Rat Corpus Luteum Research Articles

Intracellular regulation of progesterone secretion by the superoxide radical in the rat corpus luteum

Intracellular regulation of progesterone secretion by the superoxide radical in the rat corpus luteum

Prolactin-Induced Regression of the Rat Corpus Luteum: Expression of Monocyte Chemoattractant Protein-1 and Invasion of Macrophages1

Monocyte chemoattractant protein-1 (MCP-1) is a potential mediator of the recruitment of monocytes/macrophages into the regressing corpus luteum (CL). We investigated whether the luteolytic effect of prolactin in the rat is associated with the expression of MCP-1 and an invasion of monocytes/macrophages. Ovulation was induced in immature female rats by injection of eCG (5 IU, s.c.) at 30 days of age. All rats were hypophysectomized 3 days later. Rats received injections of ovine prolactin (250 micrograms, s.c.) at 12-h intervals on Day 9, 10, and 11 posthypophysectomy; controls received injection of vehicle. Rats were killed by decapitation 24, 48, or 72 h after the first injection of prolactin or vehicle. In rats treated with prolactin, immunoreactive MCP-1 was detected in the CL at 24 h after the first injection, and a consistent level of staining was reached by 72 h with immunodetectable MCP-1 diffused throughout individual CL. The number of monocytes/macrophages in the CL (mean +/- SEM) increased significantly after prolactin treatment, from 3.1 +/- 1.8 at 24 h to 49.3 +/- 8.2 at 72 h (p < 0.05), and the number of monocytes/macrophages was different from that in control, vehicle-treated rats at 72 h (10.3 +/- 4.1; p < 24 and 72 h in prolactin-treated rats (p < 0.05). It is concluded that a potentially important component of the luteolytic effect of prolactin in the rat is the expression of MCP-1 and invasion of monocytes/macrophages into the CL.

Changes in the gene expression of a protein with the cdc10/SWI6 motif, V-1, during rat follicular development and corpus luteum formation

Changes in the gene expression of a protein with the cdc10/SWI6 motif, V-1, during rat follicular development and corpus luteum formation

Changes in the gene expression of a protein with the cdc10/SWI6 motif, V-1, during rat follicular development and corpus luteum formation.

We examined the gene expression of V-1, a novel soluble protein with the cdc10/SWI6 motif, in pseudopregnant rat ovaries. Northern blot analysis on days 1, 5, and 11 of pseudopregnancy revealed an approximately 2-fold increase in the V-1 messenger RNA (mRNA) expression level on day 5 to that on day 1, and no significant change was observed between those on day 5 and day 11. An injection of human CG on days 5 further increased th V-1 mRNA level to about 1.6-fold of that of the untreated control. Western blot analysis showed higher V-1 protein expression on days 5 and 11 of pseudopregnancy than that on day 1. In situ hybridization and immunohistochemistry with ovaries on day 3 of pseudopregnancy showed that luteal cells of corpora lutea and also cells of the coexisting follicles including oocytes express V-1 mRNA and the protein, with apparent rank order of the expression: oocytes > luteal cells > follicular cells >> atretic follicular cells including oocytes. These data indicate the dynamic change in the V-1 gene expression in the ovarian steroidogenic cells and oocytes and suggest potential roles of the V-1 protein in ovarian functions including corpus luteum formation and folliculogenesis.

Prolactin induction of the alpha 2-Macroglobulin gene in rat ovarian granulosa cells: stat 5 activation and binding to the interleukin-6 response element.

