Expression of two progesterone receptor isoforms in cumulus cells and their roles during meiotic resumption of porcine oocytes

The present study aimed to investigate progesterone receptor (PR) gene expression in cumulus cells and their roles during meiotic resumption of porcine oocytes. The amount of PR-A or PR-B mRNA was analyzed by RT-PCR using primer sets for the PR-B region or the PR-A/B common region. The level of PR-B mRNA in cumulus cells was up-regulated by FSH and LH during the first 8 h of cultivation but the level significantly decreased at 12 h. However, a high level of total PR mRNA was maintained up to a cultivation period of 20 h. The level of PR-B protein in cumulus cells reached its maximum at 4 to 12 h, whereas PR-A predominated in cumulus cells of cumulus-oocyte complexes (COCs) at 20 h. Accompanying the shift in expression of PR isoforms, progesterone production in cumulus cells was significantly increased, and both the proliferative activity of cumulus cells during a 10- to 20-h cultivation period and the level of connexin-43, a major component of the gap junction, in cumulus cells significantly decreased. When COCs were cultured with FSH and LH for 10 h and then further cultured with additional RU486, there was a significant suppression in the shift in PR isoforms and in progesterone production, a loss of proliferative activity, and a decrease in connexin-43 mRNA in cumulus cells. Moreover, treatment with RU486 after 10-h cultivation of COCs inhibited the meiotic resumption of oocytes and cumulus cell expansion. These results suggest that the induction of PR isoforms in cumulus cells and their binding to progesterone appear to impact on proliferation and differentiation in a time-dependent manner, and the shift from PR-B to PR-A may help mediate certain events.     

Ovulation: new factors that prepare the oocyte for fertilization

Ovulation is a complex LH-induced process that allows the release of a fertilizable oocyte. Critical to ovulation is the proper formation of an extracellular hyaluronan (HA) rich matrix by the cumulus oocyte complex (COC), a process called expansion. During expansion genes associated matrix formation such as hyaluronan synthase 2 (HAS-2) are induced rapidly in COCs. To stabilize the long hyaluronan polymers, various HA binding proteins are covalently (or non-covalently) linked with hyaluronan. Some of the hyaluronan binding factors that have been identified in the COC matrix are the serum derived factor inter-alpha trypsin inhibitor (IalphaI) and tumor necrosis factor stimulated gene-6 (TSG-6). The latter is dependent on the induction in cumulus cells of cyclooxygenase-2 (COX-2) the limiting enzyme in the synthesis of prostaglandins (primarily PGE) that bind the PG receptor subtype EP2, leading to increased cAMP. TSG-6 and the heavy chains of I(alpha)I interact with each other and HA in a manner that is critical for the formation and/or stabilization of the expanded matrix. Another hyaluronan binding component of the expanded COC is the proteoglycan versican. Versican is induced by the LH surge and is a preferred substrate of the protease, a disintegrin and metalloproteinase with thrombospondin like repeats (ADAMTS-1), which co-localizes with versican and is coordinately induced in granulosa cells and COCs of ovulating follicles by LH and the progesterone receptor (PR). Mice null for COX-2 and EP2 fail to ovulate and exhibit impaired COC expression of TSG-6. Progesterone receptor knockout (PRKO) mice are also anovulatory and present impaired expression of ADAMTS-1. Thus, HA binding proteins and associated factors appear to be essential components of the matrix that is obligatory for release of the COCs through the ovulation pore.     

Luteinizing hormone induces progesterone receptor gene expression in cultured porcine granulosa cells.

