Rat Corpus Luteum Research Articles

Insulin-like growth factor binding protein-3 mRNA expression in endothelial cells of the primate corpus luteum.

Luteinization is associated with endothelial cell proliferation as part of the extensive angiogenesis necessary to maintain corpus luteum function. However, following luteal demise, the vasculature regresses and the endothelial cells disappear. In the rat corpus luteum, the endothelial cells express high concentrations of insulin-like growth factor-binding protein-3 (IGFBP-3) during luteolysis, suggesting a role of IGFBP-3 during endothelial cell loss. The aim of the present study was to determine the occurrence and location of the messenger ribonucleic acid (mRNA) for IGFBP-3 in the primate corpus luteum, and to determine whether or not induction of luteal regression is associated with changes in localization of the message. Marmoset corpora lutea were studied throughout the cycle. The effects of induced luteolysis were examined 12 h or 24 h after treatment with either a gonadotrophin-releasing hormone antagonist or a prostaglandin F2alpha analogue, administered during the mid-luteal phase. High IGFBP-3 expression was recorded in the endothelial cells of the majority of microvessels and a minority of capillaries surrounding the lutein cells in all functionally active corpora lutea. Expression declined markedly in regressing corpora lutea of the late follicular phase. Expression of the IGFBP-3 mRNA in lutein cells in the control corpus luteum was extremely rare. There were no major differences in the degree and pattern of IGFBP-3 expression as a consequence of induced luteal regression although there was an apparent increase in the number of capillary endothelial cells expressing. Induction of luteolysis resulted in expression in a minority of lutein cells. These results support the concept that IGFBP-3 has an autocrine/paracrine role in regulating various cell types in the primate corpus luteum, including endothelial cells. However, expression of IGFBP-3 mRNA throughout the luteal phase suggests it may regulate angiogenesis and luteal function rather than endothelial cell death and luteolysis.

The expression of interleukin-6 in the pregnant rat corpus luteum and its regulation by progesterone and glucocorticoid.

Interleukin (IL)-6, a multifunctional cytokine originally described as a T cell-derived factor, is also produced by different cell types, and it influences a wide variety of physiological and pathophysiological processes. Recent studies further suggest that IL-6 has a role in down-regulating hormone production by endocrine organs and can negatively affect the steroidogenic capacity of both ovaries and testes. Thus, the aims of this investigation were to examine whether IL-6 plays a role in luteolysis and, more specifically, to determine whether luteal cells express the IL-6 gene, whether this expression is developmentally and hormonally regulated in pregnancy, and whether the corpus luteum could be a target for IL-6 action. Using semiquantitative RT-PCR, messenger RNA (mRNA) encoding both components of the IL-6 receptor [the ligand-binding subunit (IL-6 R) and the IL-6 R-associated signal transducer (gp130)] were found to be highly expressed in corpora lutea throughout pregnancy. In contrast, IL-6 mRNA expression was barely detectable from day 4 through the end of pregnancy, whereas a sharp and abrupt expression of IL-6 mRNA occurred immediately after parturition. Although the corpus luteum does not express IL-6 mRNA during most of pregnancy, it could be induced to express this gene with an in vivo injection of the bacterial endotoxin, lipopolysaccharide. In addition, when corpora lutea from day-15 pregnant rats were isolated and maintained in culture, IL-6 mRNA that was undetectable at 0 h increased in a time-related manner and reached significant levels after 4 h of incubation, followed by a similar increase in IL-6 protein secreted in the culture media. Isolation of the small and large luteal cells by elutriation indicated that both cell populations can secrete IL-6 in culture. The apparent ability of luteal cells to spontaneously express IL-6 in vitro, together with the lack of IL-6 expression during most of pregnancy, led us to examine whether the IL-6 gene is silenced throughout pregnancy by luteotropic hormones. Corpora lutea from day-15 pregnant rats were cultured in the presence of different doses of progesterone; the synthetic glucocorticoid, dexamethasone; 17beta-estradiol; and PRL. Progesterone and dexamethasone markedly inhibited IL-6 mRNA expression, whereas 17beta-estradiol had a minimal inhibitory effect, and PRL did not affect IL-6 mRNA expression. In summary, results of this investigation have revealed that the rat corpus luteum expresses the IL-6 receptor system and that luteal cells are able to secrete IL-6. However, IL-6 gene expression is silenced during most of pregnancy, probably by the high levels of progesterone locally produced in the corpus luteum. The salient finding that progesterone and glucocorticoid strongly inhibit the expression of IL-6 in the corpus luteum suggests that one important luteotropic role of progesterone and glucocorticoids could be to prevent the expression of IL-6, which might have a deleterious effect on luteal function.