alpha 2-Macroglobulin (alpha 2M) is expressed at high levels in the corpus luteum of pregnant rats in response to PRL and rat placental lactogens. These studies document that PRL induction of alpha 2M mRNA occurs rapidly in granulosa cells differentiated to the preovulatory phenotype in the presence of FSH and steroid, is hormone specific [induced by PRL but not by LH or interleukin-6 (IL-6)], and involves tyrosine kinase activity. To analyze the cellular signaling events stimulated by PRL, transient transfections of granulosa cells and electrophoretic mobility shift assays were done using the IL-6 response element (IL-6RE) of the alpha 2M promoter. The IL-6RE consists of two gamma-activating like sequences (GAS) that bind the acute phase response factor (APRF/Stat 3) in rat liver and the mammary gland factor (MGF/Stat 5) from mammary tissue. By transfecting various alpha 2M promoter-luciferase reporter transgenes into the granulosa cell cultures, we show that the GAS-like sites together with the minimal -48 base pairs of the alpha 2M promoter can confer PRL inducibility to the luciferase reporter gene. These same GAS-like sequences of the alpha 2M promoter were used to analyze the DNA-binding activity of proteins in whole cell extracts prepared from differentiated granulosa cells exposed to PRL for 0.25, 0.5, 4, and 20 h. PRL rapidly stimulated the binding of a specific protein to labeled alpha 2M GAS-like oligonucleotide, and this PRL-induced binding activity was shown to contain Stat 5 but not Stat 1 or Stat 3, using specific antibodies in the electrophoretic mobility shift assays. Because both Stat 5 and Stat 3 proteins are present in the whole cell extracts of differentiated granulosa cells, PRL appears to activate detectable amounts of Stat 5 (and not Stat 3). Thus, the initial induction of the alpha 2M gene by PRL in differentiated rat granulosa cells involves, at least in part, the activation (tyrosine phosphorylation?) of Stat 5.

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.

Heat shock protein-70 induction mediates luteal regression in the rat.

Induction of heat shock protein 70 (HSP-70) is associated with inhibition of hormone-sensitive steroidogenesis and interruption of cholesterol translocation to or into the mitochondria. A number of pharmacological and physiological inhibitors of luteal cell function stimulate HSP-70 synthesis via activation of the heat shock transcription factor (HSF). In the present study we address the following questions: 1) is HSP-70 synthesis increased during prostaglandin F2 alpha (PGF2 alpha)-induced luteolysis or natural luteal regression? 2) Does blocking HSP-70 synthesis reverse the inhibition of hormone-sensitive steroidogenesis induced by heat stress and PGF2 alpha? Gel-retardation assays demonstrated HSF activation within 7.5 min after PGF2 alpha (400 micrograms) administered in vivo. Western blotting revealed HSP-70 synthesis by 1 h with higher levels seen at 2 h. The stress response was similarly activated during natural regression of the corpus luteum in rats. Gel retardation assays demonstrated maximal HSF activation 10 days after ovulation. Western blotting showed that HSP-70 levels increased dramatically on this same day and were sustained for several days after the initiation of luteal regression. Inhibition of HSP-70 synthesis was investigated in isolated luteal cells using a cholesteryl-conjugated phosphorothioate antisense oligodeoxynucleotide. The control was an oligodeoxynucleotide with the same base composition, but with scrambled base sequence. Incubation with 3 microM antisense oligodeoxynucleotide for 2 h before a 42 C heat shock prevented synthesis of HSP-70 while up to 4.5 microM control oligodeoxynucleotide had no effect on HSP-70 synthesis in response to heat shock. Preincubation with antisense oligodeoxynucleotide partially reversed heat stress-induced inhibition of LH-stimulated steroidogenesis. More importantly, preincubation with antisense oligodeoxynucleotide also significantly reversed inhibition of cAMP-stimulated steroidogenesis induced by PGF2 alpha. Treatment with control oligodeoxynucleotide did not reverse heat shock or PGF2 alpha inhibition of hormone-dependent steroidogenesis. The findings that the synthesis of HSP-70 coincides with the loss of luteal function and that blocking its synthesis reverses inhibition of hormone-dependent steroidogenesis strongly suggest a role for HSPs as physiological mediators of luteal regression.

Studies on the role of plasminogen activators and plasminogen activator inhibitor type-1 in rat corpus luteum of pregnancy.

