Reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55 deficient mice.

Endometriosis is a chronic gynecological disease, characterized by growth of endometrial tissue in ectopic sites due to alteration of peritoneal homeostasis and deregulation of apoptosis. Here we have examined whether TNFRp55 deficiency modulates the pro-inflammatory state and the reinnervation of endometriotic-like lesions in mice. Two-month-old female C57BL/6 mice, eight wild type (WT) and eight TNFRp55-/- (KO) were used in the study. Endometriotic-like lesions were induced experimentally. The right uterine horn was removed from the animal, divided longitudinally, cut in three square pieces and sutured to the intestine mesentery. After 4 weeks, the lesions and the peritoneal fluid were collected. The level of TNFα in the peritoneal fluid was evaluated by enzyme-linked immunosorbent assay (EIA). The expressions of COX2, GRα and GRß were evaluated in the lesions by western blot and immunohistochemistry. ß-III TUBULIN, BDNF and NGF protein concentrations were evaluated in the lesions by western blot. Gene expression of Pgp 9.5, SP and Th was analyzed by RT-PCR, whereas relative concentrations of TRKA, NTRp75, phosphorylated NFκB (pNFκB) and total NFκB in lesions were measured by EIA. Compared with the WT group, the KO mice showed lower TNFα levels in the peritoneal fluid and lower numbers of COX2 immunoreactive cells along with increased expression of GRα, ß-III TUBULIN, Pgp 9.5, SP, Th, BDNF, NGF, NTRp75 and pNFκB in the lesions. Future histological studies will be necessary to confirm the sensory/sympathetic imbalance in the endometriotic-like lesions of the KO mice. Our results suggest that a reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55-/- mice. Chronic deregulation of TNF receptors can have serious consequences for women with advanced endometriosis.

Ubiquitin-like (UBX)-domain-containing protein, UBXN2A, promotes cell death by interfering with the p53-Mortalin interactions in colon cancer cells.

Mortalin (mot-2) induces inactivation of the tumor suppressor p53's transcriptional and apoptotic functions by cytoplasmic sequestration of p53 in select cancers. The mot-2-dependent cytoprotective function enables cancer cells to support malignant transformation. Abrogating the p53-mot-2 interaction can control or slow down the growth of cancer cells. In this study, we report the discovery of a ubiquitin-like (UBX)-domain-containing protein, UBXN2A, which binds to mot-2 and consequently inhibits the binding between mot-2 and p53. Genetic analysis showed that UBXN2A binds to mot-2's substrate binding domain, and it partly overlaps p53's binding site indicating UBXN2A and p53 likely bind to mot-2 competitively. By binding to mot-2, UBXN2A releases p53 from cytosolic sequestration, rescuing the tumor suppressor functions of p53. Biochemical analysis and functional assays showed that the overexpression of UBXN2A and the functional consequences of unsequestered p53 trigger p53-dependent apoptosis. Cells expressing shRNA against UBXN2A showed the opposite effect of that seen with UBXN2A overexpression. The expression of UBXN2A and its apoptotic effects were not observed in normal colonic epithelial cells and p53-/- colon cancer cells. Finally, significant reduction in tumor volume in a xenograft mouse model in response to UBXN2A expression was verified in vivo. Our results introduce UBXN2A as a home defense response protein, which can reconstitute inactive p53-dependent apoptotic pathways. Inhibition of mot-2-p53 interaction by UBXN2A is an attractive therapeutic strategy in mot-2-elevated tumors.

Involvement of Akt, Ras and cell cycle regulators in the potential development of endometrial hyperplasia in women with polycystic ovarian syndrome.

