Inhibitory Effect Of Progesterone Research Articles

Characteristic impairment of progesterone response in cultured cervical fibroblasts obtained from patients with refractory cervical insufficiency

Preterm birth (PTB) is the leading cause of neonatal mortality, and reducing the PTB rate is one of the most critical issues in perinatal medicine. Cervical insufficiency (CI), a major cause of PTB, is characterised by premature cervical ripening in the second trimester, followed by recurrent pregnancy loss. Although multiple clinical trials have suggested that progesterone inhibits cervical ripening, no studies have focused on progesterone-induced molecular signalling in CI. Here, we established a primary culture system for human uterine cervical fibroblasts using a sample of patients with refractory innate CI who underwent transabdominal cervical cerclage and patients with low Bishop scores who underwent elective caesarean section as controls. RNA sequencing showed that the progesterone response observed in the control group was impaired in the CI group. This was consistent with the finding that progesterone receptor expression was markedly downregulated in CI. Furthermore, the inhibitory effect of progesterone on lipopolysaccharide-induced inflammatory stimuli was also impaired in CI. These results suggest that abnormal cervical ripening in CI is caused by the downregulation of progesterone signalling at the receptor level, and provide a novel insight into the molecular mechanism of PTB.

89 Does progesterone block the nerve growth factor–induced luteinizing hormone surge in llamas?

The central inhibitory effects of progesterone on gonadotrophin secretion have been well documented in several species, including camelids. Nerve growth factor (NGF) in seminal plasma triggers ovulation in camelids and is thought to act at the level of the hypothalamus. The objective of the study was to determine the effect of progesterone on NGF-induced LH release in llamas. In Experiment 1, llamas were assigned to a low, medium, or high progesterone group (n=4 per group). The low progesterone group consisted of non-mated (non-ovulatory) llamas, the medium progesterone group consisted of mated llamas (luteal phase; 3–4 weeks pregnant), and the high progesterone group consisted of non-mated llamas given a single intramuscular (IM) dose of progesterone (300mg IM, Progesterone BioRelease LA). A jugular catheter was placed, and the following day llamas were given an intravenous dose of 1mg of NGF isolated from seminal plasma. Blood samples were taken every 30min from 1h before to 5.5h after NGF treatment. In Experiment 2, the pituitary LH response to gonadotrophin-releasing hormone (GnRH) was compared between llamas treated with either Progesterone BioRelease LA or saline (n=4 per group). Sixteen hours later, llamas in both groups were given 50µg of gonadorelin (GnRH) IV, and blood samples were collected by jugular puncture at 0.5h before and 0, 1, 2, and 4h after GnRH. Blood samples were centrifuged, and plasma was stored frozen until radioimmunoassay for LH and progesterone. Data are presented as mean±s.e.m. Data were analysed by one-way ANOVA for single-point and repeated-measures, and independent or paired t-tests. In Experiment 1, plasma progesterone concentrations in the low, medium, and high progesterone groups were 0.6±0.3, 8.2±0.4, and 14.9±1.2ng mL−1, respectively, at the time of NGF treatment (P<0.05). Circulating concentrations of LH did not differ among progesterone groups (treatment, P=0.49; time, P<0.01; treatment×time interaction, P=0.65). In all groups, LH concentrations were elevated within 30min of NGF administration, reached a peak by 2h, and remained elevated beyond the sampling period. Comparison of samples collected during the pretreatment period (i.e. −60, −30, and 0min), however, revealed that plasma LH concentrations in the high progesterone group were half that of the low and medium progesterone groups (P<0.03). In Experiment 2, plasma progesterone concentrations in the progesterone- and saline-treated groups were 12.7±2.2 and 1.3±0.3ngmL−1, respectively (P<0.01). Despite the difference in circulating progesterone concentrations, the LH response to GnRH treatment was not different between groups (treatment group, P=0.43; time, P<0.01; treatment×time interaction, P=0.84). Results demonstrate a suppressive effect of progesterone on basal LH release in llamas, but no suppressive effect on NGF- or GnRH-induced surge release from the pituitary gland. Results are consistent with the hypothesis that the site of action of NGF is downstream of the hypothalamic site of action of progesterone. This research was supported by NSERC Canada.

