Rat Corpus Luteum Research Articles

Effects of arachidonate metabolism inhibitors on basal and human chorionic gonadotropin-stimulated progesterone secretion by rat corpus luteum cells in vitro

Arachidonic acid (AA) and its metabolites mediate many physiological processes including reproduction and endocrinology. The current study investigated effects of several inhibitors of AA cascade on steroidogenesis by rat corpus luteum cells in vitro. Dispersed luteal cells prepared from rat corpus luteum on day 6 of pseudopregnancy secreted progesterone (P4) in time-dependent and human chorinonic gonadotropin (hCG)-dependent fashion. Arachidonyl trifluoromethyl ketone, a preferential inhibitor of the type IVA phospholipase A 2 (PLA 2-IVA), stimulated basal P4 secretion and had no influence on hCG-stimulated steroidogenesis. A novel and more specific inhibitor pyrrophenone inhibited hCG-induced P4 secretion. A cyclooxygenase inhibitor indomethacin did not affect basal secretion but inhibited hCG-stimulated secretion. Nordihydroguaiaretic acid tended to decrease basal P4 secretion and completely inhibited hCG-stimulated secretion. Obtained results suggest that AA metabolic cascade, which is triggered at least in part by PLA 2-IVA activity, is potentially implicated in hCG-stimulated P4 secretory response in the rat corpus luteum.

Platelet-Derived Growth Factors and Receptors in the Rat Corpus Luteum: Localization and Identification of an Effect on Luteogenesis1

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) play a vital role in regulating cell growth and angiogenesis. In this study, the expression of the family of PDGFs and PDGFRs in the ovarian corpus luteum were identified and characterized, and an effect of their activity on development of the corpus luteum revealed. Gonadotropin-stimulated immature rats were utilized as a model of induced ovulation, luteogenesis, and pseudopregnancy. Levels of ovarian mRNA for Pdgfb and Pdgfd, and their receptor, Pdgfrb, increased significantly as early as 4 h after human chorionic gonadotropin (hCG) injection in immature rats primed with equine chorionic gonadotropin (eCG). Gonadotropin regulation of Pdgfb expression was confirmed by in vitro promoter-reporter assays, which showed a 2- to 3-fold increase in Pdgfb promoter activity in response to luteinizing hormone (LH). Inhibition studies implicated protein kinase A, phosphatidylinositol 3-kinase and mitogen activated protein kinase signaling pathways in the LH-induced upregulation. In the corpus luteum, PDGFA, PDGFB, PDGFC, and PDGFRA were localized to a population of luteal parenchymal/steroidogenic cells. PDGFRB was expressed primarily in what appeared to be cells of the luteal microvasculature. Intraovarian injection of an inhibitor of PDGF receptor activity, the tyrphostin AG1295, prior to injection of hCG in eCG-primed immature rats resulted in a significant 21.86%+/-11.15% decrease in corpora lutea per treated ovary in comparison to the contralateral vehicle-injected control ovary. In addition, the treated ovary of 3 of 16 rats showed widespread hemorrhage throughout the entire ovary, indicating a possible role for PDGF receptor activity in maintenance of the ovarian vasculature.

The Adenosine 5′-Triphosphate-Sensitive Potassium Channel in Endocrine Cells of the Human Ovary: Role in Membrane Potential Generation and Steroidogenesis

ATP-sensitive potassium (K(ATP)) channels couple the metabolic state with the membrane potential in several cell types, and recently evidence for K(ATP) channels was given in rat corpus luteum, a fast-growing and metabolically highly active tissue. We studied whether K(ATP) channels are present in the human ovary and luteinized granulosa cells (GCs). Human GCs were examined regarding functionality and physiological role of the channel. Human GCs were obtained from in vitro fertilization patients. K(ATP) channels are involved in membrane potential generation in human GCs because application of the K(ATP) blocker glibenclamide resulted in depolarization as monitored by fluorescence microscopy. Furthermore, glibenclamide significantly attenuated human chorionic gonadotropin-induced progesterone production. The channel pore is composed of K(ir)6.1, but not K(ir)6.2, as indicated by RT-PCR. K(ir)6.1 subunit protein was detected in human follicular and luteal cells by immunohistochemistry and localized to the plasma membrane of human GCs by immunogold staining. RT-PCR experiments revealed the sulfonylurea receptor subunit SUR2B as part of the K(ATP) channel. In addition, mRNAs encoding SUR1 and SUR2A were detected in some preparations. There is no evidence for mitochondrial K(ATP) channels in human GCs because we detected neither K(ir)6.1 protein in mitochondrial membranes nor alterations of mitochondrial membrane potential by glibenclamide or the K(ATP) opener diazoxide. Endocrine cells of the human ovary possess functional K(ATP) channels, which are linked to both plasma membrane potential generation and progesterone production. Our results may provide new insights into human ovarian physiology and raise the possibility of pharmacological targeting.