Luteal development and demise are characterized by substantial tissue destruction and remodeling, which is associated with local production of plasminogen activation. Recently we reported involvement of tissue-type plasminogen activator (tPA) in luteolysis in rhesus monkeys. In this study, we further investigated changes in expression of both tPA and urokinase-type plasminogen activator (uPA) activity during various developmental stages of rat corpus luteum (CL) of pregnancy and their possible physiological roles in luteolysis. Rat CL or dispersed luteal cells in vitro are capable of producing both tPA and uPA, and a plasminogen activator inhibitor type-1, in a stage-dependent manner. However, only tPA activity significantly increases in late phases of CL development. Furthermore, the increase in tPA activity in the CL is well correlated with a sharp decrease in luteal progesterone production. Addition of exogenous tPA to the luteal culture considerably decreases progesterone production. In contrast, immunoneutralization of endogenously produced tPA activity by inclusion of tPA monoclonal antibody in the culture results in a significant increase in luteal progesterone production. It is therefore suggested that tPA may also be involved in suppression of rat luteal function. This hypothesis is further supported by the findings that interferon-gamma significantly inhibits luteal basal and hCG-stimulated progesterone production and also stimulates basal and hCG-induced tPA activity. On the basis of the data provided in this study and similar findings in monkeys, we conclude that endogenously produced tPA in late phase of CL development may regulate luteal regression through local autocrine or paracrine action.

In vitro effects of interactions of follicle-stimulating hormone, luteinizing hormone, and prolactin on progesterone synthesis by rat luteal cells during pregnancy.

The in vitro ability of ovine (o) follicle-stimulating hormone (FSH), (o)luteinizing hormone (LH), (o)prolactin (PRL), and recombinant human FSH (rhFSH) to stimulate progesterone (P4) synthesis by rat corpora lutea on Day 4 of pregnancy was investigated. Dispersed luteal cells (large + small cells) were incubated in the presence of the gonadotropins (1-100 ng) alone or in various combinations (10 ng each) for 4 or 24 hr. Given alone, all the ovine preparations stimulated P4 in a dose-dependent manner with even 1 ng of each hormone significantly enhancing P4 production. Significantly, rhFSH--which is devoid of LH contamination--at 10 and 100 ng also stimulated P4 production, thus clearly establishing for the first time that FSH is a luteotropic hormone in the rat. The combination of oFSH + LH + PRL (10 ng each) significantly stimulated P4 synthesis to a greater extent than the combination of any two hormones or individual hormones at both 4 hr or an additional 24 hr of incubation (P < 0.05). This verified in vitro a previously established in vivo luteotropic complex. One hundred nanamolars of phorbol 12-myristate 13-acetate (PMA) did not affect basal P4 secretion but inhibited cAMP, oFSH, and oLH stimulation of P4. Thus, the luteotropic effects of FSH, LH, and activators of protein kinase A are antagonized by the protein kinase C pathway.

Expression of insulin-like growth factor I mRNA in rat luteal tissue increases with functional regression of the Corpus Luteum

The expression of insulin-like growth factor I (IGF-I) messenger ribonucleic acid (mRNA) in rat corpora lutea (CL) was determined during the second half of pregnancy and early lactation, and the effect of prostaglandin F 2α on expression was examined during late pregnancy. Northern blot analysis revealed 4 species of transcripts in CL. Radioactivity of dot blots was measured directly for quantitative analysis of expression. The expression in CL increased during the second half of pregnancy, peaked around parturition, and declined thereafter. The injection of 1 mg of prostaglandin F 2α at 1300 and 1800 h on day 18 of pregnancy, which decreased plasma progesterone concentration, doubled luteal expression of IGF-I mRNA 1 day later. These results suggest that functional regression of the corpus luteum is correlated with the increased expression of IGF-I mRNA in luteal tissue in rats.

