Physiological Concentrations Of Progesterone Research Articles

Progesterone induces sperm release from oviductal epithelial cells by modifying sperm proteomics, lipidomics and membrane fluidity

The sperm reservoir is formed after insemination in mammals, allowing sperm storage in the oviduct until their release. We previously showed that physiological concentrations of progesterone (P4) trigger in vitro the sperm release from bovine oviductal epithelial cells (BOECs), selecting a subpopulation of spermatozoa with a higher fertilizing competence. Here, by using Western-Blot, confocal microscopy and Intact Cell MALDI-TOF-Mass Spectrometry strategies, we elucidated the changes derived by the P4-induced release on sperm cells (BOEC-P4 spz). Our findings show that, compared to controls, BOEC-P4 spz presented a decrease in the abundance of Binder of Sperm Proteins (BSP) −3 and −5, suggesting one mechanism by which spermatozoa may detach from BOECs, and thus triggering the membrane remodeling with an increase of the sperm membrane fluidity. Furthermore, an interesting number of membrane lipids and proteins were differentially abundant in BOEC-P4 spz compared with controls.

Steroids affect gene expression, ciliary activity, glucose uptake, progesterone receptor expression and immunoreactive steroidogenic protein expression in equine oviduct explants in vitro.

The oviduct undergoes dramatic functional and morphological changes throughout the oestrous cycle of the mare. To unravel the effects of steroids on the morphology, functionality and gene expression of the equine oviduct, an in vitro oviduct explant culture system was stimulated with physiological concentrations of progesterone and 17β-oestradiol. Four conditions were compared: unsupplemented preovulatory explants, preovulatory explants that were stimulated with postovulatory hormone concentrations, unsupplemented postovulatory explants and postovulatory explants that were stimulated with preovulatory hormone concentrations. The modulating effects of both steroids on oviduct explants were investigated and the following parameters examined: (1) ciliary activity, (2) glucose consumption and lactate production pattern, (3) ultrastructure, (4) mRNA expression of embryotrophic genes, (5) steroidogenic capacities of oviductal explants and (6) progesterone receptor expression. The present paper shows that the equine oviduct is an organ with potential steroidogenic capacities, which is highly responsive to local changes in progesterone and 17β-oestradiol concentrations at the level of morphology, functionality and gene expression of the oviduct. These data provide a basis to study the importance of endocrine and paracrine signalling during early embryonic development in the horse.

Immunosuppressive Biological Mechanisms Support Reassessment of Use of the Injectable Contraceptive Medroxyprogesterone Acetate

Whether or not hormonal contraceptives increase the risk of HIV-1 acquisition and transmission and AIDS disease progression is a crucial public health question. There is good reason to focus in particular on the effects of the injectable contraceptive medroxyprogesterone acetate (MPA) which is administered as a 150-mg 3-monthly intramuscular injection and is referred to as Depo-Provera or depot MPA (DMPA). DMPA is used by over 50 million women worldwide particularly in areas with high HIV-1 prevalence. The central hypothesis examined in this paper is that MPA more so than physiological concentrations of progesterone (P4) is likely to repress immune function at doses within the range found in the serum of DMPA users due to its potent glucocorticoid-like immunosuppressive properties which could influence the pathogenicity of infectious diseases. Taken together the key results from the study provide important insights into biological mechanisms of MPA in primary human cells and are likely to be highly significant for HIV-1 infectivity and transmission and AIDS progression. The results provide motivation for further research into this important question.

Regulation of ERα Protein Expression by 17β-Estradiol in Cultured Neurons of Hypothalamic Ventromedial Nucleus