Cloning and characterization of a novel zinc finger protein (rZFP96) in the rat corpus luteum

The corpus luteum (CL) is a temporary organ involved in the maintenance of pregnancy. In the course of its life-cycle, the CL undergoes two distinct and consecutive processes for its inevitable removal through apoptosis: functional and structural luteolysis. We isolated a gene encoding for a novel rat zinc finger protein (ZFP), named rat ZFP96 (rZFP96) from an ovarian lambda cDNA library. Sequence analysis revealed close sequence and structural similarity to mouse ZFP96 and human zinc finger protein 305 (ZNF305). Quantitative reverse transcription-polymerase chain reaction analysis revealed a positive correlation with the end of pregnancy, that is, the onset of structural luteolysis of the CL. Messenger RNA levels increased 3-fold ( P < 0.01) between days 13 and 22 of pregnancy and 8-fold ( P < 0.01) between day 13 of pregnancy and day 1 post-partum. In addition, we detected rZFP96 expression in mammary, placenta, heart, kidney and skeletal muscle. Sequence analysis predicted that rZFP96 has a high probability of localizing to the nuclear compartment. The presence of both a perfect consensus TGEKP linker sequence between zinc fingers 2 and 3 as well as several similar sequences between the other zinc fingers suggests physical interaction with DNA. Speculatively, rZFP96 may therefore function as a transcription factor, switching-off pro-survival genes and/or upregulating pro-apoptotic genes and thereby contributing to the demise of the CL.

Expression and Regulation of Progestin Membrane Receptors in the Rat Corpus Luteum

Despite evidence strongly supporting progesterone's autocrine actions in the rat corpus luteum (CL), classical progesterone receptors (PR) have not been detected in this gland. Alternatively, in several other systems, progestins have been reported to activate nongenomic pathways via putative progestin membrane receptors (PMRs). The aim of this investigation was to determine whether rat CL membranes bind progestins and contain PMR homologs and whether these proteins are expressed during CL development in a manner that parallels luteal function. We found that luteal cell membranes specifically bind progesterone. Low levels of progesterone and 20alpha-dihydroprogesterone decreased binding of [(3)H]progesterone, whereas androstenedione, 17alpha-hydroxyprogesterone, and pregnenolone were less potent. Other steroids, including corticosterone, mifepristone, and estradiol, were ineffective. We found that the rat CL expresses five genes previously postulated to encode for putative PMRs: PMRalpha, PMRbeta, PMRgamma, PR membrane component 1 (PRMC1), and Rda288. Pmralpha, Pmrgamma, and Prmc1 transcripts rose steadily during pregnancy whereas Pmrbeta and Rda288 remained constant. Just before parturition, concomitant with falling progesterone levels, Pmralpha, Pmrbeta, and Prmc1 decreased. Luteal PMRalpha and PRMC1 protein levels were lower in samples taken at the end of pregnancy compared with midpregnancy samples. Ergocriptine, which inhibits the secretion of prolactin, the primary luteotrophic hormone in the rat CL, reduced Pmralpha, Pmrbeta, and Prmc1 expression significantly. Ergocriptine effects were prevented by coadministration of prolactin. These findings provide evidence for the expression and regulation of putative membrane-bound progestin-binding proteins in the rat CL, a tissue that does not express detectable levels of nuclear progesterone receptors.