Status of hCG/LH receptor and G proteins in human endometrium during artificial cycles of hormone replacement therapy

We examined the existence of hCG/LH receptors and associated GTP-binding (G) proteins in membrane fractions of nonpregnant human endometrium and investigated whether their expression is affected, in vivo, by estrogen and progesterone replacement therapy. A pool of normal endometrial biopsy specimens (n = 5) was initially used to characterize receptors and G proteins. Subsequently, biopsy specimens (n = 22) were obtained from 11 patients undergoing evaluation cycles of hormone replacement therapy (HRT). From each patient, two specimens were collected on successive cycle days: on day 0 (last day of estrogen) and on either day 3, 6, or 9 of progesterone supplementation. Both hCG/LH receptor and G proteins were determined in membrane (10,000 x g) fractions by immunoblot analysis using specific polyclonal antibodies against synthetic fragments of hCG/LH receptor and against G proteins. Membrane fractions from rat brain and rat corpus luteum were used as controls. Proteins were loaded on the gel under reducing conditions. The receptor antibody immunoreacted with a protein of approximately 68 kd in endometrial membranes. A similar protein was detected in rat corpus luteum. The G-protein antibodies detected Gs alpha, Gi3 alpha, Gi1 alpha/Gi2 alpha, and common beta subunits in endometrial membranes with a molecular weight of 48-42 kd, 41 kd, 40 kd, and 37 kd, respectively. Analysis of membranes obtained during HRT indicated that levels of hCG/LH receptors remained fairly constant throughout the cycle days (days 0, 3, 6, and 9). Similar results were observed for Gi1 alpha/Gi2 alpha and Gi3 alpha. In great contrast, Gs alpha was low at day 0 but increased with the administration of progesterone (days 3, 6, and 9). Human endometrium contains both membrane-bound hCG/LH receptors and associated G proteins. During HRT, progesterone supplementation to estrogen therapy enhances the expression of Gs alpha protein subunit, but not hCG/LH receptors.

Inducible expression of cellular retinoic acid-binding protein II in rat ovary: gonadotropin regulation during luteal development.

Two members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular retinoic acid-binding protein and cellular retinoic acid-binding protein II [CRABP(II)]. CRABP is found in many adult tissues, whereas CRABP(II) is more restricted and is reported as abundant primarily in skin. Here we report a much greater expression of CRABP(II) in rat corpus luteum than in any other organ/tissue examined, including skin. A rat complementary DNA clone encoding CRABP(II) was isolated and the ovarian expression followed during gonadotropin induction of follicular development in the pseudopregnant rat. The pattern of rat CRABP(II) messenger RNA and protein expression correlated with the appearance of corpora lutea and the rise in progesterone production as the corpora lutea developed, and was similar to the induction of 3 beta-hydroxysteroid dehydrogenase. Immunohistochemical localization revealed that CRABP(II) appeared in luteal cells and was dramatically restricted to their cytoplasmic compartment, with no apparent presence in the nucleus. This suggests that CRABP(II) may be expressed to restrict retinoic acid from occupying nuclear retinoic acid receptors, implying that the differentiation and maintenance of the rat corpus luteum may involve in part a release of certain pathways from retinoid suppression.

Localization of epidermal growth factor (EGF) receptor in the rat corpus luteum, and EGF and transforming growth factor-α stimulation of luteal cell steroidogenesis in vitro

Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) have potent mitogenic effects on granulosa and theca cells. However, their effects on steroidogenesis by these cells is controversial, and there is limited information regarding their effects on luteal cell steroidogenesis. The present study investigated the cellular distribution of the EGF receptor (EGF-R) in the rat corpus luteum (CL) by immunocytochemical staining, and the effects of EGF and TGF-α on progesterone and 20α-dihydroprogesterone (20α-OH-P) production in cultures of luteal cells. Using a primary antibody directed against the human EGF-R peptide, specific EGF-R staining was obtained in the CL. Both small and large luteal cells had EGF-R staining. In initial cell culture experiments, treatment of freshly isolated luteal cells with EGF or TGF-α (0.5–50 ng/ml) for 24 h had no effect on progesterone and 20α-OH-P accumulation. Addition of LH (250 ng/ml) alone caused a 3.5-fold increase in both progestins, but co-treatment with EGF or TGF-α produced no further enhancement of progestin accumulation. However, when cells were seeded overnight and the attached cells were washed prior to growth factor treatment for 3 days with media change every 24 h, both EGF and TGF-α caused dose-dependent increases in progesterone accumulation/24 h period (up to 2-fold at 50 ng/ml growth factor) on days 1 and 2 but not day 3 of treatment. 20α-OH-P accumulation was similarly stimulated (up to 2.5-fold) by EGF and TGF-α under these conditions. In addition, LH-induced increases in progestin accumulation were further enhanced dose-dependently by TGF-α. These data confirm the presence of EGF-R in rat CL, and suggest a physiological role for EGF and TGF-α in luteal steroidogenesis.

Does the rat corpus luteum express the progesterone receptor gene?

To determine whether the progesterone receptor (PR) gene is expressed in rat corpora lutea (CL), PR messenger RNA (mRNA) and protein levels were examined in CL of both cycling and pregnant rats using in situ hybridization, reverse transcription-polymerase chain reaction, and Western blotting analyses. During the estrous cycle, levels of luteal PR mRNA were below the sensitivity of in situ hybridization. Although no signal could be detected in luteal cells, granulosa cells of preovulatory follicles of the same ovary expressed PR mRNA at high levels during the evening of proestrus. Likewise, CL of pregnant rats expressed undetectable levels of PR mRNA, which remained unchanged throughout pregnancy, as determined by in situ hybridization and reverse transcription-polymerase chain reaction. In addition, no PR protein could be detected in rat CL by Western analysis, indicating that luteal expression of the PR gene, if any, is negligible. To determine whether the lack of PR mRNA in the rat CL is due to progesterone-induced down-regulation of PR mRNA or to low levels of estrogen, aminoglutethimide was used to block the synthesis of progesterone in the presence and absence of exogenous estrogen, and PR mRNA levels were examined in the CL as well as the placenta. Inhibition of progesterone synthesis did increase PR mRNA levels in the placenta, and additional estrogen treatment further increased PR mRNA levels in this tissue. In contrast, neither aminoglutethimide alone nor aminoglutethimide plus estrogen induced PR mRNA expression in CL of the same rats. The temperature-sensitive luteal cell line derived from the rat CL, which produces very low levels of progesterone, also did not express PR mRNA. These results indicate that the rat corpus luteum expresses undetectable levels of PR mRNA and protein, which is not attributable to progesterone-induced down-regulation of PR mRNA or to a lack of estrogen-induced up-regulation of PR mRNA in this tissue.

Presence of the intermediate filaments cytokeratins and vimentin in the rat corpus luteum during luteal life-span.

The presence of the intermediate filament (IF) proteins cytokeratins and vimentin in corpus luteum (CL) and other parts of the ovary from adult pseudopregnant rats was investigated using immunohistochemistry and immunoblot analysis. To induce pseudopregnancy, female rats were mated with sterile male rats. The mating procedure induces a prolonged luteal life-span of 13 +/- 1 days. Positive staining for cytokeratin could be seen in CL, in the theca layer of follicles, and the ovarian surface epithelium with the broad-spectrum monoclonal antibody cocktail AE1/AE3. Weak staining was also seen in CL with antibodies against cytokeratins 8 and 18. A similar distribution was also seen for vimentin, which furthermore was detected in blood vessels. No changes in staining intensity was seen in CL of different luteal age. The strong staining for vimentin in CL was confirmed by immunoblot analysis, where one main band of 57 kDa was observed from day 1 to day 19 of pseudopregnancy. Expression of the IF proteins investigated seems to start in the newly formed CL and the continuous expression indicates that they are not directly regulated by luteal steroids.