We have examined the effect of LH on the regulation of the progesterone receptor (PR) in cultured porcine granulosa cells. In this study we used the RNase protection assay to evaluate the PR mRNA levels with a porcine cDNA clone isolated by the polymerase chain reaction (PCR) method. This clone was regarded as part of the porcine PR cDNA because of its 98.3% and 95.7% homology to the hormone-binding domain of human PR cDNA in amino acid and nucleotide sequences, respectively. Treatment with LH (500 ng/ml) increased porcine PR mRNA to a maximum level of 8.6 +/- 1.1-fold (mean +/- SE) after 3-h exposure. This induction was mimicked by (Bu)2cAMP as well as by FSH and hCG, and the increased PR caused by LH and (Bu)2cAMP occurred in a dose-dependent manner. Basal and LH-induced PR mRNA levels were not affected by progesterone (100 ng/ml), estrogen (100 ng/ml), and RU 486 (10 ng/ml) at 3 h. The mechanism of the increased PR mRNA levels was studied in the presence of actinomycin-D and cycloheximide. While inhibition of RNA synthesis with actinomycin-D blocked LH-induced PR mRNA expression, inhibition of protein synthesis with cycloheximide increased basal and LH-induced PR mRNA levels. These results indicate that the expression of PR mRNA is positively regulated by LH, and this induction does not require ongoing protein synthesis. There may be a cycloheximide-sensitive mechanism that modulates PR mRNA stability. From our results we suspect that progesterone modulates ovarian function through LH-induced PR in granulosa cells.     

Hormone-induced expression of tumor necrosis factor alpha-converting enzyme/A disintegrin and metalloprotease-17 impacts porcine cumulus cell oocyte complex expansion and meiotic maturation via ligand activation of the epidermal growth factor receptor

The epidermal growth factor (EGF)-like growth factors, amphiregulin (AREG) and epiregulin (EREG), are expressed in murine cumulus oocyte complexes (COCs) where they impact the function of cumulus cells and oocyte maturation during LH-mediated ovulation. Because TNFalpha-converting enzyme (TACE)/a disintegrin and metalloprotease-17 (ADAM17) is essential for ectodomain shedding of AREG and EREG from the surface of other cell types, the expression and function of TACE/ADAM17 was analyzed in a porcine COC culture system in which FSH- and LH-mediated expansion and oocyte meiotic maturation have been well characterized and shown to occur between 20 and 40 h. In this model, Areg, Ereg, and Tace/Adam17 mRNAs increased significantly with maximal levels observed between 5 and 20 h of culture with FSH plus LH. TACE/ADAM17 protein and protease activity were up-regulated markedly at 10 h and maintained to 40 h. Treatment of COCs with the TACE/ADAM17-selective inhibitor TNFalpha-processing inhibitor-2 (TAPI-2) significantly suppressed in a time-dependent manner downstream targets of EGF receptor activation such as ERK1/2 phosphorylation, Ptgs2, Has2, and Tnfaip6 mRNA expression, hormone-induced COC expansion, and meiotic maturation of the oocytes. Addition of EGF to COCs cultured in the presence of FSH/LH reversed the inhibitory effects of TAPI-2 on these ovulation-related processes. Gonadotropin-induced phosphorylation of ERK1/2 was also inhibited in rat granulosa cells treated with TAPI-2 or after transfection with Tace/Adam17 small interfering RNA. Induced expression of Tnfaip6 mRNA was also reduced by Tace/Adam17 small interfering RNA. Thus, TACE/ADAM17 is induced and the activity is involved in porcine COC expansion as well as oocyte meiotic maturation through the activation of EGF receptor in cumulus cells.     

