day s.c. restored the magnitude of nocturnal PRL surges. These data suggest that GnRH-Ag may act either at the level of the brain to suppress the nocturnal PRL surge, resulting in a fall in serum P4 levels or at the level of the corpus luteum itself or at both sites simultaneously to terminate pregnancy.