Immunoreactive (ir)-transforming growth factor (TGF)-beta in rat corpus luteum: ir-TGF beta is expressed by luteal macrophages.

Western analyses and immunohistochemistry of transforming growth factor beta 1 (TGF beta 1) and TGF beta 2 were performed in rat luteal tissue at either the functional or regressing stage. Anti-TGF beta 1 or TGF beta 2 was localized simultaneously to macrophages within cultured luteal cells and frozen ovarian sections. By Western analysis, an active form of TGF beta (25 kDa) was detected clearly with anti-TGF beta 2 but only faintly with anti-TGF beta 1, and the former band from functional corpora lutea was more intense than that from regressing ones. Treatment with prolactin (PRL), a luteotropic hormone in rodents, also increased the intensity of 25 kDa TGF beta 2 in the corpus luteum. Several specific bands with higher molecular weights than 25 kDa were also recognized with anti-TGF beta 1 or TGF beta 2; they are probably ascribed to latent TGF beta s and/or TGF beta precursor proteins. In cultured luteal cells and frozen ovarian sections, many anti-TGF beta 1 or anti-TGF beta 2 positive cells from functional corpora lutea of pseudopregnant rats were double-stained with anti-macrophage. There were numerous macrophages in structurally regressing corpora lutea of pseudopregnant rats, but most of them were not stained with anti-TGF beta s. These results suggest that the expression of TGF beta, at least for TGF beta 2, is under the influence of PRL in the rat corpora lutea, and that macrophages are responsible for some, if not all, of the immunoreactive-TGF beta in rat luteal tissue.

Inhibin is present in the corpus luteum of pseudopregnant rats and can interact with progesterone to inhibit resumption of estrous cycles.

The corpora lutea (CL) of ovaries of adult pseudopregnant (PSP) rats were stained by immunohistochemistry for inhibin alpha subunit using sheep alpha-chain1-27 antibody as a measure of inhibin content. Staining was light in estrous cycle CL (stage III), increased in Day 2 and Day 4 PSP CL and reached maximal density on day 6 PSP when both large cells and many small cells stained. Staining was decreased in CL from 8 and 10 day PSP. Granulosa cells of growing follicles showed light stain on days 8 and 10 of PSP. Adult female rats were treated for 5 days beginning at estrous cycle Stage III (cornified masses). In Experiment 1, three groups of rats were used: 1. implanted with 30mm progesterone capsules and injected with saline, 2. implanted with 30mm progesterone capsules and injected with 10 ng rh inhibin A daily, 3. implanted with empty capsules and injected with 10 ng rh inhibin A daily. Both groups of rats with progesterone implants had significantly slower return of cycling after removal of the capsules than did the rats injected with 10ng rh inhibin A alone. In Experiment 2, the same treatments were given except that 20ng of rh inhibin A (10ng 2x/day) was given to both inhibin treatment groups. Rats treated with progesterone and 20ng inhibin had a significantly slower return of cyclic activity (4.4 days) over that found in the other two treatment groups. We conclude that the absence of cyclic activity in PSP rats is possibly due to the secretion of both inhibin and progesterone by the CL.

Cloning of the rat and human prostaglandin F 2α receptors and the expression of the rat prostaglandin F 2α receptor

We have cloned the FP receptor from rat corpus luteum and human uterus cDNA libraries, respectively. The coding DNA sequence in the rat cDNA is 1101 bp and is similar to the mouse cDNA coding for a receptor protein of 366 amino acids. The human sequence shows a 5 bp deficiency in the 3′ region, truncating the coding sequence to 359 amino acids. Northern blot analysis indicates highest expression in the ovary. Cell lines have been established giving stable expression of the FP receptor. Activation of the cloned FP receptor gave an increase in intracellular calcium, indicating signaling via phospholipase C-mediated phosphoinositide turnover. Using [ 3H]PGF 2α, binding of PGs showed the rank order of fluprostenol > PhXA70 > PGF 2α >- PhXA85 > PGD 2 > PGE 2.