The Expression of Interleukin-6 in the Pregnant Rat Corpus Luteum and Its Regulation by Progesterone and Glucocorticoid

The Expression of Interleukin-6 in the Pregnant Rat Corpus Luteum and Its Regulation by Progesterone and Glucocorticoid

Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase in the rat corpus luteum: induction of manganese superoxide dismutase messenger ribonucleic acid by inflammatory cytokines.

This study was undertaken to investigate the regulation of mitochondrial manganese superoxide dismutase (Mn-SOD) and cytosolic copper-zinc SOD (Cu,Zn-SOD) in the corpus luteum by inflammatory cytokines. We first examined the developmental expression of both SOD mRNAs in the rat corpus luteum throughout pregnancy. SOD mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction. Whereas Cu,Zn-SOD mRNA levels decreased during late pregnancy, Mn-SOD mRNA levels remained elevated. We secondly examined the effects of inflammatory reaction on luteal SODs. Rats received injections of lipopolysaccharide (LPS; 5 mg, i.p.) on Day 15 of pregnancy, and corpora lutea were removed 2 h later. LPS caused an increase in Mn-SOD mRNA levels in the corpus luteum and a decrease in serum progesterone levels, but neither in levels of Cu,Zn-SOD mRNA. To further study the effects of LPS or LPS-induced cytokines, we incubated either whole corpora lutea obtained on Day 15 of pregnancy or a temperature-sensitive simian virus-40 transformed luteal cell line (GG-CL; derived from large luteal cells of the corpus luteum of pregnant rats) in serum-free medium with LPS, interleukin-1alpha (IL-1alpha), IL-beta, IL-6, and tumor necrosis factor alpha. LPS and these cytokines induced a remarkable increase in Mn-SOD mRNA levels in both corpora lutea and GG-CL cells but had no effect on Cu,Zn-SOD mRNA expression. In conclusion, Cu,Zn-SOD and Mn-SOD mRNAs are differently expressed and regulated in the corpus luteum of pregnancy. Mn-SOD mRNA, but not Cu,Zn-SOD mRNA, is highly induced by inflammatory cytokines and may play an important role in protecting luteal cells from inflammation-mediated oxidative damage.

Differential expression of the estrogen receptors alpha and beta in the rat corpus luteum of pregnancy: regulation by prolactin and placental lactogens.

Estradiol, together with PRL and placental lactogens, regulates steroidogenesis and cell hypertrophy in the rat corpus luteum of pregnancy. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat corpus luteum of pregnancy expresses the estrogen receptor (ER) genes. In this investigation, we have analyzed the expression of the two ER genes (ER alpha and ER beta) (by RT-PCR and in situ hybridization) in the rat corpus luteum, studied their developmental changes throughout pregnancy, and investigated the regulation of ER alpha and ER beta messenger RNA (mRNA) expression by PRL and placental lactogens. The RT-PCR studies showed that both ER mRNA species (ER alpha and ER beta) are coexpressed in the rat corpus luteum during pregnancy. Whereas ER alpha mRNA increased from early pregnancy, reached a maximum at midpregnancy, and had a remarkable decline before parturition; ER beta mRNA remained constant throughout pregnancy, with a significant decline at parturition. Examination of ER alpha and ER beta mRNA expression at the cellular level, by in situ hybridization, showed ER alpha expressed in both follicles and corpus luteum, with maximal expression at midpregnancy. In parallel with the RT-PCR studies, ER beta mRNA was similarly expressed throughout pregnancy in the corpus luteum, but it was less abundant when compared with small and growing follicles. Western blot analysis revealed two ER immunoreactive proteins in the nuclear fraction obtained from pregnant rat corpus luteum: a 67-kDa moiety, highly expressed at midpregnancy but barely detectable in early and late gestation; and a 61-kDa form that remained developmentally unchanged. Hypophysectomy, performed early in pregnancy, induced a sharp decline in ER alpha mRNA expression but a less-marked reduction in ER beta mRNA levels. PRL treatment reverted the inhibition induced by hypophysectomy in both receptor subtypes. When primary luteinized cells were used to test the effect of PRL, rat placental lactogen I, and rat placental lactogen II on the expression of ER alpha and ER beta mRNA, all these lactogenic hormones stimulated both ER mRNA species in a dose-dependent manner. The regulation of ER mRNA expression was further evaluated in a luteal cell line, termed GG-CL, which apparently expresses only the ER beta mRNA species. Culture of the GG-CL cells, in the presence of PRL, resulted in a dose-related up-regulation of ER beta mRNA expression. In addition, PRL treatment enhanced the binding activity of GG-CL cell nuclear proteins to a classical estrogen response element. Furthermore, in these cells, estradiol treatment induced a dose-dependent up-regulation of the mRNA encoding protein kinase C delta isoform, a well-known estrogen target gene in the corpus luteum of the pregnant rat.