Identical changes in Bax expression, but not Fas ligand expression, occur in structural luteolysis in gonadotropin releasing hormone agonist- and prolactin-treated superovulated rats

Structural luteolysis induced by gonadotropin releasing hormone agonist (GnRHa) or prolactin (PRL) is defined as histological involution of the corpus luteum. We reported that one of the mechanisms of structural luteolysis induced by PRL was tissue remodeling by matrix metalloproteinase (MMP) and also apoptosis in superovulated rats. We also reported that GnRHa induced structural luteolysis with elevation of MMP. In this study, we investigated whether GnRHa caused apoptosis in mature corpus luteum of superovulated rats and also examined the expression of apoptosis - related molecules (Fas, Fas ligand (FasL), Bcl - 2, Bax). We gave 4 - day GnRHa treatment 5 days after hCG injection to immature female rats treated with pregnant mare surum gonadotrophin (PMSG) and hCG to induce structural involution of mature corpus luteum. PMSG - hCG - treated rats without GnRHa treatment, rats treated with bromocryptine (Brom) to induce functional luteolysis and rats treated with Brom followed by PRL (Brom + PRL) to mimic the PRL surge to induce structural luteolysis as we previously reported were used for comparison. GnRHa treatment caused structural luteolysis characterized by structural involution, a decrease in the serum progestin level, and apoptotic bodies as well as structural luteolysis induced by Brom + PRL. FasL expression in corpora lutea was elevated after Brom treatment, but there was no elevation of FasL after GnRHa treatment started. FasL expression decreased and Bax expression increased in structural luteolysis induced by GnRHa as well as Brom + PRL treatment, although Fas and Bcl-2 expression did not change throughout the luteal phase. In summary, both GnRHa and Brom+PRL caused structural luteolysis, one of whose mechanisms was apoptosis with an increase in Bax expression, but not with an identical change in FasL expression. It is speculated that the significance in alteration of FasL may involve some mechanism other than apoptosis.

A Quantitative Study of Blood Capillary Formation (Angiogenesis) Concomitant with Parenchymal Tissue Differentiation

Examination of the rat corpus luteum (CL) provided quantitative data supporting adaptation of the developing vasculature to maximise efficient acceptance of steroids secreted from the luteal cells. Numbers of endothelial cells (ECs) significantly increased during the initial formation of the CL, followed by a further significant proliferation from day 10 to day 16 when there was maximal growth of the CL. As a consequence, there was significant growth of the vascular compartment during this time interval. The final phase of expanding endothelium (days 10 to 16) was a result of increased ECs volume with elongation of the EC in the direction of growth. Continued increase in capillary surface area and a corresponding marked reduction in diffusion distance between LC and ECs evidenced adaptation of the developing microvasculature to enable efficient endocrine function by day 16, when steroid secretion is maximal. Furthermore, from day 1 to day 3 there was close apposition of pericytes to the endothelium, suggesting the important role of pericytes in the initiation of angiogenesis. However, this degree of association was reduced from day 10 to day 16 and was a consequence of expansion of the EC cytoplasm to provide a greater surface area for transport of steroids.

Vascular endothelial growth factor increases messenger RNAs encoding cyclooxygenase-II and membrane-associated prostaglandin E synthase in rat luteal cells

Vascular endothelial growth factor (VEGF) is known to be necessary for the vascularization of the developing corpus luteum. Our recent data suggested that cyclooxygenase-II (COX-II) may play a role in the formation of vascular plexuses in developing corpora lutea of the rat. Here we examined the relationship between VEGF and the expression of prostaglandin (PG)- metabolizing enzymes in rat ovarian luteal cells. VEGF treatment caused a dose-dependent increase in the expression of COX-II and membrane-associated PGE synthase (mPGES) mRNA in cultured rat luteal cells. However, pretreatment of the luteal cells with a selective COX-II inhibitor, NS-398, abolished the VEGF-enhanced mPGES mRNA expression. VEGF also increased PGE2 secretion. Conversely, PGE2 dose-dependently stimulated VEGF mRNA expression. Furthermore, VEGF induced VEGF mRNA expression, but this effect was abolished by NS-398 pretreatment. These findings suggest that VEGF enhances PGE2 production by stimulating COX-II and mPGES expression in rat corpus luteum and that the effect of VEGF on luteal cells may be partially mediated by this stimulation of PGE2 production.