Progesterone receptor and the cell cycle modulate apoptosis in granulosa cells

Our previous studies showed that exposure of bovine preovulatory follicles to the LH surge-induced resistance of granulosa cells, but not theca cells, to apoptosis. Here, the temporal development of resistance to apoptosis and potential roles of progesterone receptor (PR) and alterations in the cell cycle in mediating this effect were examined. Injection of cows with GnRH induced an LH surge within 2 h. Granulosa cells isolated 0, 6, and 10 h after GnRH were sensitive to Fas ligand-induced apoptosis, but cells isolated at 14 h were resistant. PR was first detectable in granulosa cells at 10 and 14 h and was not detectable in theca. Treatment of granulosa cells isolated 14 h after GnRH with the PR antagonist, RU486, induced susceptibility to apoptosis, an effect mediated by PR and not glucocorticoid receptor. After GnRH treatment, granulosa cells, but not theca cells, exited the cell cycle, expression of cyclin D2 was reduced, and p27(Kip1) was elevated. Treatment of granulosa cells isolated from small antral follicles with the G1 phase blocker, mimosine, reduced Fas ligand-induced killing, suggesting that nonproliferating cells are resistant to apoptosis. Treatment of granulosa cells isolated 14 h after GnRH with RU486 induced reentry of some cells into the cell cycle and reversed resistance to apoptosis, suggesting that cycling cells became susceptible to apoptosis. Treatment with mimosine prevented the ability of RU486 to promote susceptibility to apoptosis. In summary, the LH surge induces expression of PR by granulosa cells and withdrawal from the cell cycle, and these events promote resistance to apoptosis.     

Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes

In this study, we investigated the mechanisms of protein kinase B (PKB) activation and its role in cumulus cells during in vitro meiotic resumption of porcine oocytes. PKB activity in cumulus cells was significantly decreased by 12 h cultivation of cumulus-oocyte complexes (COCs) in basic medium. However, the addition of phosphodiesterase inhibitors, hypoxanthine or 3-isobutyl-1-methylxanthine, maintained the level of PKB activity in cumulus cells at comparable with that in cumulus cells just after collection from their follicles. When COCs were cultured with phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, PKB activity was significantly decreased, and both caspase 3 activity and the proportion of apoptotic cells were significantly increased as compared with those in cumulus cells just after collection from their follicles. Moreover, the inhibitory effect of hypoxanthine on spontaneous meiotic resumption was overcome by addition of LY294002. On the other hand, markedly high activity of PKB and high intensity of the phosphorylated PKB band were observed in cumulus cells of COCs which were cultured with FSH. The addition of 20 microM LY294002 to FSH-containing medium induced an apoptosis of cumulus cells, whereas little apoptotic-positive signal was detected in COCs cultured with 5 microM LY294002 and FSH. However, the inhibitory effects of LY294002 on progesterone production by cumulus cells and germinal vesicle breakdown in oocytes reached a maximum at 5 microM. Thus, high activity of the PI 3-kinase-PKB pathway in cumulus cells plays an important role in FSH regulation of cell function. Judging from these results, it is estimated that PI 3-kinase in cumulus cells is required for both the suppression of spontaneous meiotic resumption and the induction of gonadotropin-stimulated meiotic resumption.     

Preovulatory secretion of steroids by cultured cumulus oophorus complexes of the rat: effects of FSH and LH.

The main objective of the present study was to establish whether a shift in steroid production, previously observed for preovulatory follicles, also takes place in preovulatory cumulus oophorus complexes (COCs). Female Wistar rats, displaying a regular 4-day oestrous cycle, were killed in succession every 2 or 3 h on the day of prooestrus and oestrus until ovulation (11.00-24.00 h). From excised ovaries preovulatory follicles were isolated. After puncturing cumuli oophori were aspirated and subsequently cultured for 24 h either in hormone-free or FSH- or LH- or FSH plus LH-supplemented medium. Cultured COCs released only a small amount of androgens, the main steroid produced being oestradiol. Its secretion decreased before ovulation (24.00 h). Relatively high progesterone release occured only in cultures set up at 22.00 and 24.00 h, thus during cumulus expansion. FSH and LH present in the medium effected above all oestradiol release, stimulating it before the presumptive endogenous gonadotrophin surge and inhibiting it thereafter. The activity of delta5-3beta-hydroxysteroid dehydrogenase (3betaHSD) investigated in cryostat sections appeared in COCs at 22.00h and was still present at 24.00 h. However, the activity was much weaker than in the granulosa cells lining the basal lamina. This in vivo study confirms in vitro results on more intense progesterone synthesis during cumulus expansion. The results indicate that in preovulatory COCs a shift in steroid production occurs after which the main steroid synthesized is progesterone, while oestradiol secretion decreases. However, this switch in COCs takes place later than the previously established shift in whole follicles.     