OBJECTIVE: To examine whether the abundance, localization, and/or activity of cell cycle regulators CDK2, Cyclin E, p27, and survival proteins AKT and Ras in PCOS-associated endometria (with and without hyperplasia) differ from non-PCOS endometria. METHODS: The expression of CDK2, Cyclin E, p27, AKT and Ras was measured by immunohistochemistry and/or Western blot in 9 normal endometria (NE), 12 endometria from PCOS patients without endometrial hyperplasia (PCOSE), 7 endometria from PCOS women with endometrial hyperplasia (HPCOSE), and 9 endometria from patients with endometrial hyperplasia (HE). The activity of CDK2 was assessed by an in vitro kinase assay. RESULTS: CDK2, Cyclin E and p27 proteins were expressed mainly in the endometrial epithelial cells of the studied groups. No change in the activity of CDK2 was observed in total extracts obtained from the tissue samples. However, the nuclear expression of CDK2 in epithelial cells was slightly elevated in PCOSE and significantly increased in HPCOSE when compared to NE. Higher expression of p27 was detected in the cytoplasm of epithelial cells of PCOSE and HPCOSE when compared to NE. Also, we found an increment in Ser473-AKT phosphorylation and an over-expression of the Ras oncogene in endometria of patients with PCOS. CONCLUSION: The PCOS condition is associated with increased Ser473-AKT phosphorylation, elevated expression of Ras, increased cytoplasmic abundance of p27, and increased nuclear abundance of CDK2 in the endometrial epithelial cells. These biological events could potentially provide a chance for endometrial cells from PCOS patients to exit the controlled cell cycle and become hyperplastic at a later stage.

Involvement of nuclear factor kappa B in the regulation of rat luteal function: potential roles as survival factor and inhibitor of 20alpha-hydroxysteroid dehydrogenase.

Nuclear factor kappa B (NFkappaB) is an important intracellular conveyor of extracellular signals and modulates a number of gene responses. Due to the potential significance of NFkappaB in regulating ovarian gene expression, we examined in the rat: (i) whether NFkappaB is activated and developmentally regulated in the corpus luteum (CL) throughout pregnancy; (ii) the proteins forming the NFkappaB complex in luteal cells; and (iii) the role of this transcription factor in luteal function. Western analysis and immunohistochemistry revealed that p65 and p50 were highly expressed throughout pregnancy and were located in both the nucleus and cytoplasm of luteal cells. In addition, because NFkappaB is maintained in the cytoplasm bound to IkappaB, whose phosphorylation allows NFkappaB translocation to the nucleus, we studied the developmental expression of phosphorylated and nonphosphorylated forms of IkappaBalpha. Western analysis revealed that IkappaBalpha was present and phosphorylated throughout pregnancy in the CL whereas by protein/DNA array and electromobility shift assays we found that luteal nuclear extracts bind to an NFkappaB consensus sequence, and that the binding activity decreased along pregnancy. The specific binding was supershifted only by an anti-p65 antibody and not by antibodies against p50, p52, cRel, or RelB. Using day 4 postpartum ovaries, we found higher NFkappaB binding activity in the newly formed CL than in old CL of pregnancy. Furthermore, NFkappaB DNA binding activity was enhanced by prolactin in luteinized granulosa cells. In our first functional study, blockade of NFkappaB/p65 binding to DNA with the sesquiterpene lactone helenalin in luteinized granulosa cells correlated with induction of cell death in a dose-dependent manner. In a second functional study, overexpression of NFkappaB/p65 in luteal cells resulted in inhibition of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD) promoter activity as well as endogenous 20alphaHSD mRNA expression. In summary, we have shown that: (i) NFkappaB is expressed within the CL, primary luteinized granulosa cells, and a rat luteal cell line; (ii) NFkappaB activation within the CL is developmentally regulated in pregnancy, depends on the age of the gland, and can be upregulated by prolactin; (iii) inhibition of NFkappaB/p65 binding to an NFkappaB DNA consensus sequence correlates with induction of cell death in ovarian luteinized granulosa cells; and (iv) overexpression of NFkappaB in luteal cells inhibits 20alphaHSD gene expression. The results further support a role for NFkappaB as a survival factor in the CL.

Differential roles for signal transducers and activators of transcription 5a and 5b in PRL stimulation of ERalpha and ERbeta transcription.