Progesterone Antagonizes Dexamethasone-Regulated Surfactant Proteins In Vitro

Pregnant women at risk of preterm labor routinely receive glucocorticoids (GCs) and frequently also progesterone. Administration of GCs accelerates intrauterine surfactant synthesis and lung maturation, thereby reducing the incidence of neonatal respiratory distress syndrome; progesterone has the potential to prevent preterm birth. Little is known about possible interactions of GCs and progesterone. Our aim was to clarify whether progesterone can affect dexamethasone (DXM)-regulated expression of surfactant protein A (SP-A), SP-B, and SP-D in lung epithelial cells. H441 cells were exposed to DXM and progesterone and expression of SPs was analyzed by quantitative real-time polymerase chain reaction and immunoblotting. Although progesterone had no direct effect on the expression of SP-B, DXM-mediated induction was inhibited dose dependently on the transcriptional (64 μM [P <.0001], 32 μM [P -.0005], 16 μM [P -.0019]) and the translational level. Furthermore, progesterone inhibited stimulatory effects of other GCs as well. While exogenous tissue growth factor β1 (TGF-β1) inhibited DXM-induced SPB expression (messenger RNA [mRNA]: P -.0014), progesterone itself did not influence TGF-β1 mRNA expression and/or TGFβ1/ Smad signaling, demonstrating that TGF-β1 and/or Smad activation is not involved. The inhibitory effect of progesterone could be imitated by the GC and progesterone receptor (PR) antagonist RU-486, but not by the specific PR antagonist PF-02413873, indicating that progesterone acts as a competitive antagonist of DXM. The effect of progesterone on DXM-regulated genes was not specific for SP-B, as expression of SP-A and SP-D mRNAs was also antagonized. The present study highlights a new action of progesterone as a potential physiological inhibitor of GC-dependent SP expression in lung epithelial cells. The clinical relevance of this in vitro finding is currently unknown.

Luteal peptides and intercellular communication

The variety of peptides synthesized by the corpus luteum (relaxin, vasopressin, oxytocin and oxytocin-related neurophysin) and their possible intracellular effects are reviewed. After luteinization of the granulosa cells and in response to LH and FSH, the output of oxytocin is increased. In addition, insulin-like growth factor is a very potent stimulus of oxytocin secretion. Although luteal cells respond to gonadotrophins by increased production of progesterone, there is no further secretion of oxytocin. Oxytocin is localized in large luteal cells which seem not to be under the direct control of gonadotrophins. Synthesis of luteal oxytocin seems to occur during the early luteal phase according to measurements of oxytocin mRNA. Highest tissue concentrations and secretion under in-vitro conditions were observed during the mid-luteal phase, and so synthesis, storage and secretion are unlikely to occur concomitantly. Under in-vitro conditions, oxytocin is secreted concomitantly with neurophysin and progesterone, and there appears to be some form of communication between small and large luteal cells for the secretion of progesterone and oxytocin under in-vivo conditions. Evidence has been obtained that oxytocin may have local effects in the ovary by inhibition of secretion (synthesis ?) of progesterone, especially during the early luteal phase. A mechanism can be suggested whereby, under physiological conditions, oxytocin may delay the increase of progesterone by inhibition of progesterone secretion and therefore delay down regulation of its own receptor. This would prolong the life-span of the CL and the oestrous cycle. Exogenous progesterone given on Days 1-4 shortens the cycle to about 12 days. The best evidence that oxytocin may be involved in controlling luteolysis comes from immunization experiments in ewes and goats, but there is no clear evidence of this type for cattle. Basal concentrations of oxytocin at the end of the luteal phase may interact with oxytocin receptors after the inhibitory effect of progesterone in the uterus is reduced, thus initiating synthesis of PGF-2 alpha.

Participation of membrane progesterone receptor α in the inhibitory effect of progesterone on prolactin secretion.