The activation of the subtype α of estrogen receptors (ERα) in the hypothalamic ventromedial nucleus (VMNvl) is required to stimulate female sexual receptivity. Moreover, the hormone was found to govern the expression of the receptor. Its removal due to ovariectomy and subsequent substitution suggest that the hormone down-regulates the expression of ERα. In contrast, in normally cycling animals the expression of the receptor peaks at proestrus, the phase of highest concentration of 17β-estradiol in estrous cycle. Therefore, in this study we examined the influence of the hormone on ERα expression in primary dissociated cultures of neurons isolated from the VMNvl of young adult female rats. Measurements of ERα immunofluorescence revealed that both supraphysiological and physiological concentrations of 17β-estradiol increase the expression of ERα. Analyses with selective agonists showed that both nuclear ERs are able to mediate the action of the hormone. However, the activation of ERα had a stronger effect on the expression of its own receptor than the activation of ERβ. Simultaneous activation of both receptors attenuated the influence of ERα alone. Physiological concentrations of progesterone were found to revoke the effect of 17β-estradiol, whereas the expression of ERα is up-regulated by progesterone alone. These data indicate that the expression of ERα in VMNvl neurons is under the control of both types of nuclear ERs and, in addition, progesterone receptors (PRs). The particular contribution of the receptors is dependent on their level of expression and the hormonal context. In neurons expressing high quantity of ERα, ERβ attenuates the overall expression of the receptor, whereas in cells containing mostly ERβ it contributes to the up-regulation of ERα synthesis. Simultaneous activation of ERs and PRs reverses the influences of the receptors due to inter-inhibition of their transcriptional activities.

Progesterone modulates aggression in sex-role reversed female African black coucals.

Testosterone is assumed to be the key hormone related to resource-defence aggression. While this role has been confirmed mostly in the context of reproduction in male vertebrates, the effect of testosterone on the expression of resource-defence aggression in female vertebrates is not so well established. Furthermore, laboratory work suggests that progesterone inhibits aggressive behaviour in females. In this study, we investigated the hormonal changes underlying territorial aggression in free-living female African black coucals, Centropus grillii (Aves; Cuculidae). Females of this sex-role reversed polyandrous bird species should be particularly prone to be affected by testosterone because they aggressively defend territories similar to males of other species. We show, however, that territorial aggression in female black coucals is modulated by progesterone. After aggressive territorial challenges female black coucals expressed lower levels of progesterone than unchallenged territorial females and females without territories, suggesting that progesterone may suppress territorial aggression and is downregulated during aggressive encounters. Indeed, females treated with physiological concentrations of progesterone were less aggressive than females with placebo implants. This is one of the first demonstrations of a corresponding hormone-behaviour interaction under challenged and experimental conditions in free-living females. We anticipate that our observation in a sex-role reversed species may provide a more general mechanism, by which progesterone--in interaction with testosterone--may regulate resource-defence aggression in female vertebrates.

Identification of Friend murine retrovirus-infected immune cells and studies of the effects of sex and steroid hormones in the early phase of infection.

Male mice are more susceptible than female mice to the murine retrovirus FIS-2. We previously reported that sex-related factors influence early virus replication via mechanisms involving a glucocorticoid response element (GRE) in the long terminal repeat (LTR) enhancer region. In the present study, we investigated further the influence of sex and steroid hormones on early murine retrovirus dissemination and immune functions. In male mice we found a correlation between an early expansion of the CD8+ cell subset and rapid infection of lymphocytes, including CD8+ cells. Virus load in blood declined faster in females than in males, and the postpeak declines coincided with more rapidly generation of antibodies against virus-positive cells. Moreover, female-derived T-cells responded better to in vitro mitogen stimuli than male-derived T-cells. Physiological concentrations of progesterone and dexamethasone induced a dose-dependent inhibition of T-cell proliferation. Administration of progestin in vivo did not modify early FIS-2 production in female mice. Male castrated mice, who were notably less involved in aggressive behaviour and fighting compared to male control mice, had a significant delay of virus dissemination. We suggest that testosterone-dependent aggression, with successive enhanced stress hormone levels, may influence both FIS-2 replication and immune responses during infection.

Oestradiol Stimulates Morphological and Functional Differentiation of Human Villous Cytotrophoblast