PRL has been shown to stimulate mRNA expression of both ERalpha and ERbeta in the rat corpus luteum and decidua of pregnancy. To investigate whether PRL may stimulate ER expression at the level of transcription and which transcription factors may mediate this stimulation, we have cloned the 5'-flanking regions of both rat ER genes. A constitutively active PRL receptor (PRL-R(CA)) stimulated both ERalpha and ERbeta promoter activity, indicating that PRL is acting to stimulate ER transcription. Putative signal transducer and activator of transcription (Stat)5 response elements were identified at -189 in the ERalpha promoter and at -330 in the ERbeta promoter. Mutation of these response elements or overexpression of dominant negative Stat5 prevented stimulation of ERalpha and ERbeta promoter activity, indicating that PRL regulation of ER expression requires both intact Stat5 binding sites as well as functional Stat5. Interestingly, either Stat5a or Stat5b could stimulate ERalpha transcription while stimulation of ERbeta occurred only in the presence of Stat5b. Through mutational analysis, a single nucleotide difference between the ERalpha and ERbeta Stat5 response elements was shown to be responsible for the lack of Stat5a-mediated stimulation of ERbeta. These findings indicate that PRL stimulation of ER expression occurs at the level of transcription and that PRL regulation of ERalpha can be mediated by either Stat5a or Stat5b, while regulation of ERbeta appears to be mediated only by Stat5b.

Apoptosis induced by antigestagen RU486 in rat corpus luteum of pregnancy.

Administration of RU486 to late pregnant rats results in preterm delivery 24 h after treatment and the induction of a luteolytic process after labor. We investigated whether functional changes occurring within the corpora lutea after RU486 treatment were associated with morphologic features of apoptotic cell death. Rats on d 18 of pregnancy were treated with RU486 (5 mg/kg) at 10:00 am and killed 72 h after. We studied the number of apoptotic cells in paraffin sections of the corpora lutea by routine hematoxylin and eosin (H&E) staining, and by in situ 3' end labeling (TdT-mediated dUTP nick-end labeling [TUNEL]). The corpora lutea were also processed for electron microscopy to study ultrastructural changes after RU486 treatment. The number of cells showing apoptotic nuclei in H&E-stained sections was higher in RU486-treated animals than in controls (vehicle-treated rats). The quantification of the number of apoptotic nuclei within the corpora lutea performed by TUNEL confirmed the higher number of apoptotic nuclei in animals receiving the antigestagen compared with controls. Ultrastructurally, the luteal cells undergoing apoptosis presented a highly deteriorated cytoplasmic organization The nuclei, in an initial step of regression, displayed condensation of the chromatin, a prominent nucleolus, and a perinuclear space. In an advanced step of degeneration, the nuclei showed evidence of large irregular aggregates of condensed chromatin. Prostaglandin F2,alpha(PGF2alpha), which mediates the luteolytic action of RU486, mimicked the effect of the antigestagen on the induction of apoptosis when administered to rats on d 18 of pregnancy (100 microg at 9:00 am and 1:00 pm), which were killed 72 after the last injection. In conclusion, the present results indicate that functional luteolysis in rats is associated with structural luteal regression with the morphologic features of apoptotic cell death, as demonstrated by studying the luteolytic process induced by the administration of the antigestagen RU486.

Downregulation of long-form prolactin receptor mRNA during prolactin-induced luteal regression.

