G. Gibori

Differential Expression of the Estrogen Receptors α and β in the Rat Corpus Luteum of Pregnancy: Regulation by Prolactin and Placental Lactogens1

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 (ERα and ERβ) (by RT-PCR and in situ hybridization) in the rat corpus luteum, studied their developmental changes throughout pregnancy, and investigated the regulation of ERα and ERβ messenger RNA (mRNA) expression by PRL and placental lactogens. The RT-PCR studies showed that both ER mRNA species (ERα and ERβ) are coexpressed in the rat corpus luteum during pregnancy. Whereas ERα mRNA increased from early pregnancy, reached a maximum at midpregnancy, and had a remarkable decline before parturition; ERβ mRNA remained constant throughout pregnancy, with a significant decline at parturition. Examinati...

Progesterone Inhibits 20α-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 20α-hydroxysteroid dehydrogenase (20α-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 20α-HSD but lacks the PR. Treatment with different doses of either progesterone or dexamethasone caused a dose-related decrease in 20α-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 20α-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 ...

Estrogen Receptors α and β in Rat Decidua Cells: Cell-Specific Expression and Differential Regulation by Steroid Hormones and Prolactin1

Estradiol is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. In particular, this hormone with progesterone is necessary for blastocyst implantation and subsequent decidualization in the rat. Although binding experiments have demonstrated the presence of estrogenbinding sites, no evidence exists as to whether the rat decidua expresses both isoforms of the estrogen receptor (ER), a and b. In this investigation, we analyzed the expression of decidual ERa and ERb, studied their regulation by PRL and steroid hormones and examined the ability of decidual ERb to transduce the estradiol signal to the progesterone receptor. Immunocytochemistry, RT-PCR, and Northern blot analysis showed that both ER species are coexpressed in the decidua during pseudopregnancy. Interestingly, these genes were preferentially found in a cell population localized in the antimesometrial site of the uterus where blastocyst implantation takes place. Using decidual cells in primary culture obtained from pseudopregnant rats and a decidua-derived cell line (GG-AD), we show a differential regulation of ERa and ERb by PRL and ovarian steroid hormones. Whereas PRL, estradiol, and progesterone all increased ERb messenger RNA (mRNA) expression in a dose-dependent manner, only PRL up-regulated the mRNA levels of ERa. Estradiol had no effect on ERa expression, whereas progesterone markedly decreased its mRNA levels. Interestingly, progesterone, which up-regulates the ability of PRL to signal to a PRL-regulated gene in mammary-gland derived cells, prevented PRL stimulation of decidual ERa and had no synergistic effect on ERb expression. The use of GG-AD cells, which express only ERb, allowed us to demonstrate that this receptor subtype is functional and transduces estradiol signal to the progesterone receptor. In summary, the results of this investigation have revealed that ERb is expressed in addition to ERa in the rat decidua, and that the expression of both ERs are cell specific and differentially regulated by PRL and steroids. One salient finding of this investigation is that progesterone down-regulates ERa, but concomitantly increases the expression of a functional ERb that mediates estradiol up-regulation of the decidual progesterone receptor. (Endocrinology 141: 3842‐3851, 2000)

The Expression of Interleukin-6 (IL-6), IL-6 Receptor, and gp130-Kilodalton Glycoprotein in the Rat Decidua and a Decidual Cell Line: Regulation by 17β-Estradiol and Prolactin1

The cytokine interleukin 6 (IL-6), a major mediator of immune and acute phase responses of the liver, has been implicated in the termination of pregnancy once expressed in the uterus. This study was undertaken to investigate the expression and regulation of genes encoding IL-6 and IL-6 receptor (IL-6R) in rat decidual tissue. Total RNA obtained from rat decidual tissue on different days of pseudopregnancy was analyzed by RT-PCR using specific primers for IL-6, IL-6R, and 130-kDa glycoprotein (gp130). Ribosomal L19 primers served as an internal control. IL-6R and gp130 were found to be expressed in the decidua throughout development, while no messenger RNA (mRNA) for IL-6 was detected. Interestingly, within several hours of culture, decidual explants acquired the ability to express IL-6. The apparent ability of decidual cells to express IL-6 and its lack of expression in vivo led us to examine whether the IL-6 gene is actively inhibited. Primary decidual cells were cultured in the presence of estradiol, pr...