Dickkopf-1, an inhibitor of Wnt signaling, is regulated by progesterone in human endometrial stromal cells

CONTEXT: Some members of the Wnt family, including ligands, receptors, inhibitors, and signaling components, are expressed in human endometrium. Dickkopf-1 (Dkk-1), a potent inhibitor of the Wnt signaling pathway, was recently found to be up-regulated in decidualizing endometrial stromal cells during the secretory phase of the menstrual cycle, suggesting regulation by progesterone. OBJECTIVES: To test the hypothesis that progesterone regulates Dkk-1 expression in human endometrial stromal cells, we investigated the following effects on stromal cell expression of Dkk-1 mRNA and protein: decidualizing stimuli (progesterone or cAMP), RU486 (an inhibitor of progesterone action), and withdrawal of progesterone. RESULTS: Short-term treatment (up to 72 h, which corresponds to the full decidualized phenotype in response to cAMP and an early response to progesterone) did not reveal regulation of Dkk-1 mRNA or protein by cAMP but did show induction of Dkk-1 expression when the cells were treated with progesterone, an effect that was blocked by RU486. In long-term cultures (from 14 to 23 d, which corresponds to the full decidualized phenotype in response to progesterone), a significant increase in Dkk-1 mRNA and protein production was observed. Addition of RU486 or withdrawal of progesterone after long-term decidualization resulted in a decrease of Dkk-1 mRNA and protein to control levels. Estradiol alone had no effect on stromal Dkk-1 expression. CONCLUSIONS: These data strongly support regulation by progesterone of Dkk-1 mRNA synthesis and protein expression in human endometrial stromal cells and that the response is specific for progesterone and independent of cAMP and estradiol.     

Regulation of estrogen receptor alpha and progesterone receptor (isoform A and B) expression in cultured human endometrial cells.

The effects of RU 486 together with estradiol and progesterone on estrogen receptor alpha and progesterone receptor (isoforms A and B) expression were studied in human endometrial long term cultures at the mRNA and protein level. We asked whether ligand induced receptor regulation, found in mammals in vivo, is also found in human cultured endometrial cells with special regard to the progesterone isoforms A and B. Endometrial cultures were maintained for 27 days. Media were supplemented with progesterone and/or estradiol alone or in combination with RU 486. Receptor expression (estrogen receptor alpha and progesterone receptor isoform A and B) was examined at the mRNA level by RT-PCR and at the protein level by western blot analysis. All receptor types examined were expressed in our culture model. Estradiol led to a general increase of receptor expression whereas treatment with estradiol in combination with progesterone down regulated receptor expression. The receptor down regulation was not found when RU 486 was additionally supplemented into the medium. Activation or inhibition of expression due to these treatments was similar for both PR isoforms. Our results (1) show that in our culture system estradiol induced up regulation of estrogen receptor and progesterone receptor A and B and suggest that the estrogen induced up regulation is prevented by progesterone (2) a clear cut antigestagenic effect of RU 486 and (3) suggest that both progesterone isoforms are analogously regulated in our culture model. We conclude that human endometrial cell cultures are suitable for the study of the dynamics of steroid receptor expression.     