Effect of the pretreatment with prolactin on the distribution of immunoreactive beta-endorphin through different ovarian compartments in immature, superovulated rats

Beta-endorphin and prolactin (PRL) are natural inhibitors of ovulation via central and peripheral mechanisms, but their possible interactions within the ovary are still unknown. The aims of the present study were to determine the gene expression and the topographic distribution of beta-endorphin, and the possible changes evoked by the pretreatment with PRL on the ovarian beta-endorphin localization in immature, superovulated rats. Prepuberal female Wistar rats weighing 60-70 g were superovulated with 20 IU equine gonadotrophins and, 48 h later, 20 IU human chorionic gonalotropin (hCG). Four hours after the hCG injection, the rats received either 200 microg rat PRL .i.p. (n = 12) or saline vehicle (n = 10). In the following morning the rats were killed and their ovaries were quickly removed. Beta-endorphin localization was assessed by immunohistochemistry and proopiomelanocortin (POMC) mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Beta-endorphin was expressed mostly in the corpora lutea and perivascular stroma, but a weak to moderate immunostaining was also present in the theca cells and some granulosa cells of tertiary/antral and preovulatory follicles. The main differences observed in the distribution of ovarian beta-endorphin between the two groups were a more intense immunostaining in the granulosa cells of antral follicles, corpus luteum and stroma of PRL-treated rats. POMC gene transcripts were detected in 2/5 samples from the control group and in 3/7 samples from the PRL-treated group. Thus, the expression of beta-endorphin in tertiary/antral follicles is enhanced by PRL treatment in immature, superovulated rats, providing a putative mechanism by which PRL could inhibit the ovarian response to induced ovulation.

Electron microscope immunocytochemical localization of cyclooxygenase-1 and -2 in pseudopregnant rat corpus luteum during luteolysis

Prostaglandins converted from arachidonic acid by cyclooxygenases play an important regulatory role in regression of the corpus luteum. To reveal luteal distribution of cyclooxygenase isoforms during luteolysis, an electron microscope immunocytochemical study was performed. Cyclooxygenase-1 and -2 were found both in luteal steroid-producing and interstitial cells on days 13, 15 and 18 of the adult pseudopregnant rat. Cyclooxygenase-2 immunolabelling was predominantly seen in non-luteal cells. The two enzymes were localized in similar fashion to the plasma membrane, rough and smooth endoplasmic reticulum, lipid bodies and mitochondria, but differently in the nuclear compartment. Cyclooxygenase-1 labelling was found only in the perinuclear region, while cyclooxygenase-2 was localized to the nuclear envelope, region of condensed heterochromatin as well as at the perimeter of the heterochromatin. Nuclear residence may indicate additional roles for cyclooxygenase-2 in regulating gene expression. Identification of both enzymes on lipid bodies suggests that these inclusions may be involved in luteal prostanoid production.

Characterization and functional analysis of the 5'-flanking region of the mouse 20alpha-hydroxysteroid dehydrogenase gene.

20alpha-Hydroxysteroid dehydrogenase (20alpha-HSD), which metabolizes progesterone to an inactive steroid in the corpus luteum of mice and rats but not of humans, is thought to play a crucial role in shortening the oestrous cycles in these rodent species. We determined the nucleotide sequence of the 5'-flanking region of the mouse 20alpha-HSD gene, and examined its promoter activity using a rat luteinized granulosa cell culture. A reporter assay, using reporter constructs of various lengths of the 5'-flanking region, revealed that the region between -83 and 60 bp upstream of the transcription start site was essential for transcriptional activity. Furthermore, mutational analysis demonstrated that a putative Sp1 site in this region was critical to the expression of the reporter gene. Electrophoretic mobility-shift assays showed that the interaction of proteins in a nuclear extract from rat luteinized granulosa cells with this region was inhibited by a competitor having the wild-type Sp1 sequence in its promoter, but not a mutated Sp1 sequence. Supershift analysis confirmed that Sp1 and Sp3 were present in the nuclear extract of these cells, and that these factors bound to the element. Finally, promoter activity was elevated by the co-transfection of an Sp1 expression vector, and, to a lesser extent, by an Sp3 expression vector, supporting further the involvement of these factors in the expression of the 20alpha-HSD gene.

Expressions of VEGF and its receptors in rat corpus luteum during interferon alpha administration in early and pseudopregnancy.