Differential Expression of the Estrogen Receptors and in the Rat Corpus Luteum of Pregnancy: Regulation by Prolactin and Placental Lactogens

Differential Expression of the Estrogen Receptors and in the Rat Corpus Luteum of Pregnancy: Regulation by Prolactin and Placental Lactogens

Apoptosis and PCNA expression induced by prolactin in structural involution of the rat corpus luteum.

There are two stages of luteal regression. The first stage is functional regression that is characterized by a decreased production of progesterone secretion; the second stage of structural involution is referred to as a structural luteolysis. In rodents, prolactin has a biphasic action on the corpus luteum. It is luteotrophic, but when exposed to functionally regressed corpora lutea it causes luteolysis. The objective of the present studies was to examine mechanisms of prolactin action in structural luteolysis, whether apoptosis is involved in this process, and to examine the possible association of cell proliferation signals as mediators of structural luteolysis. Prolactin-induced structural luteolysis was associated with apoptosis verified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). Apoptotic cells made up about 3% of the cells 24 hours after the first injection of prolactin, a level that remained constant at all stages of structural luteolysis. Total ovarian weight and DNA content were decreased about 50% in 72 hours after induction of structural luteolysis by prolactin, The finding of about 3% of cells in apoptosis indicates apoptosis is a rapid process. Proliferating cell nuclear antigens (PCNA) of luteal cells were significantly decreased during functional luteal regression, but were conversely increased in structural luteolysis as shown by western blotting and immunohistochemistry. In general PCNA expression is reported to be decreased during structural involution, and there are no reports that have linked excess expression of PCNA with apoptosis and structural luteolysis. We speculate that an excessive increase in expression of PCNA which signals activation of cell proliferation creates a disorder in the signals involved with DNA synthesis. This disorder results in mitotic catastrophe and in the induction of apoptosis. Therefore the disorder of cell cycle signals in luteal cells are associated with prolactin induced apoptosis in structural luteolysis.

The Different Forms of the Prolactin Receptor in the Rat Corpus Luteum: Developmental Expression and Hormonal Regulation in Pregnancy

The Different Forms of the Prolactin Receptor in the Rat Corpus Luteum: Developmental Expression and Hormonal Regulation in Pregnancy

The different forms of the prolactin receptor in the rat corpus luteum: developmental expression and hormonal regulation in pregnancy.