Lipid hydroperoxides evoke antigonadotropic and antisteroidogenic activity in rat luteal cells.

At functional luteolysis, the rat corpus luteum generates hydrogen peroxide (H2O2), which is known to rapidly inhibit gonadotropin-sensitive cAMP and progesterone production in isolated luteal cells. Lipid peroxides also increase markedly in the rat corpus luteum with the onset of functional luteolysis, and H2O2 is a potent inducer of lipid peroxidation. However, the actions of lipid peroxides on cell function are unknown. The objective of this study was to investigate the impact of typical lipid peroxides, cumene hydroperoxide (CuOOH) and 15(S)-hydroperoxyeicosatetraenoic acid, on rat luteal cells. CuOOH inhibited both LH-sensitive cAMP accumulation (ED50, 25 microM) and progesterone production (ED50, 20 microM). 15(S)-hydroperoxyeicosatetraenoic acid also dose dependently inhibited steroidogenesis. A significant reduction of LH-stimulated progesterone production was evident within 5 min of treatment with CuOOH, whereas inhibition of cAMP accumulation was not evident until 60 min. 8-Bromo-cAMP and 22-hydroxycholesterol caused partial and complete reversal of CuOOH-inhibited progesterone secretion, respectively. Preincubation of cells with o-phenanthroline completely reversed the inhibitory effects of CuOOH on cAMP accumulation and partially reversed its effects on progesterone production. Incorporation of radiolabeled amino acids into luteal proteins was significantly inhibited by CuOOH (25 microM) within 2 min of treatment and was reduced to 40 +/- 14% of control levels at 60 min. CuOOH (25 microM) maximally stimulated PGE2 production within 30 min of treatment (180 +/- 30% of control), a response that was completely blocked by aristolochic acid (100 microM), a phospholipase-A2 inhibitor, and indomethacin (1 microgram/ml), a prostaglandin (PG) synthesis inhibitor. The present results suggest that the acute inhibitory action of lipid peroxides on LH-stimulated progesterone production occurs down-stream of cAMP synthesis and appears to be due to impaired cholesterol utilization for steroidogenesis, possibly through inhibition of protein synthesis. The stimulation of PGE2 production by CuOOH appears to involve the activation of phospholipase-A2, which is a rate-limiting step in PG synthesis. Lipid peroxides as well as H2O2 may serve as mediators of functional luteolysis.

Androstenedione stimulates progesterone production in corpora lutea of pregnant rats: an effect not mediated by oestrogen

To determine if androstenedione, an aromatizable androgen, has a direct effect on luteal progesterone secretion, collagenase-dispersed luteal cells or whole corpora lutea from pregnant rats were incubated in the presence of the androgen. Luteal cells from 15-day pregnant rats responded to androstenedione in a dose-dependent manner, with an increase in progesterone output at doses of 1 and 10 μM, but with no effect at minor doses of the androgen. Luteal cells obtained from animals on day 4, 9, 15 or 19 of pregnancy and incubated with 10 μM of androstenedione, increased progesterone production by 243, 39, 84 and 146%, respectively. Androgens (androstenedione, testosterone or dihydrotestosterone) but no oestrogens (oestradiol or diethylstilboestrol) at a dose of 10 μM, stimulated progesterone production in incubated luteal cells obtained from 15-day pregnant rats. The time-course pattern of androstenedione-induced progesterone production was studied by superfusion experiments using corpora lutea from rats on day 15 of pregnancy. A significant progesterone output was observed when androstenedione, but not oestradiol, was perfused through the luteal tissue. Intrabursal ovarian administration of androstenedione (10 μM) to 19-day pregnant rats induced a significative increase in serum progesterone levels 8 and 24 h after treatment. These in vivo results confirm the stimulatory effect of androstendione on progesterone production obtained in incubated luteal cells from pregnant rats. This study reports a direct luteotrophic effect of androstenedione in rat corpus luteum, not mediated by previous conversion to oestrogens.