The membrane progesterone receptors (mPRα, mPRβ, mPRγ, mPRδ and mPRε) are known to mediate rapid nongenomic progesterone functions in different cell types. However, the functions of these receptors in the pituitary have not been reported to date. In the present study, we show that the expression of mPRα was the highest among the mPRs in the rat anterior pituitary gland. Immunostaining of mPRα was detected in somatotrophs, gonadotrophs and lactotrophs. Interestingly, 63% of mPRα-positive cells within the pituitary were lactotrophs, suggesting that mPRα is involved in controlling prolactin (PRL) secretion in the pituitary. To test this hypothesis, rat pituitaries were incubated (1hour) with either progesterone (P4) or the mPRα-specific agonist Org OD 02-0. PRL secretion was then measured by radioimmunoassay. The results of this experiment revealed that both P4 and Org OD 02-0 decreased PRL secretion. Moreover, the results from the GH3 cell line (CCL-82.1) showed that P4 and Org OD 02-0 inhibited PRL release, although the nuclear PR agonist R5020 was ineffective. Our investigation of the cellular mechanisms behind mPRα activity indicated that both P4 and Org OD 02-0 decreased cAMP accumulation, whereas R5020 was ineffective. In addition, the Org OD 02-0-effect on PRL release was blocked by pretreatment with pertussis toxin, an inhibitor of Go/Gi proteins. Because transforming growth factor (TGF)β1 is a potent inhibitor of PRL secretion in lactotrophs, we lastly evaluated whether TGFβ1 was activated by progesterone and whether this effect was mediated by mPRα. Our results showed that P4 and Org OD 02-0, but not R5020, increased active TGFβ1 levels. This effect was not observed when cells were transfected with mPRα-small interfering RNA. Taken together, these data provide new evidence suggesting that mPRα mediates the progesterone inhibitory effect on PRL secretion through both decreases in cAMP levels and activation of TGFβ1 in the lactotroph population.

Potential inhibitory effect of progesterone on breast cancer metastasis via the regulation on protein expression of apoptosis- and EMT-related genes

Potential inhibitory effect of progesterone on breast cancer metastasis via the regulation on protein expression of apoptosis- and EMT-related genes

Inhibitory effect of progesterone during early embryonic development: Suppression of myocardial differentiation and calcium-related transcriptome by progesterone in mESCs: Progesterone disturb cardiac differentiation of mESCs through lower cytosolic Ca(2.).

Progesterone (PG) and its derivates are used in prevention of spontaneous miscarriage. However, some studies have reported that exposure to PG and its derivates during pregnancy can cause malformations and affect both blood pressure and the cardiovascular system. The effect of PG on cardiomyogenesis of mouse embryonic stem cells (mESCs) is not well known. Expression of Pgr mRNA showed an opposite pattern of beating-ratio during differentiation. PG treatment resulted in reduction of the beating ratio to 60.45±1.54% from 92.17±2.98% in normal differentiation, reduced transcripts of heart morphogenesis and Ca(2+) binding-related genes in the next generation sequencing data and significantly decreased expression levels of Ca(2+)/contraction-related genes including Ryr2, Calm2, Trpv2, and Mylk3, the intracellular Ca(2+) level, and the beating frequency. These results suggest that PG exerts inhibitory effects on differentiation of mESCs into functional cardiomyocytes.

Black cohosh inhibits 17β-estradiol-induced cell proliferation of endometrial adenocarcinoma cells

This study was conducted to investigate the effect of black cohosh (BC) extract on the proliferation and apoptosis of Ishikawa cells. Ishikawa human endometrial adenocarcinoma cells were treated with or without BC (1, 5, 10 and 25 μM) and cell proliferation and cytotoxicity were measured by CCK-8 assays and flow cytometry analysis. Additionally, Ishikawa cells were treated with 17β-estradiol (E2), E2 + progesterone and E2 + BC (5 and 10 μM) and the effect of BC and progesterone on E2-induced cell proliferation was analyzed. BC decreased the proliferation of Ishikawa cells at a dose-dependent rate compared with the control group (p < 0.05). The proliferation of Ishikawa cells increased in the presence of E2, whereas the subsequent addition of progesterone or BC decreased proliferation to the level of the control group (p < 0.05). The inhibitory effect of BC on E2-induced cell proliferation was greater than the inhibitory effect of progesterone. In conclusion, BC induces apoptosis in Ishikawa cells and suppresses E2-induced cell proliferation in Ishikawa cells. BC could be considered a candidate co-treatment agent of estrogen-dependent tumors, especially those involving endometrial cells.