The corpora lutea of pregnancy in the rat are highly dependent on the action of PRL and PRL-like hormones to hypertrophy and to produce progesterone needed for the maintenance of gestation. Two forms of the PRL receptor (PRL-R), designated as long (PRL-RL) and short (PRL-RS), have been described in rat tissues. To determine whether both forms are present in the corpus luteum during pregnancy and to examine the developmental and hormonal regulation of their expression, total RNA isolated from corpora lutea at different stages of pregnancy and from highly luteinized granulosa cells subjected to different hormonal treatments were analyzed by semiquantitative RT-PCR. Immunoblotting of luteal proteins from early and late pregnancy was also performed to determine if the pattern of PRL-R proteins follows that of PRL-R messenger RNA (mRNA) expression. In addition, the correlation between the well characterized PRL-regulated gene, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), and PRL-R gene expression was investigated during the time of luteolysis. Both PRL-RL and PRL-RS mRNA and protein were expressed in corpora lutea of pregnancy, with the long form being the most dominant at all stages. Whereas no changes in mRNA level of either PRL-RL or PRL-RS were found until day 20 of gestation, a profound decline in PRL-R mRNA and protein for both receptor types occurred at the end of pregnancy. This drop in PRL-R expression was accompanied by a sharp and abrupt expression of 20alpha-HSD mRNA. Studies performed in vivo and in luteinized cells in culture indicate that PRL can up-regulate the expression of the PRL-RL mRNA, an effect prevented by the tyrosine kinase inhibitor, genistein. PRL-RL mRNA was also selectively increased by cAMP. In summary, the results of this investigation have established that: 1) the corpus luteum of pregnancy expresses both the short and long forms of the PRL-R with the long form being more abundant; 2) the mRNA for both forms of the PRL-R remains at constant levels throughout pregnancy but drops before parturition; 3) the decline in PRL-R mRNA at the end of pregnancy is accompanied by a dramatic rise in 20alpha-HSD; 4) PRL is able to increase the expression of PRL-R mRNA; and that 5) both A kinase and tyrosine kinase mediated pathways appear to participate in the up-regulatory mechanism involved in PRL-R mRNA expression.

Progesterone inhibits 20alpha-hydroxysteroid dehydrogenase expression in the rat corpus luteum through the glucocorticoid receptor.

In this study, we investigated whether progesterone exerts an intraluteal action despite the lack of progesterone receptors (PR) in the rat corpus luteum and whether progesterone acts through the glucocorticoid receptor (GR) to enhance its own levels by down-regulating the expression of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). We first established that the corpus luteum constitutively expresses the GR throughout pregnancy and after parturition. We also generated a temperature sensitive SV-40 transformed luteal cell line (GG-CL) that expresses the GR and 20alpha-HSD but lacks the PR. Treatment with different doses of either progesterone or dexamethasone caused a dose-related decrease in 20alpha-HSD mRNA in both cultured corpora lutea and in the luteal cell line. RU486, a PR/GR antagonist, completely blocked both the progesterone and the dexamethasone mediated inhibition of 20alpha-HSD expression in GG-CL cells. In summary, this report provides the first evidence that despite the absence of the PR in the rat corpus luteum, progesterone can act through the GR to down-regulate the expression of 20alpha-HSD, an enzyme that catabolizes progesterone and reduces progesterone secretion by the corpus luteum.

Role of cytosolic phospholipase A 2 in eicosanoid generation by corpora lutea of pseudopregnant rats: effects of its specific inhibitor

This study was performed to investigate whether 85 kDa cytosolic phospholipase A 2 (cPLA 2) functions in eicosanoid generation in rat corpora lutea (CL) using its specific inhibitor, arachidonyl trifluoromethyl ketone (ATK). In both immature and adult pseudopregnant rats, PLA 2 activity in the cytosol of CL, measured by the liposome-vesicle assay, increased from day 6 of pseudopregnancy (PSP6) to PSP12. 10 μM ATK potently inhibited all of these activities to 10–20% and the rate of the inhibition by ATK was much higher on PSP12. ATK also reduced arachidonic acid (AA) release from luteal cells of PSP12 prelabelled with 3H-AA. Furthermore, the production of prostaglandin E 2 by cultured luteal cells was mostly suppressed by the drug. These results suggest the augmentation of cPLA 2 activity with the luteal age of pseudopregnant rats and its principal role in eicosanoid generation in CL.

PRAP, a prolactin receptor associated protein: its gene expression and regulation in the corpus luteum.