Prolactin Receptor-Associated Protein/17β-Hydroxysteroid Dehydrogenase Type 7 Gene (Hsd17b7) Plays a Crucial Role in Embryonic Development and Fetal Survival

Our laboratory has previously cloned and purified a protein named PRAP (prolactin receptor-associated protein) that was shown to be a novel 17beta-hydroxysteroid dehydrogenase (HSD) enzyme with dual activity. This enzyme, renamed HSD17B7 or PRAP/17beta-HSD7, converts estrone to estradiol and is also involved in cholesterol biosynthesis. The major site of its expression is the corpus luteum of a great number of species including rodents and humans. To examine the functional significance of HSD17B7 in pregnancy, we generated a knockout mouse model with targeted deletions of exons 1-4 of this gene. We anticipated a mouse with a severe fertility defect due to its inability to regulate estrogen levels during pregnancy. The heterozygous mutant mice are normal in their development and gross anatomy. The females cycle normally, and both male and female are fertile with normal litter size. To our surprise, the breeding of heterozygous mice yielded no viable HSD17B7 null mice. However, we found HSD17B7 null embryo alive in utero on d 8.5 and d 9.5. By d 10.5, the fetuses grow and suffer from severe brain malformation and heart defect. Because the brain depends on in situ cholesterol biosynthesis for its development beginning at d 10, the major cause of fetal death appears to be due to the cholesterol synthetic activity of this enzyme. By ablating HSD17B7 function, we have uncovered, in vivo, an important requirement for this enzyme during fetal development.

Decidual Activin: Its Role In the Apoptotic Process and Its Regulation by Prolactin

Abstract Successful pregnancy requires profound differentiation and reorganization of the uterine tissues including, as pregnancy progresses, extensive apoptosis of decidual tissue to accommodate the developing conceptus. We have previously shown a positive correlation between expression of activin A and apoptosis in the decidua and have also shown that expression of activin A occurs at the time when prolactin (PRL) receptors disappear from decidual cells. The goals of this study were to examine whether activin A plays a role in decidual apoptosis and whether expression of activin A in the decidua is regulated by PRL and placental lactogens. Studies were carried out using primary rat decidual cells, a decidual cell line (GG-AD), and PRL null mice. Treatment of decidual cells with activin A significantly increased DNA degradation, caspase 3 activity, and caspase 3 mRNA expression. However, this effect was observed only in the absence of endogenous activin production by these cells. Addition of follistatin to decidual cells that were producing activin A decreased both caspase 3 activity and mRNA expression. Similarly, addition of activin-blocking antibodies to cultures of GG-AD cells, which also produce activin A, caused a reduction in both DNA degradation and caspase 3 activity. PRL and placental lactogens caused an inhibition of activin A mRNA expression in primary decidual cells. Even more convincingly, decidua of PRL null mice expressed abundant activin A at a time when no expression of this hormone is detected in wild-type mice and treatment of PRL null mice with PRL caused a profound inhibition of activin A mRNA expression. In summary, our investigations into the role and regulation of decidual activin have revealed that activin A can induce cell death in the decidua and that its expression is under tight regulation by PRL and placental lactogens.

Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes

To determine if calcium‐dependent secretagogues directly act on epithelial cells to elicit CI− secretion, their effects on CI− transport and intracellular Ca2+ concentrations ([Ca2+]i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl− sensitive fluorescent probe, 6‐methoxyquinolyl acetoethyl ester, MQAE and the Ca2+‐sensitive fluorescent probe, fura‐2AM were used to assess Cl− transport and [Ca2+]i, respectively. Basal Cl− transport (0.274 ± 0.09 mM/sec) was inhibited significantly by the Cl− channel blocker diphenylamine‐2‐carboxylate (DPC, 50 μM, 0.068 ± 0.02 mM/sec; P < 0.001) and the Na+/K+/2Cl− cotransport inhibitor furosemide (1 μM, 0.137 ± 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be I− > F− ≥ Cl− > Br−. DPC inhibited I− influx by ∼50%, F− influx by 80%, Cl− influx by 85%, and Br− influx by 90%. Furosemide significantly inhibited influx of Br− (84%) and Cl− (81%) but not of F− and I−. The effects of agents known to alter biological response by increasing [Ca2+]i in other epithelial systems were used to stimulate Cl− transport. Cl− influx in mM/second was stimulated by 1 μM histamine (0.58 ± 0.05), 10 μM neurotensin (2.07 ± 0.32), 1 μM serotonin (1.63 ± 0.28), and 0.1 μM of the Ca2+ ionophore A23187 (2.05 ± 0.40). The Cl− permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine‐induced Cl− influx was significantly inhibited by only furosemide. Indomethacin blocked histamine‐stimulated Cl− permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl− transport by activating the Na+/K+/2Cl− cotransporter via a cyclooxygenase‐dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl− channels and the cotransporter, and their actions are cyclooxygenase‐independent.