Progesterone rapidly down-regulates the biosynthesis of 5-lipoxygenase products in human primary monocytes

5-Lipoxygenase (5-LO), the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) from arachidonic acid, is regulated by androgens in human neutrophils and monocytes accounting for sex differences in LT formation. Here we show that progesterone suppresses the synthesis of 5-LO metabolites in human primary monocytes. 5-LO product formation in monocytes stimulated with Ca2+-ionophore A23187 or with lipopolysaccharide/formyl peptide was suppressed by progesterone at concentrations of 10–100nM in cells from females and at 1μM in cells from males. Progesterone down-regulated 5-LO product formation in a rapid and reversible manner, but did not significantly inhibit 5-LO activity in cell-free assays using monocyte homogenates. Also, arachidonic acid release and its metabolism to other eicosanoids in monocytes were not significantly reduced by progesterone. The inhibitory effect of progesterone on LTs was still observed when mitogen-activated protein kinases were pharmacologically blocked, stimulatory 1-oleoyl-2-acetyl-sn-glycerol was exogenously supplied, or extracellular Ca2+ was removed by chelation. Instead, suppression of PKA by means of two different pharmacological approaches (i.e. H89 and a cell-permeable PKA inhibitor peptide) prevented inhibition of 5-LO product generation by progesterone, to a similar extent as observed for the PKA activators prostaglandin E2 and 8-Br-cAMP, suggesting the involvement of PKA. In summary, progesterone affects the capacity of human primary monocytes to generate 5-LO products and, in addition to androgens, may account for sex-specific effects on pro-inflammatory LTs.

Progesterone-induced cyclin G1 inhibits the proliferation of endometrial epithelial cell and its possible molecular mechanism.

Under normal conditions, progesterone inhi-bits the estrogen-induced proliferation of endometrial epithelium. Our previous studies have shown that cyclin G1 was progesterone-dependent in mouse endometrial epithelium at peri-implantation, and exogenous cyclin G1 suppressed the proliferation of endometrial cancer cells. The objectives of this study are to determine whether cyclin G1, as a negative regulator of the cell cycle, is involved in the antiproliferative action of progesterone on endometrial epithelial cells, and to explore the possible molecular mechanism of cyclin G1 inhibition. The siRNA-mediated elimination of cyclin G1 attenuated the antiproliferative action of progesterone on endometrial epithelial cells. Immunoprecipitation showed that progesterone-induced cyclin G1 could interact with PP2A to mediate its phosphatase activity. The block of PP2A activity also attenuated the antiproliferative action of progesterone on endometrial epithelial cells and increased the phosphorylated Rb. In conclusion, progesterone-induced cyclin G1 mediates the inhibitory effect of progesterone on endometrial epithelial cell proliferation possibly through the recruitment of PP2A to dephosphorylate Rb.

Inhibitory Effect of Progesterone on Cervical Tissue Formation in a Three-Dimensional Culture System with Human Cervical Fibroblasts1

Progesterone supplementation is recommended to prevent preterm birth in women with a short cervix, but the mechanism is unclear. We hypothesize that progesterone acts by altering the composition of the cervical extracellular matrix (ECM). We tested this hypothesis using human cervical fibroblasts in both two-dimensional (2D) and three-dimensional (3D) cultures. For 2D culture, cells were seeded in 6-well plates and cultured with media supplemented with estradiol (10(-8) M), progesterone (10(-7) or 10(-6) M), and vehicle. For 3D culture, the cells were cultured on a porous silk protein scaffold system. Progesterone and estrogen receptors were documented by immunohistochemistry and Western blot analysis. In both 2D and 3D cultures, decreased collagen synthesis was seen with increased progesterone concentration. Three-dimensional cultures could be maintained significantly longer than 2D cultures, and the morphology of 3D cultures appeared similar to native cervical tissue. Thus, further studies were performed in 3D culture. To determine the effect of progesterone concentration, the 3D scaffolds were cultured with estradiol (10(-8) M) and five conditions: vehicle; 10(-9), 10(-8), or 10(-7) M progesterone; or 10(-7) M progesterone plus 10(-6) M mifepristone. The highest progesterone concentration correlated with the least amount of collagen synthesis. Collagen synthesis progressively increased as progesterone concentration decreased. This effect was partially antagonized by mifepristone, suggesting the mechanism is mediated by the progesterone receptor. This hormonally responsive 3D culture system supports the hypothesis that progesterone has a direct effect on remodeling cervical ECM during pregnancy. The 3D culture system could be useful for studying the mechanism of progesterone effects on the cervix.