OBJECTIVE: Prolactin is capable of both trophic and lytic actions in rat corpora lutea. In corpora lutea responding to a trophic prolactin signal, the long form of the prolactin receptor is the dominant form and is upregulated by prolactin. We investigated whether mRNA for the short form of the prolactin receptor was dominant in corpora lutea responding to a lytic prolactin signal, and whether the relative concentrations of the mRNAs for both forms of the prolactin receptor were changed during this response. DESIGN AND METHODS: Immature rats were ovulated by injection of 5 IU equine chorionic gonadotrophin and 5 IU human chorionic gonadotrophin, and were hypophysectomized shortly after ovulation. Nine days after hypophysectomy, rats were injected with prolactin (500 microg/day) or vehicle for 24 (n=6, n=6) or 72 h (n=13, n=5). Total RNA was isolated from corpora lutea and mRNA for both types of prolactin receptor were analyzed by semiquantitative RT-PCR using the ribosomal protein S16 as the internal control. RESULTS: The intensities of the long- and short-form prolactin receptor signals were normalized to the S16 internal control and expressed as relative densitometric units. The normalized values at 24h for prolactin-treated vs vehicle-treated rats were 0.23 +/- 0.05 vs 0.49 +/- 0.15 (P>0.05) for the short form and 4.04 +/- 0.8 vs 4.23 +/- 0. 6 (P>0.05) for the long form. The values for 72 h were 0.30 +/- 0.05 vs 0.24 +/- 0.05 (P>0.05) for the short form and 2.76 +/- 0.4 vs 5. 53 +/- 0.3 (P<0.01) for the long form respectively. CONCLUSION: The long form of the prolactin receptor is the dominant form at both time-points; however, the concentration of mRNA for this receptor isoform was specifically downregulated by prolactin treatment. Our results suggest that the short form of the prolactin receptor alone is unlikely to mediate the luteolytic action of prolactin, but that luteolytic events may be influenced via a change in the ratio of the two receptor isoforms.

Progesterone receptor is not required for progesterone action in the rat corpus luteum of pregnancy.

In this study, we investigated whether progesterone exerts a local action regulating the function of the corpus luteum of pregnancy in rats. The luteal activities of the enzymes 3beta-hydroxysteroid dehydrogenase (3beta-HSD), involved in progesterone biosynthesis, and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), that catabolizes progesterone and reduces progesterone secretion by the corpus luteum, were evaluated after intrabursal ovarian administration of progesterone in pregnant rats that had received a luteolytic dose of prostaglandin F2alpha (PGF2alpha). Luteal 3beta-HSD activity decreased and 20alpha-HSD activity increased after PGF2alpha treatment (100 microg x 2 intraperitoneally on Day 19 of pregnancy at 12:00 p.m. and 4:00 p.m.) when compared with controls sacrificed at 8:00 p.m. on Day 20 of pregnancy. This effect of PGF2alpha on the luteal 3beta-HSD and 20alpha-HSD activities was abolished in animals that also received an intraovarian dose of progesterone (3 microg/ovary on Day 19 of pregnancy at 8:00-9:00 a.m.). In a second functional study, luteal cells obtained from 19-day pregnant rats responded to the synthetic progestin promegestone (R5020) in a dose-dependent manner, with an increase in the progesterone output. In addition, the glucocorticoid agent hydrocortisone did not affect progesterone accumulation in the same luteal cell culture. We also examined by immunocytochemistry the expression of progesterone receptors (PR) in the corpora lutea during pregnancy and demonstrated the absence of PR in this endocrine gland in all the days of pregnancy studied. In the same pregnant rats, positive staining for PR was observed in cells within the uteroplacental unit, such as cells of the decidua basalis and trophoblast giant cells of the junctional zone. In addition, positive PR staining was observed in the ovarian granulosa and theca cells of growing follicles, but not in corpora lutea of ovaries obtained from cycling rats at proestrus. In summary, this report provides further evidence of a local action of progesterone regulating luteal function in the rat despite the absence of a classic PR.

Regulation and role of the insulin-like growth factor I system in rat luteal cells.

The relationship between insulin-like growth factor I (IGF-I), a hormone which has potent metabolic effects and stimulates protein synthesis, and prolactin and oestradiol was examined to investigate a possible mechanism for the luteal cell hypertrophy that is responsible for the increase in size of the corpus luteum. A luteal cell line (GG-CL) derived from large luteal cells of the pregnant rat corpus luteum was used. IGF-I, IGF-I receptor and oestrogen receptor beta mRNA contents were determined by semiquantitative RT-PCR. The results revealed that prolactin upregulates the expression of IGF-I mRNA in luteal cells, but not that of its receptor. IGF-I had no effect on the expression of its receptor but caused a dose-related increase in the expression of oestrogen receptor beta. Furthermore, whereas IGF-I upregulated oestrogen receptor beta expression, oestradiol downregulated expression of mRNA for both IGF-I and its receptor. This effect of oestradiol is not mediated through progesterone which is stimulated by oestradiol in the corpus luteum. The developmental studies indicate that mRNA for IGF-I and its receptor are not expressed in tandem throughout pregnancy. Whereas the receptor mRNA is expressed at higher concentrations in early pregnancy, that of its ligand is highly expressed close to parturition. Collectively, the results indicate that prolactin stimulates luteal IGF-I production, which in turn acts on the luteal cell to stimulate expression of oestrogen receptor beta. Luteal cells with increased oestrogen receptor beta can respond fully to oestradiol, leading to cell hypertrophy.