We have recently identified, characterized, and cloned a luteal microsomal 32-kDa phosphoprotein that we named PRAP (for PRL-receptor associated protein), and we have demonstrated that PRAP binds to the intracellular domain of the short but not the long form of the PRL receptor. In this study, we used PRAP cDNA to examine the tissue specificity, the developmental expression, and the hormonal regulation of PRAP gene expression. Northern blot analysis revealed that in the corpus luteum, PRAP cDNA hybridized to multiple transcripts (5.5 kb, 4.3 kb, and 1.8 kb), with the smallest transcript (1.8 kb) corresponding to the size of the cDNA clone. However, none of these transcripts were detected in any other tissues examined. PRAP appears to be tightly regulated by steroids and PRL. When pregnant rats were treated with aminoglutethimide, a steroid synthesis inhibitor, all three PRAP transcripts became barely detectable. Similar results were obtained when all luteotropic support was removed by hypophysectomy and hysterectomy. Estradiol up-regulated PRAP expression and, more specifically, the two lower transcripts. PRL had no stimulatory effect on PRAP messenger RNA (mRNA) expression but caused a substantial increase in the level of PRAP protein when administered to hypophysectomized pregnant rat, suggesting that PRL may stabilize this protein. Similar dissociation between levels of mRNA and protein were observed during luteal development. Although both PRAP mRNA and protein were barely detectable in early pregnancy, their expression increased abruptly from midpregnancy; however, whereas levels of PRAP mRNA declined from day 18, those of the protein remained elevated until parturition. In summary, results of this study have defined the tissue specificity and developmental expression of PRAP mRNA during pregnancy. The data have also revealed that the gene expression of this protein is up-regulated by estradiol, suggesting a pivotal role for PRAP in the synergistic action of estradiol and PRL on the function of the rat corpus luteum.

The role of p53 tumor suppressor gene and bcl-2 protooncogene in rat corpus luteum death

OBJECTIVE: The purpose of this study was to find out whether the mammalian corpus luteum undergoes genetically programmed cell death as evidenced by the positive or negative expression of specific biochemical markers of apoptosis (p53 and bcl-2). STUDY DESIGN: Twenty-six immature 28-day-old female Sprague-Dawley rats were given 10 IU of pregnant mare's serum gonadotropin to induce ovulation and corpus luteum formation. Corpora lutea were collected on postovulatory days 8, 10, 12, and 14 and snap-frozen immediately. Determinations of apoptotic fragmentation of deoxyribonucleic acid were performed with use of samples radiolabeled at 3′-ends with deoxynucleotide α-phosphorus 32–deoxycytidine triphosphate (3000 Ci/mmol). Transcription of p53 and bcl-2 was determined by Northern blot analysis of total ribonucleic acid. Protein expression of p53 and bcl-2 was determined by Western blot analysis with a monoclonal rat antibody for p53 and a polyclonal rabbit antibody for bcl-2. RESULTS: The nuclear fragmentation assay revealed formation of oligonucleosomes resulting in typical laddering of the deoxyribonucleic acid (corpus luteum) consistent with programmed cell death. Northern blot analysis of total ribonucleic acid prepared from immature (28-day-old) rat corpus luteum revealed the presence of a single p53 messenger ribonucleic acid transcript (2.1 kb) in all ages of corpus luteum studied from day 8 to day 14. Western blot analysis for p53 revealed a gradual reduction in p53 protein in corpus luteum from day 8 to day 12 until it became undetectable on day 14. Western blotting revealed expression of specific protein for bcl-2. CONCLUSIONS: It appears that programmed cell death, as evidenced by formation of oligonucleosomes, occurs during mammalian luteal regression. The patterns of p53 ribonucleic acid expression in the corpus luteum suggest that the protein products of p53 and bcl-2 do not act in a diametric manner to regulate programmed cell death in the corpus luteum. The current results suggest that the mechanisms leading to programmed cell death in the mammalian corpus luteum may differ considerably from those in other organ systems. (Am J Obstet Gynecol 1997;177:327-32.)

Evidence for steroidogenic luteal cell hypertrophy and hyperplasia during pregnancy in the rat.