223: Progesterone inhibits cervical tissue formation in a 3D culture system in a dose-dependent fashion

3D culture system in a dose-dependent fashion Michael House, Serkalem Tadesse, Errol Norwitz, David Kaplan Tufts Medical Center, Maternal Fetal Medicine, Boston, MA, Tufts University, Biomedical Engineering, Medford, MA, Yale University, Obstetrics and Gynecology, New Haven, CT OBJECTIVE: Progesterone supplementation can prevent preterm birth in some women with cervical shortening, suggesting that progesterone may directly affect remodeling of cervical tissue. A previously described 3D culture system was used to test progesterone effects on cervical tissue synthesis. STUDY DESIGN: Fibroblasts were obtained from cervical biopsies of non-pregnant premenopausal women undergoing hysterectomy for benign disease. Fibroblasts from passage 5 were seeded on silk sponge scaffolds and cultured in spinner flasks using previously validated culture system (House, Tissue Eng Part A, 2012). Culture media included DMEM 5% charcoal-stripped FBS estradiol 10-8M. Five progesterone conditions were used: 0 (vehicle), 10-9M, 10-8M, 10-7M and 10-7M 10-6M mifepristone. Scaffolds were cultured for 4 weeks and assayed for collagen production (hydroxyproline), histology and immunohistochemistry (IHC). Cervical-like tissue was assayed for estrogen and progesterone receptors using IHC and western blot. Experiments were repeated in triplicate with cells from 3 different women. RESULTS: Cervical cells reliably synthesized cervical-like tissue in 3D. Both progesterone and estrogen receptors were documented by IHC and western blot. A dose-dependent inhibitory effect of progesterone was observed. The highest progesterone concentration (10-7M) was associated with the least amount of collagen synthesis. Collagen synthesis increased progressively as progesterone concentration decreased (p .01). This effect was abrogated by mifepristone (p .05). Differences in tissue morphology on histology and IHC correlated with collagen synthesis. CONCLUSION: Progesterone inhibited the synthesis of cervical-like tissue in 3D culture from cervical fibroblasts in a dose-dependent manner. This effect was abrogated by mifepristone, a progesterone receptor antagonist. This hormonally-responsive in vitro culture system could be used to study the mechanism of progesterone effects on the cervix. 224 Role of (pro)rennin and its receptor in pathogenesis of preeclampsia: a rat model and human patients studies Kelsey Kelso, Russell Fothergill, Steven Allen, Richard Jones, Thomas Kuehl, Mohammad Uddin Scott & White Memorial Hospital / Texas AM placental (pro)renin for NP:152 79 and PreE:302 42 ng/g tissue). In addition to serving as a source of (pro)renin, the placenta is also a site for signaling as ERK1/2 phosphorylation is greater (p 0.05) in placental tissue of preE rats. CONCLUSION: Together with the upregulation of ERK1/2 phosphorylation in placenta of the rat model, there is now evidence of (pro)renin and its receptor associated novel RAS activation to play a role in preE pathogenesis through (pro)renin receptor-mediated detrimental cellular signaling at the placental boundary. This offers an opportunity for interventional treatments with signal inhibitors.

Progesterone and calcitriol attenuate inflammatory cytokines CXCL1 and CXCL2 in ovarian and endometrial cancer cells

Cytokines/chemokines are key players in cancer-related inflammation. Increasing evidence suggests that chemokines produced by tumor cells are the mediators of metastasis. Thus, agents that can downregulate chemokines expression have potential against cancer metastasis. We have previously shown inhibition of ovarian and endometrial cancer cell growth with progesterone and calcitriol. In the present study, we evaluated the effect of these two agents on the expression of inflammatory genes. Using a RT-PCR array of inflammatory cytokines/chemokines and their receptors, we found a marked attenuation of CXCL1 and CXCL2 (GRO-α and -β) in cancer cells by both treatments. Knockdown of NFκB resulted in a reduced expression of CXCL1 and CXCL2 and the inhibitory effect of progesterone and calcitriol on the expression of chemokines was abrogated in NFκB-silenced cancer cells. Silencing of IκBα increased the expression of CXCL1 and CXCL2 in cancer cells, which can be attributed to the increased activation of NFκB-p65, caused by the lack of its inhibitor. Progesterone and calcitriol-induced inhibition was abolished in IκBα-knockdown cells. Our results demonstrate that suppression of IκBα phosphorylation by progesterone and calcitriol contributes to the reduced expression of CXCL1 and CXCL2. Downregulation of CXCL1 and CXCL2 was associated with a marked inhibition of metastasis-promoting genes. Overall, our results indicate that progesterone and calcitriol inhibit IκBα phosphorylation, NFκB activation, and the expression of NFκB regulated metastasis promoting genes. These results provide attractive data for the possible use of progesterone and calcitriol in the management of endometrial and ovarian tumors.