Hormonal regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase messenger ribonucleic acid in the rat corpus luteum: induction by prolactin and placental lactogens.

The corpus luteum expresses two enzymes that scavenge superoxide radicals and protect the cells from their toxic activities: cytosolic copper, zinc-superoxide dismutase (Cu,Zn-SOD) and mitochondrial manganese-SOD (Mn-SOD). The present study was undertaken to investigate whether the mRNA expression of each of these enzymes is regulated by luteotropic hormones. Cu,Zn-SOD and Mn-SOD mRNA levels were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). We first examined the effects of prolactin (PRL) on Cu,Zn-SOD and Mn-SOD mRNA expression in the corpus luteum. Hypophysectomy of Day 3 pregnant rats caused a sharp decline in both Cu,Zn-SOD and Mn-SOD mRNA levels, which was completely reversed by PRL administration. To further examine the effects of PRL and rat placental lactogen (rPL) on the expression of these enzymes, either primary luteinized granulosa cells or temperature-sensitive simian virus-40 transformed luteal cells (GG-CL) were cultured with different doses of PRL or rPL. These hormones induced a remarkable increase in Cu,Zn-SOD and Mn-SOD mRNA levels in both primary luteinized granulosa cells and GG-CL cells. Interestingly, whereas PRL up-regulated the expression of the SOD in luteal cells, other luteotropic hormones such as estradiol and dexamethasone inhibited both SOD mRNA expression while progesterone had no effect. In conclusion, PRL and PRL-like hormones induce a protective ability against toxic oxygen radicals by stimulating the expression of SODs, a phenomenon that may play an important role in maintaining luteal cell integrity and steroidogenic capacity.

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.

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.

Establishment and characterization of a simian virus 40-transformed temperature-sensitive rat luteal cell line.

The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the possibility for the study of gene regulation in luteal cells. The goal of this study was to develop a cell line to serve as a model by which to study the expression and regulation of various genes specific to luteal cells. We attempted to develop a luteal cell line by transformation of large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive mutation required for the maintenance of cell transformation. We report here the successful establishment of such a cell line, designated GG-CL cells. Large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of day 14 pregnant rats, cultured for 24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of transformed cells were isolated and passaged. They multiplied at 33 C and formed multilayers. At the nonpermissive temperature (40 C), cells reverted to the normal differentiated phenotype similar to the primary luteal cells in culture. To determine whether GG-CL cells express the genes found in normal luteal cells, messenger RNA (mRNA) expression was examined by either Northern analysis or RT-PCR with primers specific to each mRNA. GG-CL cells were found to express receptors for interleukin-6 and glucocorticoid, as well as the newly discovered estrogen receptor-beta (ER-beta) and the orphan nuclear receptor nur 77. No receptors for ER-alpha, progesterone, LH, or PRL could be detected. This cell line also expressed 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), but not cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase, or aromatase cytochrome P450 (P450arom). Although the cells did not express the PRL receptor, they did express Janus kinase (JAK2) and signal transducers and activators of transcription (Stat5b), and, when transfected with the PRL receptor, they responded to PRL with a marked inhibition in 20alpha-HSD mRNA expression. In addition, estradiol enhanced ER-beta expression in a dose-dependent manner whereas cAMP stimulation caused a marked and rapid increase in the expression of the orphan receptor nur 77. In summary, a temperature-sensitive cell line was successfully established from the large luteal cells of rat corpora lutea. These cells express key genes encoding enzymes and receptors inherent to this defined luteal cell population and respond to stimulation by PRL, estradiol, and cAMP.