The proliferative activity of the rat corpus luteum was studied on days 2, 3, 6, 9, 12, 15, 17, 19 and 21 of pregnancy. Proliferating cells were detected by the immunohistochemical demonstration of DNA-incorporated 5-bromodeoxyuridine (BrdU) and by the presence of mitoses. Steroidogenic luteal cells showed two proliferative waves on days 12-15 and on day 21, when relatively abundant BrdU-labeled and mitotic cells were observed. These cells were clearly distinguishable from non-steroidogenic cells by their round nuclei and large polygonal cytoplasm. The proliferative activity on days 12-15 was coincident with an increase in the size of the cells and in progesterone concentrations. On the other hand, the proliferative activity of non-steroidogenic luteal cells (especially endothelial cells of the blood and lymphatic vessels) followed a different pattern. These cells intensely proliferated on days 2-3 of pregnancy and this proliferative activity was significantly higher than that observed in non-pregnant rats on metestrus and diestrus. A new proliferative wave was observed on days 12-15, in association with the increase in the proliferative activity of steroidogenic cells. The presence of both BrdU-labeled and mitotic steroidogenic luteal cells provides evidence that these cells do proliferate and that both hypertrophy and hyperplasia are involved in the increase in the parenchyma of the corpus luteum during pregnancy. Also, the results suggest that different mechanisms are involved in the regulation of the proliferative activity in the corpus luteum at different times during pregnancy.

Rat corpus luteum expresses both PACAP and PACAP type IA receptor mRNAs.

Both pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type I receptor gene expressions were detected in the corpus luteum of pregnant mare's serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG)-treated immature rats using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products of the poly(A)+ RNA extracted from rat corpora lutea yielded dominant DNA bands that corresponded to segments of PACAP mRNA (453 bp) and PACAP type IA receptor mRNA (290 bp) spanned by the PCR primers. The identities of the PACAP cDNA and the PACAP receptor cDNA fragments were confirmed by Southern blot hybridization analyses. Our results showed that PACAP mRNA and PACAP type IA receptor mRNA are synthesized within luteinized cells of rat ovary, and suggest that PACAP is closely linked to the reproductive process.

11beta-Hydroxysteroid dehydrogenase type 2 in the rat corpus luteum: induction of messenger ribonucleic acid expression and bioactivity coincident with luteal regression

11beta-Hydroxysteroid dehydrogenase type 2 in the rat corpus luteum: induction of messenger ribonucleic acid expression and bioactivity coincident with luteal regression

11beta-Hydroxysteroid dehydrogenase type 2 in the rat corpus luteum: induction of messenger ribonucleic acid expression and bioactivity coincident with luteal regression.

The corpus luteum (CL) is the major source of progesterone during rat pregnancy, and its regression precedes and is essential for parturition. Recent studies show that luteal regression in the rat can be blocked by the administration of synthetic glucocorticoids, yet endogenous glucocorticoids are maximal at the time of normal luteal regression in pregnancy. This suggests that endogenous glucocorticoid may be inactivated locally within the CL, presumably via the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD-2), which is known to regulate glucocorticoid access to receptors in other target tissues. This possibility was examined in the present study by measurement of 11beta-HSD-2 messenger RNA (mRNA) expression and bioactivity in rat CL over the second half of pregnancy, thus covering periods of maximal and minimal progesterone secretion. 11beta-HSD-2 bioactivity was measured in luteal homogenates obtained from rats on days 11, 16, 19, and 22 of pregnancy (term = day 23). Bioactivity was measurable in CL at each stage of pregnancy, with an apparent Km for corticosterone of approximately 100 nM. Enzyme activity was lowest on day 11 (maximum velocity, 1.0 +/- 0.6 pmol/min x mg protein), increased more than 5-fold by day 16 (6.2 +/- 0.5), then increased by an additional 4-fold by day 19 (24.3 +/- 4.3), and this high level of activity was maintained to day 22 (26.5 +/- 5.2). In kidney, the apparent Km for corticosterone was lower than that in CL, but remained unchanged throughout pregnancy (overall mean, 28.9 +/- 1.9 nM) as did the maximum velocity (overall mean, 25.4 +/- 1.3 pmol/min x mg protein). Consistent with the pattern of bioactivity in CL, mRNA for 11beta-HSD-2 was not detectable in CL by Northern analysis on either day 11 or day 16, but was clearly evident on days 19 and 22. In situ hybridization also revealed a substantial up-regulation of 11beta-HSD-2 expression specifically within the CL on days 19 and 22, whereas glucocorticoid receptor mRNA expression was consistent across all stages. In contrast, there was no detectable mRNA expression in CL for either 11beta-HSD-1 or the mineralocorticoid receptor at any stage. These data show that a marked induction of 11beta-HSD-2 mRNA expression and bioactivity occurs within the CL late in rat pregnancy and thus suggest that local inactivation of endogenous glucocorticoids facilitates luteal regression.