Abstract 1412: Progesterone and calcitriol attenuate inflammatory cytokines CXCL1 and -2 in endometrial and ovarian cancer cells

Abstract Chronic inflammation has been shown to play a critical role in growth of cancers. NF-κB is a transcriptional factor that regulates diverse biological processes, including cell proliferation, survival and inflammation. We have previously shown inhibition of endometrial and ovarian cancer cell growth with progesterone and calcitriol. In the present study, we evaluated the effect of progesterone and calcitriol on the expression of inflammatory genes. Using RT-PCR array of inflammatory cytokine and receptor genes, we found two inflammatory cytokines, CXCL1 and -2 (GRO-A and -B), downregulated by both treatments. PCR array results were validated by Western blotting. Knockdown of NF-kB resulted in a reduced expression of CXCL1 and -2 and the inhibitory effect of progesterone and calcitriol on the expression of cytokines was abrogated in NF-kB-silenced cancer cells. Silencing of IkBα increased the expression of CXCL1 and -2 in cancer cells, which can be attributed to the increased activation of NF-kB-p65, caused by the lack of its inhibitor. Progesterone and calcitriol-induced inhibition was abolished in IkBα-knockdown cells. Our results demonstrate that suppression of IkBα phosphorylation by progesterone and calcitriol contributes to the reduced expression of CXCL1 and -2. We investigated the effect of CXCL1 and -2 downregulation on metastasis-promoting genes and found a marked inhibition of CXCR4, COX2 and matrix metalloproteinases (MMP2 and MPP-9). Overall, our results indicate that progesterone and calcitriol downregulate NF-kB in cancer cells, leading to the suppression of metastasis, thus providing the molecular basis for the treatment of endometrial and ovarian cancer patients with progesterone and calcitriol. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1412. doi:1538-7445.AM2012-1412

The inhibitory effect of progesterone on lactogenesis during pregnancy is already evident by mid- to late gestation in rodents

Lactogenesis is a very complex process highly dependent on hormonal regulation. In the present study the time-course of the inhibitory actions of progesterone on prolactin secretion, mammary gland morphology and lactogenesis from mid- to late gestation in rodents was investigated. Groups of pregnant rats were luteectomised or administered with mifepristone on Day 10, 13, 15 or 17 of gestation and decapitated 28 or 48h later. Whole-blood samples and the inguinal mammary glands were taken for determinations of hormone levels and for measurement of mammary content of casein and lactose and for tissue morphology analyses, respectively. Luteectomy or mifepristone evoked prolactin increases only after Day 17 of gestation. Mammary content of casein was increased by both treatments regardless of timing or duration. Mifepristone was less effective than luteectomy in inducing lactose production and the effect was only observed after Day 15 of gestation. Analysis of mammary gland morphology confirmed the observed effect of progesterone on lactogenesis. Both treatments triggered remarkable secretory activity in the mammary gland, even without a parallel epithelial proliferation, demonstrating that the mammary epithelium is able to synthesise milk compounds long before its full lobulo-alveolar development is achieved, provided that progesterone action is abolished. Thus, the present study demonstrates that progesterone is a potent hormonal switch for the prolactin and prolactin-like effects on mammary gland development and its milk-synthesising capacity during pregnancy, and that its inhibitory action is already evident by mid-pregnancy in rodents.

Neither the Basic Helix-Loop-Helix Transcription Factor Hand2 Nor the MAP Kinase Pathway Are Involved in Progesterone Effects on Cervical Epithelium in the Mouse.