Fertility impairment after mifepristone treatment to rats at proestrus. Actions on the hypothalamic-hypophyseal-ovarian axis.

Accumulated evidence indicates that the antigestagen mifepristone affects the reproductive axis acting on hypothalamic, pituitary, ovarian, and uterine tissues. The purpose of this study was to further investigate which reproductive functions are impaired by the antagonist, critically compromising the reproductive process, leading to unsuccessful pregnancy. Circulating pituitary and ovarian hormones, sexual receptivity, ovulation, and implantation rates were studied in cycling rats receiving a single dose of mifepristone (1 or 10 mg/kg subcutaneously) at 12:00 proestrus, before luteinizing hormone (LH) stimulation of the ovulatory process. Mifepristone-treated rats had decreased preovulatory surges of LH and prolactin (PRL), and hypersecretion of LH, PRL, and progesterone at estrus. The sexual receptivity was dramatically affected by the antagonist as indicated by the profound decrease in the lordosis response evaluated on the night of proestrus. The number of ovulating animals and the number of oocytes recovered from the oviduct on the morning of estrus were not affected by mifepristone. The low number of rats that succeeded in mating with potent males became pregnant. However, they delivered an average of only two pups at parturition, indicating a failure in the implantation of the fertilized ova, as ovulation was not affected by the antagonist at the dose used. We conclude that a dramatic inhibition of the sexual receptivity and unsuccessful implantation, preceded by a reduction on LH and PRL secretion, are the major components leading to fertility impairment after a single dose of mifepristone administered before the preovulatory surge of LH.

PIP: Mifepristone has been demonstrated to act on hypothalamic, pituitary, ovarian, and uterine tissue. To further investigate impairments in reproductive function triggered by this antagonist, circulating pituitary and ovarian hormones, sexual receptivity, ovulation, and implantation rates were studied in cycling Wistar rats receiving a single dose (1 or 10 mg/kg subcutaneously) of mifepristone at 12:00 proestrus, before luteinizing hormone (LH) stimulation of the ovulatory process. Treated rats had decreased preovulatory LH and prolactin (PRL) surges and hypersecretion of LH, PRL, and progesterone as estrus. A profound decrease in the lordosis response on the night of proestrus indicated a dramatic effect on sexual receptivity. There was no affect on the number of ovulating animals and the number of oocytes recovered from the oviduct on the morning of estrus. The few rats who succeeded in mating with potent males became pregnant, but they delivered an average of only two pups, indicating a failure in the implantation of the fertilized ova. These findings suggest that the dramatic inhibition of sexual receptivity and unsuccessful implantation, preceded by a reduction in LH and PRL secretion, are the major factors producing fertility impairment after a single dose of mifepristone before the preovulatory LH surge. factors such as smoking and parity.

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.

Reproductive function in rats after mifepristone-induced termination of pregnancy.

The effect of mifepristone, a potent progesterone receptor antagonist, on the reproductive function during early pregnancy in rats was examined. A single dose of this drug (10 mg/kg) was injected s.c. at 1200 h on day 4 (Group 1), day 7 (Group II) or day 10 (Group III) of pregnancy. Gestation was interrupted and the vaginal cytology showed a typical proestrus condition two days after mifepristone treatment in all groups. When compared with cycling proestrus rats, serum LH concentrations at 1800 h in the mifepristone-induced proestrus were lower in Group I, similar in Group II and higher in Group III, and serum prolactin (PRL) values were lower in Group I, but not different in Groups II and III. Serum progesterone levels were higher in the three experimental groups when compared with cycling proestrus rats, and similar to that of pregnant rats. Rats in Group I showed a significantly lower sexual receptivity and ovulation rate when compared with Groups II and III or cycling proestrus rats. Most of the mifepristone-treated rats that copulated during the night of the induced proestrus did not become pregnant and showed a delayed pseudopregnancy-like condition. These results indicate that mifepristone administered in a single dose to early pregnant rats terminates pregnancy and induces a proestrus condition two days after treatment followed by successful postovulatory contraception. The mifepristone-induced proestrus is characterized by a differential pattern of serum LH, PRL and progesterone concentrations, mating behavior and ovulation rates, depending on the day of pregnancy when mifepristone is administered.