It is accepted that angiogenesis plays an important role in the development of the corpus luteum (CL) and is probably necessary for normal lutein cell function. A number of drugs currently being tested in clinical trials as possible angiogenesis inhibitors were not originally developed with the intention of suppressing tumor angiogenesis. Interferon alpha (IFN-alpha) is one of the notable examples of such 'accidental angiogenesis inhibitors' and daily administration of IFN-alpha is known to suppress tumor growth, tumor vascularization, and down-regulation of various growth factors. We investigated the effects of IFN-alpha treatment on the expression of vascular endothelial growth factor (VEGF), and its receptors KDR and Flt-1, and CD34 in CL during the first week of pseudopregnancy and pregnancy in hormonally induced rat ovaries by immunohistochemistry and Western blot techniques. Basal body temperatures of the drug-treated rats, as an indicator of treatment effect, were determined daily and were increased significantly when compared to controls (38.03 +/- 0.18 vs. 36.6 +/- 0.1 degrees C), respectively. The effect of IFN-alpha treatment was minimal when the entire week was evaluated, however, the expression of VEGF decreased at 3rd, 5th, and 7th days of both pregnancy and pseudopregnancy, when compared to the 1st day, whereas there was not a such alteration in the untreated rats regarding these days. The daily subcutaneous administrations of 672.500 U IFN-alpha2b had minimal effects on the expressions of VEGF, and its two receptors KDR and Flt-1 in either pregnant or pseudopregnant corpora lutea utilizing HSCORE.

Plasminogen activator/plasminogen activator inhibitors in ovarian physiology.

The target extracellular matrix (ECM) degradation generated by plasminogen activator (PA) and regulated by plasminogen activator inhibitor (PAI) is an event that affects a wide variety of physiological and pathological processes in the ovary. Studies carried out over the past 25 years in a number of laboratories have elucidated some of the biochemical events related to the function and regulation of the PA system in the ovary. Hormone-induced coordinated expression of tissue-type PA (tPA) produced mainly by granulosa cells and its inhibitor PAI-1 secreted by theca cells in the preovulatory follicles is responsible for a controlled and directed proteolysis leading to the rupture of selected follicles in the rat, monkey and other mammals. Increase in tPA and PAI-1 expression in corpus luteum (CL) of rat and monkey at a later stage is well correlated with a sharp decrease in CL progesterone production, indicating its important role in the initiation of luteal regression. In contrast, the urokinase-type PA (uPA) may play an essential role in the early growing follicles during cell proliferation and migration, and in the early CL formation related to ECM degradation and angiogenesis. Ovarian function is also modulated by endogenously-produced local factors that regulate expression of the PA activator and inhibitor, and the MMP system. Thus, the next challenge is to identify the interrelationship between multiple paracrine and autocrine factors and the PA system, and to know how they regulate the protease and the protease inhibitor in the ovary.

Luteal 3beta-hydroxysteroid dehydrogenase and 20alpha-hydroxysteroid dehydrogenase activities in the rat corpus luteum of pseudopregnancy: Effect of the deciduoma reaction

BackgroundIn the rat, the maintenance of gestation is dependent on progesterone production from the corpora lutea (CL), which are under the control of pituitary, decidual and placental hormones. The luteal metabolism of progesterone during gestation has been amply studied. However, the regulation of progesterone synthesis and degradation during pseudopregnancy (PSP), in which the CL are mainly under the control of pituitary prolactin (PRL), is not well known. The objectives of this investigation were: i) to study the luteal metabolism of progesterone during PSP by measuring the activities of the enzymes 3beta-hydroxysteroid dehydrogenase (3betaHSD), involved in progesterone biosynthesis, and that of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD), involved in progesterone catabolism; and ii) to determine the role of decidualization on progesterone metabolism in PSP.MethodsPSP was induced mechanically at 10:00 h on the estrus of 4-day cycling Wistar rats, and the stimulus for decidualization was provided by scratching the uterus on day 4 of PSP. 3betaHSD and 20alphaHSD activities were measured in the CL isolated from ovaries of PSP rats using a spectrophotometric method. Serum concentrations of progesterone, PRL, androstenedione, and estradiol were measured by radioimmunoassay (RIA).ResultsThe PSP stage induced mechanically in cycling rats lasted 11.3 ± 0.09 days (n = 14). Serum progesterone concentration was high until day 10 of PSP, and declined thereafter. Serum PRL concentration was high on the first days of PSP but decreased significantly from days 6 to 9, having minimal values on days 10 and 11. Luteal 3betaHSD activities were elevated until day 6 of PSP, after which they progressively declined, reaching minimal values at the end of PSP. Luteal 20alphaHSD activities were very low until day 9, but abruptly increased at the end of PSP. When the deciduoma was induced by scratching the uterus of pseudopregnant animals on day 4 (PSP+D), PSP was extended to 18 ± 2.2 days (n = 8). In PSP + D rats, serum progesterone and PRL levels, and luteal 3betaHSD activities were higher than in pseudopregnant rats on day 11. Decidualization also prevented the increase in luteal 20alphaHSD activities observed on day 11 of PSP. Administration of the dopaminergic agonist CB154 in PSP + D rats on day 10 of PSP induced a decline in both serum PRL and progesterone on day 11 of PSP, values that were not different from that of pseudopregnant controls.ConclusionsWe have established that during the final period of PSP a decline in progesterone biosynthesis occurs before the increase in progesterone catabolism. We have also shown that decidualization in pseudopregnant rats extends the life of the CL by prolonging the production of pituitary PRL, and by maintaining high 3betaHSD and low 20alphaHSD activities within the CL leading to sustained production of progesterone.