Mouse calbindin-D(9k) gene expression in the uterus during late pregnancy and lactation

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium binding protein mainly expressed in duodenum, placenta and uterus. In order to understand the expression pattern and regulation of uterine CaBP-9k gene, the expression of CaBP-9k mRNA and its regulation by estrogen (E2) and progesterone (P4) were investigated in the mouse uterus during late pregnancy (from day 12 to 18) and lactation. The expression levels of uterine CaBP-9k, estrogen receptor alpha (ERalpha) and progesterone receptor (PR) mRNAs were measured by Northern blot analysis. The expression levels of mouse uterine CaBP-9k mRNA gradually increased from pregnancy day 16 (P16), peaked at P18 (6.0-fold vs. P12) and declined at birth and during lactation. The expression levels of ERalpha and PR mRNAs indicated a similar fluctuation as CaBP-9k mRNA, suggesting the role of sex steroids/receptors in the regulation of CaBP-9k gene. To investigate effect of steroid hormone on CaBP-9k mRNA expression, three groups of animals were injected (s.c) with steroid hormone antagonists (RU486, tamoxifen, and ICI182780), respectively. RU486, a P4 antagonist, induced a significant decrease in CaBP-9k mRNA expression at 48 (3.2-fold) and 72 h (3.8-fold). However, tamoxifen and ICI182780, E2 antagonists, had no effect on CaBP-9k mRNA expression. Combined treatment with RU486 and ICI182780 did not further decrease the expression level of CaBP-9k mRNA when compared with RU486 treatment at 48 and 72 h. In addition, the treatment with RU40555, a glucocorticoid/progesterone antagonist, resulted in a decrease at 48 and 72 h following treatment. These results indicate that E2 is not likely involved in the regulation of CaBP-9k gene in the mouse uterus during late pregnancy and lactation. In conclusion, the present results suggest that P4, not E2 is a key regulator of CaBP-9k mRNA expression during late pregnancy and lactation.     

Effect of human chorionic gonadotropin on the expression of progesterone receptors and estrogen receptors in rabbit ovarian granulosa cells and the uterus.

To investigate whether LH/human CG (hCG) or progesterone acts as a regulator of estrogen receptors (ER) and progesterone receptors (PR) in granulosa cells, we studied the immunohistochemical expression of both ER and PR in the ovary and the uterus of mature rabbits, during the induction of ovulation by FSH followed by administration of hCG, progesterone, or a progesterone antagonist (RU486) and hCG. Granulosa cells pretreated with FSH for 3 days showed ER staining, but negligible PR staining. The staining pattern for ER and PR changed in animals pretreated with FSH followed by hCG injection; by 6 h after hCG injection, we observed the disappearance of ER and the appearance of PR, and by 3 days after hCG injection, we observed the reappearance of ER and the disappearance of PR. However, the expression of ER and PR in the granulosa cells of animals pretreated with FSH followed by progesterone administration instead of hCG was almost the same as that of animals pretreated with FSH alone. In addition, the expression of ER and PR in the granulosa cells of animals pretreated with FSH followed by RU486 and hCG was almost the same as that of animals pretreated with FSH followed by hCG administration. The uterine glandular epithelium, in contrast, began to show decreased appearance of ER and PR by 48 h after hCG injection, and we observed the disappearance of both receptors by 3 days after hCG administration. These results suggest that the expression of ER and PR in granulosa cells is not regulated by the action of progesterone, but by that of LH/hCG.     

Effects of RU486 on cyclooxygenase-2 gene expression, prostaglandin F2α synthesis and ovulation in Xenopus laevis

RU486 is a synthetic analog of progesterone and functions as a progesterone receptor antagonist. It binds to the progesterone receptor to prevent progesterone from occupying its receptor in many cellular systems. Early studies from our laboratory have shown that in Xenopus laevis ovarian follicles progesterone stimulates the expression of cyclooxygenase-2 (COX-2) gene which leads to a rapid increase in the production of prostaglandin F2α (PGF2α) and subsequent ovulation. In this study, we examined the effect of RU486 on the synthesis of COX-2 mRNA, production of PGF2α and ovulation in X. laevis. Ovarian tissue fragments were primed with human chorionic gonadotropin (hCG) and then incubated with progesterone (P4) alone or in the presence of varying concentrations of RU486 over a period of 12 h. After the incubation ovulated oocytes were counted, COX-2 expression and synthesis of PGF2α were measured. Results demonstrated that RU486 attenuated the expression of COX-2 gene, reduced the synthesis of PGF2α, and inhibited ovulation in a dose-dependent manner. This finding suggests that progesterone receptor is an important regulator in the progesterone-cyclooxygenase-prostaglandin-mediated ovulation in amphibians.     