Dual regulation of luteal progesterone production by androstenedione during spontaneous and RU486-induced luteolysis in pregnant rats.

The effect of androstenedione on luteal progesterone production was studied during luteolysis preceding parturition as well as that induced by the antiprogestin RU486 in late pregnant rats. Luteal cells from animals on days 19, 20 or 21 of pregnancy and incubated with 10 microM androstenedione increased progesterone production by 99, 136, and 277%, respectively. The animals receiving androstenedione (10 mg/rat s.c.) on day 19 of pregnancy showed an increase in serum progesterone levels, a decline in luteal 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity and an increase in corpus luteum weight without modifying 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity on day 21 of pregnancy. Androstenedione and testosterone but not dihydrotestosterone were able to prevent the decrease in serum progesterone concentration and corpus luteum weight observed 58 h after treatment with RU486 (2 mg/kg) on day 18 of pregnancy. However, the three androgens studied inhibited the luteal 3 beta-HSD activity but 20 alpha-HSD activity was not affected, when compared with animals receiving RU486 alone. The co-administration of androstenedione with the aromatase inhibitor 4-hydroxyandrostenedione or with the specific antioestrogen ICI 164,384 did not modify the effects induced by androstenedione in RU486-treated rats, indicating that the action of androstenedione on progesterone production and secretion at the time of luteolysis seems to occur through an androgenic mechanism and is not mediated by previous conversion of the androgens to oestrogens. In all experiments the high luteal 20 alpha-HSD activity, that characterizes a luteolytic process, was not modified by androgens. Androstenedione administered to adrenalectomized rats was also able to prevent the decrease in serum progesterone concentration observed in spontaneous or RU486-induced luteolysis. The administration of androstenedione to RU486-treated rats induced a decrease in luteal progesterone content concomitant with an increase in serum progesterone levels. These studies demonstrate that androgens during luteolysis, are able to stimulate luteal progesterone secretion, prevent the loss in corpora lutea weight and enhance the decrease in 3 beta-HSD activity, without affecting the increase in 20 alpha-HSD activity.

Luteolytic action of RU486: modulation of luteal 3 beta-hydroxysteroid dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities in late pregnant rats.

The effect of the synthetic antiprogestin RU486 on luteal function in late pregnant rats was studied by evaluating the activities of the enzymes 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD). RU486 (2 mg/kg) administered to rats on day 18 of pregnancy at 10.00 h induced preterm delivery 26.4 +/- 0.35 h (n = 8) after treatment. Luteal 3 beta-HSD activity increased 24 and 34 h after RU486 injection, but a significant and progressive decrease started at 48 h with the maximal reduction 72 h after RU486 treatment, when compared with controls. Serum progesterone concentration decreased at the time of 3 beta-HSD activity reduction. Interestingly, 20 alpha-HSD activity started to increase 58 h after RU486 injection. The administration of the cyclooxygenase inhibitor, diclofenac (1.3 mg/kg), on days 17-19 of pregnancy to RU486-treated rats, delayed abortion and the duration of delivery, and prevented the decrease in 3 beta-HSD and the increase in 20 alpha-HSD activities observed 58 h after antiprogesterone treatment. RU486 administered intrabursally (1 microgram per ovary) on day 20 (14.00-15.00 h) increased 3 beta-HSD and decreased 20 alpha-HSD luteal activities at 18.00 h on day 21 of pregnancy, without modifying serum progesterone concentration, when compared with normal pregnant rats. In conclusion, the luteolytic process after preterm delivery induced by RU486 administration in late pregnant rats is characterized by a decrease in luteal 3 beta-HSD activity and circulating progesterone, which may trigger the increase in luteal 20 alpha-HSD activity. Prostaglandins seems to be involved in the increase of 20 alpha-HSD activity and therefore, in the demise of corpora lutea.