Endothelin-I interaction with the ET-A receptor in the regression of the rat corpus luteum

Objective: The process of luteolysis is rather complex and only partially understood. Endothelin I (ET-1), a peptide released by the vascular endothelium and identified as a potent vasoconstrictor appears to play an important role. The purpose of our study is to evaluate the effects of ET-1 on progesterone (P) and prostaglandin F2 a (PGF2a) production by the ovary and the regulation by its principal receptor (ET-A) in rodents.

Concomitant induction of apoptosis and expression of monocyte chemoattractant protein-1 in cultured rat luteal cells by nuclear factor-kappaB and oxidative stress.

It has previously been shown that expression of monocyte chemoattractant protein (mcp)-1 and apoptosis of luteal cells occur concomitantly during the estrous cycle in the rat corpus luteum; however, luteal cells containing mcp-1 mRNA did not seem to be apoptotic. In the present study, the relationship between the induction of apoptosis and mcp-1 expression in cultures of dispersed rat luteal cells was examined. Both apoptosis and mcp-1 expression were spontaneously induced in cultured luteal cells in a manner inhibitable by antioxidative reagents or an inhibitor of nuclear translocation of nuclear factor-kB. However, the cells containing mcp-1 mRNA were distinct from those undergoing apoptosis, and the inhibition of apoptosis by the pan-caspase inhibitor z-VAD-fmk did not influence the induction of mcp-1 expression. These results collectively indicate that oxidative stress simultaneously, but independently, induces apoptosis and mcp-1 expression in luteal cells through the activation of nuclear factor-kB. This phenomenon might help to explain how monocytes/macrophages accumulate in regressive corpora lutea where their target apoptotic cells exist.

Prolactin Inhibits Annexin 5 Expression and Apoptosis in the Corpus Luteum of Pseudopregnant Rats: Involvement of Local Gonadotropin-Releasing Hormone

We investigated a specific relationship between the expression of annexin 5 and prolactin in the corpus luteum of pseudopregnant rats, with particular interest in GnRH and apoptosis of luteal cells. The expression of ovarian annexin 5 mRNA was significantly decreased at mid-pseudopregnancy and recovered at the end, whereas it remained low on the corresponding day of pregnancy. The dopamine agonist CB-154, administered at mid-pseudopregnancy (d 5), increased ovarian annexin 5 mRNA, whereas prolactin, given daily for 3 d to cycling rats, decreased it. An immunocytochemical study also showed that annexin 5 increased in the corpus luteum on d 6 and 7 of pseudopregnancy after treatment with CB-154 on d 5. The distribution of annexin 5-positive cells was not uniform in the corpus luteum and matched that of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells. Because GnRH stimulates annexin 5 mRNA expression in the gonadotropes, involvement of the GnRH receptor was examined. Local administration of a GnRH antagonist, Cetrorelix, to hemilateral ovarian bursa of pseudopregnant rats simultaneously receiving CB-154 abrogated both the expression of annexin 5 and the TUNEL reaction. The present results clearly demonstrate that prolactin decreases annexin 5 mRNA in the luteal cells during pseudopregnancy. Prolactin is suggested to suppress the local action of GnRH, which stimulates annexin 5 synthesis and apoptosis of functional luteal cells during pseudopregnancy.