Stroma-epithelial crosstalk is necessary for growth, regulation of proliferation and normal function of reproductive tissues. A recent study reported that the basic helix-loop-helix transcription factor Hand2 is stimulated by progesterone in uterine stroma, and is essential for progesterone-mediated inhibition of estrogen-induced epithelial proliferation in mouse uterus. Progesterone-induced inhibition of epithelial proliferation by Hand2 was mediated through suppression of members of the fibroblast growth factor family, which led to inactivation of the Erk/MAP kinase signaling pathway normally stimulated by 17β-estradiol. During the first half of pregnancy, the uterine cervix remains rigid and compact, but grows extensively during the second half, a process regulated by 17β-estradiol and progesterone. In this study, we investigated the role of Hand2 in the mouse cervix and determined whether the effects of progesterone in the cervix were dependent on this transcription factor as they are in the uterus. To evaluate whether Hand2 was involved in progesterone-mediated inhibition of cervical growth, reproductive tracts from female mice were collected on day 8 of pregnancy and from ovariectomized virgin females that received various regimens of hormone replacement. For both groups, immunohistochemistry was performed to assess expression of the following proteins: Hand2, phospho-ERK (an indication of MAP kinase activation), and MKi-67 (a marker for cell proliferation). Consistent with the previous report, Hand2 was expressed in uterine stroma during early pregnancy and in ovariectomized mice following progesterone treatment. Surprisingly, Hand2 expression in the stroma ceased abruptly and totally at the uterine-cervical junction; Hand2 was not expressed in epithelium of either organ. Similar results were seen with the ovariectomized females. Thus, Hand2 seems to be specific to the uterus, since no expression was observed in stroma at or beyond the uterine-cervical junction. Further, phospho-ERK expression was high in the cervical epithelium of ovariectomized mice treated with 17β-estradiol, and similarly high phospho-ERK expression was seen in the cervical epithelium of 17β-estradiol + progesterone treated mice. This contrasts sharply with previous results obtained in the uterus, where 17β-estradiol induces marked increases in phospho-ERK, but progesterone treatment is capable of essentially abolishing this increase. Thus, it appears that despite the anatomical proximity of the uterus and cervix, the inhibitory effect of progesterone on uterine epithelial proliferation mediated through induction of stromal Hand2 that secondarily inhibits epithelial production of phospho-ERK is totally different than what are seen in the cervix following progesterone treatment, where neither Hand2 nor phospho-ERK, and the MAP kinase signaling pathway, appear to be progesterone targets. (poster)

Evidence that the Arcuate Nucleus Is an Important Site of Progesterone Negative Feedback in the Ewe

There is now considerable evidence that dynorphin neurons mediate the negative feedback actions of progesterone to inhibit GnRH and LH pulse frequency, but the specific neurons have yet to be identified. In ewes, dynorphin neurons in the arcuate nucleus (ARC) and preoptic area (POA) are likely candidates based on colocalization with progesterone receptors. These studies tested the hypothesis that progesterone negative feedback occurs in either the ARC or POA by determining whether microimplants of progesterone into either site would inhibit LH pulse frequency (study 1) and whether microimplants of the progesterone receptor antagonist, RU486, would disrupt the inhibitory effects of peripheral progesterone (study 2). Both studies were done in ovariectomized (OVX) and estradiol-treated OVX ewes. In study 1, no inhibitory effects of progesterone were observed during treatment in either area. In study 2, microimplants of RU486 into the ARC disrupted the negative-feedback actions of peripheral progesterone treatments on LH pulse frequency in both OVX and OVX+estradiol ewes. In contrast, microimplants of RU486 into the POA had no effect on the ability of systemic progesterone to inhibit LH pulse frequency. We thus conclude that the ARC is one important site of progesterone-negative feedback in the ewe. These data, which are the first evidence on the neural sites in which progesterone inhibits GnRH pulse frequency in any species, are consistent with the hypothesis that ARC dynorphin neurons mediate this action of progesterone.