The role of estrogen-dependent progesterone receptor in protein kinase C-mediated LH secretion and GnRH self-priming in rat anterior pituitary glands

The aim of the present study was to explore the involvement of pituitary progesterone receptor (PR) in PKC-mediated LH secretion and LHRH self-priming and the role of the estrogen (E) environment. Eight randomly selected hemipituitaries from adult female rats in proestrus or from 2 weeks ovariectomized (OVX) rats were incubated, in the absence of progesterone (P), over 3 h in Dulbecco's modified Eagle's medium (DMEM). In the first experiment, hemipituitaries were incubated continuously with: medium alone, GnRH (10 nM), the PKC stimulator PMA (100 nM), the PKC inhibitor staurosporine (100 nM), the antiprogestin at the receptor RU486 (10 nM), LHRH+staurosporine, GnRH+RU486 or PMA+RU486. In the second experiment, hemipituitaries were incubated, one h apart, with GnRH to determine the GnRH self-priming and this was compared with the priming effect of PMA. Also, the effect of staurosporine and RU486 during the induction period (1st h) on GnRH and PMA priming was evaluated. Medium was aspirated at the end of each h to determine LH accumulation and to evaluate GnRH self-priming. Both GnRH and PMA stimulated LH secretion. Staurosporine and RU486 reduced basal and GnRH-stimulated LH secretion, and RU486 reduced PMA-stimulated LH secretion from proestrus pituitaries. The stimulating effect of GnRH and PMA on LH secretion and the inhibitory action of staurosporine and RU486 on basal or stimulated LH secretion were significantly reduced in OVX-rats. Both GnRH and PMA induced GnRH priming. Staurosporine during the induction h reduced GnRH self-priming while RU486 reduced both GnRH self-potentiation and PMA priming. The magnitude of these inhibitory effects was blunted in OVX-rats. These results showed that PKC signaling pathway in the gonadotrope mediates, at least in part, basal and GnRH-stimulated LH secretion and GnRH self-priming. Also, the results are suggestive of an interaction of PKC signaling pathway with E-dependent PR in a ligand-independent activation manner in the gonadotrope.     

Effects of testosterone, FSH, and LH on oestradiol and progesterone secretion by preovulatory cumulus oophorus complexes of the rat.

Female Wistar rats exhibiting a regular 4-day oestrous cycle were included in this study. They were killed in succession on the day of pro-oestrus at 11.00, 18.00, and 22.00 h. From ovarian preovulatory follicles cumulus oophorus complexes (COCs) were isolated and subsequently cultured with or without testosterone (T), T plus FSH, or T plus LH. In control cultures COCs isolated at all investigated hours released similar amounts of oestradiol. T stimulated this basal secretion and the effect was usually enhanced in the presence of FSH or LH. In control cultures the amount of released progesterone was greatest when expanded COCs were isolated (22.00 h). T present in culture media diminished the amount of secreted progesterone. However, when T was added with FSH or LH a distinct stimulatory effect was observed, except in cultures with T plus FSH set up at 22.00 h. Previously, gonadotrophins alone did not effect progesterone secretion. The results suggest that T can regulate steroid, and especially progesterone secretion by COCs. Until the preovulatory gonadotrophin surge T can inhibit luteinization of COCs, while afterwards, acting synergestically with gonadotrphins (especially with LH), T can stimulate progesterone production in the cumulus granulosa cells.