Neovascularization of the corpus luteum of rats during the estrus cycle.

In order to elucidate the chronological morphological changes of the corpus luteum (CL) of rats, as a physiological angiogenesis model, the CL of rat ovaries was studied light microscopically using periodic acid methenamine silver staining (PAM) and immunostaining for type IV collagen, laminin, thrombomodulin (TM), factor VIII related antigen (factor VIII) and alpha-smooth muscle actin (alpha-SMA). The CL was also studied electron microscopically. Female Wistar-Imamichi rats were used, which have a regular 4-day estrous cycle. The histological changes of the CL were observed in 6-hour intervals from 4 h before the ovulation to 28 h post-ovulation during the estrous cycle. Once the basement membrane (BM) of the follicle disintegrated following ovulation, developing capillaries entered into the CL and formed a vascular lumen with a surrounding BM, which showed positive for PAM staining, type IV collagen and laminin. The developing capillaries in the CL showed a weakly positive reaction for TM and factor VIII, but were negative for alpha-SMA. However, the appearance of immature pericytes around the well-developed capillary was obvious with electron microscopy. The study reported here provides detailed descriptions of angiogenesis during luteinization. It is concluded that the angiogenesis of the CL begins at the time of destruction of the BM of the ovarian follicle, and that the capillary BM appears when the capillary forms its lumen. Moreover, it was demonstrated that the capillary does not develop into an arteriole during luteinization.

Cloning and Characterization of an Ovarian-specific Protein That Associates with the Short Form of the Prolactin Receptor

Prolactin (PRL) is essential for progesterone biosynthesis and luteal cell hypertrophy of the rat corpus luteum during pregnancy. Both the long and short form of the PRL receptor have been identified in the corpus luteum of pregnant rat. The long form has been shown to transduce PRL signal in other cells, whereas no information is available on the role of the short form, especially in the corpus luteum. In the present study, we have cloned a rat ovarian-specific phosphoprotein, PRAP (PRL Receptor Associated Protein), which has no significant homology to other known proteins. We have demonstrated that this protein is immunoprecipitated by anti-PRL receptor and anti-phosphotyrosine antibodies. To determine whether PRAP associates with either the long or the short form of the PRL receptor, fusion proteins with glutathione S-transferase containing the cytoplasmic domain of the long or short form of the PRL receptor were produced, purified, and incubated with luteal proteins. Our results indicate that PRAP preferentially binds to the short form of the PRL receptor. Thus, the long form and short forms of the PRL receptor may signal through distinct pathways. These data provide evidence for the involvement of a novel protein in PRL signal transduction and suggest that PRAP may contribute to the luteotropic effects of PRL on the corpus luteum during pregnancy.

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

In this study we examined the prospect that the superoxide radical (SOR) is involved in the mechanism by which LH stimulates progesterone secretion in the rat corpus luteum (CL). Treatment of dispersed CL cells with low doses of LH or a SOR-generating system (xanthine-xanthine oxidase) resulted in a significant increase in progesterone release and SOR production. High doses of each treatment were inhibitory. SOR generation also decreased hCG binding. To determine whether SOR may be required for progesterone secretion, dispersed cells were electroporated with antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)] and treated with either low (50 ng) stimulatory or high (20 micrograms) inhibitory doses of LH. At 50 ng LH, insertion of SOD or CAT dose-dependently inhibited progesterone secretion. However, at high doses of LH (20 micrograms), which are associated with high levels of SOR, electroporation of SOD or CAT produced the opposite response. This stimulatory response of SOD or CAT on progesterone release was also dose related. These results indicate that SOR may be involved in the mechanisms that stimulate as well as those that inhibit progesterone release. The effect on progesterone secretion appears to be dose related, with small increases associated with stimulation and high levels involved in inhibition of secretion.