Expression of apoptosis-related genes Fas/FasL, Bax/Bcl-2 and Caspase-3 in rat corpus luteum during luteal regression

Using immunohistochemistry, in situ hybridization, Western blot and TUNEL methods, we have studied the expression of Fas/FasL, Bcl-2/Bax and caspase-3 in the corpora lutea (CL) at various stages of pseudopregnant rat induced by injection of PMSF/hCG. The results showed that no apoptotic signal could be observed until Day 14 after hCG injection. Fas weakly expressed in the CL at all the stages increased when luteolysis took place. FasL signal increased dramatically on Day 14 and reached the maximum level on Day 21. The expression of Bcl-2 and Bax was detected in a time-dependent manner. At the early stage of CL development, Bcl-2 expression was stronger, while Bax was low. The expression of Bcl-2 and Bax in the CL was completely reversed. Caspase-3 antigen could be detected throughout all the phases of CL development in a time-dependent fashion, low on Day 2 and reaching the maximum on Day 21. These results suggest that luteal regression at the late phases may be related to cell apoptosis.

In vivo hormonal environment leads to differential susceptibility of the corpus luteum to apoptosis in vitro.

We evaluated the involvement of the in vivo hormonal environment on the ability of the rat corpus luteum (CL) to undergo apoptosis. Gel electrophoretic DNA fragmentation analysis revealed no apoptosis in CL isolated either the 2 last days of pregnancy (Days 21 and 22) or throughout the 4 days following parturition, suggesting that the number of cells undergoing apoptosis at the same time is not sufficient to allow for visualization of DNA breakdown. In contrast, CL incubated in serum-free medium underwent significant apoptosis, as evaluated by chromatin condensation and DNA fragmentation, regardless of their developmental stage in pregnancy. However, CL obtained on Day 7 of pregnancy and on Day 4 postpartum demonstrated higher sensitivity to apoptosis in vitro, but lactation reduced significantly the capacity of the CL to undergo apoptosis when maintained in culture. These data suggest that the exposure of the CL to different hormonal environments throughout pregnancy and after parturition is responsible for the differential susceptibility to apoptosis observed in vitro. We have previously shown that progesterone is a direct factor for survival of the CL. Prolactin stimulates luteal progesterone production; therefore, we examined whether prolactin prevents apoptosis in luteal cells independently of its stimulatory action on progesterone production. We used a luteal cell line (GG-CL) that expresses the prolactin receptor but does not produce progesterone. These cells undergo apoptosis under conditions of serum starvation, and addition of prolactin to the culture medium significantly reduced DNA fragmentation. These results indicate that the extent of luteal cell death induced by incubation of CL under serum-free conditions depends on the hormonal environment to which this endocrine gland is exposed in vivo. These results also indicate an important role for lactation in preventing apoptosis, which is further supported by the antiapoptotic activity of prolactin observed in luteal cells.

Gonadotropin-releasing hormone-agonist inhibits synthesis of nitric oxide and steroidogenesis by luteal cells in the pregnant rat.

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo suppressed progesterone production and induced apoptosis in the corpus luteum (CL) of the pregnant rat. To investigate the mechanism(s) by which progesterone secretion is suppressed and apoptosis is induced in the luteal cells, we studied nitric oxide (NO) as a messenger molecule for GnRH action. Rats were treated individually on Day 8 of pregnancy with 5 microg/day of GnRH-Ag for 4, 8, and 24 h. GnRH-Ag decreased the production of progesterone and pregnenolone 8 and 24 h after the administration. Corresponding with the reduction in these steroid hormones, luteal NO concentrations decreased at 8 and 24 h. Western blotting and immunohistochemical studies of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) in the CL demonstrated that administration of GnRH-Ag was associated with a marked decrease in eNOS and iNOS compared with sham controls at 4 and 8 h, but nNOS did not change throughout the experimental period. We demonstrated, for the first time, the presence of nNOS protein in the CL of the pregnant rat. To determine if this suppressive action of GnRH-Ag is directly on the CL, luteal cells were treated with GnRH-Ag for 4, 8, 12, and 24 h in vitro. Progesterone and NO concentrations in the media decreased at 8 and 12 h after the treatment and recovered at 24 h. Western blots revealed that eNOS and iNOS decreased in luteal cells treated with GnRH-Ag compared with controls at 4 and 8 h. These results demonstrate that suppression of luteal NO synthesis by GnRH-Ag is direct and leads to a decrease in the luteal production and release of progesterone and pregnenolone and thus suggest that GnRH could induce luteolysis in pregnant rats via NO.