Differential Effects of Progesterone on COX-2 and Mn-SOD Expressions Are Associated with Histone Acetylation Status of the Promoter Region in Human Endometrial Stromal Cells

Progesterone differently regulates TNFα-induced gene expression of cyclooxygenase-2 (COX-2) and manganese superoxide dismutase (Mn-SOD) in human endometrial stromal cells (ESC). The present study investigated the mechanisms by which TNFα and progesterone affect the expressions of COX-2 and Mn-SOD in ESC. ESC were incubated with TNFα and progesterone. COX-2 and Mn-SOD mRNA expression was determined by real-time RT-PCR. Nuclear factor (NF)-κB binding to the promoter region or histone acetylation status of the NF-κB response element was analyzed by a chromatin immunoprecipitation assay. TNFα increased COX-2 and Mn-SOD mRNA levels. Progesterone (10(-6) M) suppressed TNFα-induced COX-2 mRNA expression, whereas TNFα-induced Mn-SOD expression was not inhibited by progesterone. The inhibitory effect of progesterone was abolished by knockdown of progesterone receptors by small interfering RNA. Chromatin immunoprecipitation assay revealed that TNFα increased NF-κB binding at both the COX-2 promoter and the Mn-SOD enhancer and that progesterone inhibited only the NF-κB binding at the COX-2 promoter. The histone acetylation level of the NF-κB response element of the Mn-SOD enhancer was lower than that of the COX-2 promoter. However, when histone acetylation was induced by histone deacetylase inhibitors, progesterone inhibited the TNFα-induced NF-κB binding to the Mn-SOD enhancer. TNFα increased COX-2 and Mn-SOD expression via NF-κB activation. Progesterone inhibited COX-2 expression by inhibiting the binding of NF-κB to its response element but did not inhibit TNFα-induced Mn-SOD expression. The gene-specific action of progesterone may be due to the difference in chromatin structure at the NF-κB response elements in the COX-2 promoter and Mn-SOD enhancer.

Abstract 1603: The VEGF, PDGF and STAT3 pathways are the potential therapeutic targets to treat endometrial cancer

Abstract Progesterone inhibits growth of endometrial cancer through its receptors A (PRA) and B (PRB) although the molecular mechanisms of inhibition have yet to be fully characterized. A global analysis of gene regulation in human endometrial cancer cells that express heterologous progesterone receptors was performed to identify the candidate genes that mediate inhibitory effects of progesterone in endometrium. Data indicate that progesterone down-regulates the expression of vascular endothelial growth factor A (VEGFA), platelet derived growth factor A (PDGFA) and signal transducer and activator of transcription 3 (STAT3). Down-regulation of VGFA is mediated by the PRA whereas both PRA and PRB are required to attenuate expression of PDGFA and STAT3 genes. These results suggest that progesterone exerts the growth-limiting effects in endometrium via inhibition of angiogenesis and proliferation. VGFA, PDGFA and STAT3 pathways may be central to the inhibitory effects of progesterone in endometrial cancer and may constitute the important targets for therapeutic interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1603.

Sexual experience and temperament affect the response of Merino ewes to the ram effect during the anoestrous season

In seasonally anoestrous ewes of many breeds, the introduction of rams triggers an increase in gonadotrophin secretion that induces ovulation, a phenomenon known as the ‘ram effect’. The ram effect is a practical method for mating ewes outside the natural breeding season, and also can provide synchronised lambing, but the variability of the response, especially in young animals, reduces its potential for widespread application. The aim of our study was to assess two factors that are thought to contribute to the variability in young ewes: temperament and sexual experience. We used anovulatory ewes from a flock that had been genetically selected for ‘calm’ or ‘nervous’ temperament and compared the endocrine and ovarian responses to the ram effect in four groups (each n = 15): ‘calm’ and parous (3–6 years old); ‘calm’ and nulliparous (2 years old); ‘nervous’ and parous; and ‘nervous’ and nulliparous. Parous ewes, independently of their temperament, exhibited a faster endocrine response and a higher proportion of females cycling after ram introduction than nulliparous ewes. ‘Nervous’ ewes exhibited a higher proportion of females cycling after ram introduction than calm ewes, but only in the nulliparous group. We conclude that temperament exerts little influence on the response to the ram effect in sexually experienced ewes, and that females of ‘nervous’ temperament appear to respond better when sexually ‘naive’. Both sexual experience and temperament need to be taken into consideration when flock management involves the ram effect. Finally, some ewes were cyclic at ram introduction, yet exhibited an increase in LH secretion even in the presence of high concentrations of progesterone. The mechanism by which the inhibitory effect of progesterone on LH secretion was bypassed needs to be clarified.