Trophoblast differentiation is a complex process involving interactions of cytotrophoblastic cells with their evolutive milieu. During pregnancy, the feto-placental unit produces large amounts of steroids. Progesterone and oestradiol are increasingly produced when the syncytiotrophoblast is highly differentiated. Furthermore, receptors to these hormones are expressed by the trophoblast. This led us to test the hypothesis that steroid production could affect the morphological and functional differentiation of the trophoblast during gestation.The fusion of cytotrophoblastic cells into syncytiotrophoblast was assessed using fluorescence recovery after photobleaching for gap junctional communication analysis (gap-FRAP), desmoplakin immunostaining and connexin 43 expression. In parallel, functional differentiation was assessed by β-human chorionic gonadotrophin (βhCG) production and human chorionic somatomammotropin (hCS) expression analysis. The presence of oestradiol, 1μm, increased the percentage of coupled cells (3.8-fold), connexin 43 expression and stimulated the syncytium formation. In parallel, oestradiol (1, 3 and 5μm) induced a significant increase in the daily hCG production. The steroid action was specific, as the stimulatory effects were inhibited by tamoxifen. Oestradiol also stimulated hCS expression (51 per cent compared to control after 3 days). As trophoblastic differentiation is specifically stimulated by hCG, oestradiol could act via the stimulation of hCG production or via a direct action. In the presence of an efficient concentration of hCG antibody, oestradiol still stimulated hCS expression, suggesting a self-sufficient effect of the steroid. Physiological concentrations of progesterone were ineffective in modulating trophoblast differentiation.In conclusion, oestradiol could be implicated in the maturation and aging of the trophoblast.

Progesterone increases the production of tissue inhibitor of metalloproteinases-2 in rabbit uterine cervical fibroblasts

Rabbit uterine cervical fibroblasts in culture produces tissue inhibitor of metalloproteinases (TIMP)-l and TIMP-2. When cells were treated with physiological concentrations of progesterone, the production of two TIMPs increased, and essentially all TIMP-2 was found to be complexed with promatrix metalloproteinase 2 (proMMP-2)/progelatinase A. Progesterone did not modulate the production of proMMP-2 and resulted in the increased total amount of proMMP-2-TIMP-2 complex. These observations provide the first evidence that progesterone participates in maintaining the homeostasis of connective tissue matrix in uterine cervix by augmenting both TIMP-1 and TIMP-2 production along with the known suppressive effects on the proMMP-1 and proMMP-3 production.

Progesterone regulates the activity of collagenase and related gelatinases A and B in human endometrial explants.

Explants of human endometrium were cultured to study the release of matrix metalloproteinases (MMPs). Analysis of conditioned media by zymography revealed latent and active forms of collagenase (MMP-1, EC 3.4.24.7), 72-kDa gelatinase A (MMP-2, EC 3.4.24.24), and 92-kDa gelatinase B (MMP-9, EC 3.4.24.35). These proteinases were identified by their M(r), their inhibition by tissue inhibitor of metalloproteinases, and the activation of their zymogens by trypsin or aminophenylmercuric acetate. In the absence of sex hormone, explants released large amounts of enzyme activities, as measured by densitometry of zymograms or in soluble assays. Physiological concentrations of progesterone (10-200 nM) almost totally abolished the release of collagenase, of total gelatinase activity, and of the active form of gelatinase B and largely inhibited the release of the active form of gelatinase A. These effects, which were antagonized by mifepristone (RU 38486), suggest that progesterone restrains endometrial tissue breakdown by blocking the secretion and activation of MMPs.

Progesterone enhancement of prostaglandin E2 production by fetal placental macrophages.

The role of progesterone in immunoregulation at the fetomaternal interface at physiological concentrations (2-6 micrograms/ml) is controversial. This study examines the effect of progesterone on maternal lymphocyte proliferation in a one-way mixed lymphocytic reaction (MLR) stimulated by allogenic lymphocytes as well as on prostaglandin production and secretion by fetal placental macrophages. No suppressive effect on maternal lymphocyte proliferation was found at progesterone levels of up to 20 micrograms/ml. However, physiological concentrations of progesterone were found to induce a significant increase in the fetal macrophage release of PGE2, which is well known as a strong immunosuppressant. PGI2 production and secretion by these cells, measured by the appearance of its 6-keto-PGF1 alpha product, was not affected by incubation with progesterone. Enhancement of PGE2 secretion by progesterone may partly explain the roles of progesterone and fetal placental macrophages in immunosuppression at the fetomaternal interface.

Modulation of lactogenic receptors by progestins in cultured human breast cancer cells.

Progesterone receptors (PgR) are present in many breast cancers, but few specific actions of progestins in breast cancer cells have been reported. We now report that progestins specifically modulate lactogenic receptor expression in cultured T-47D and MCF-7 human mammary carcinoma cells. When T-47D cells were preincubated for 24 h with 1 nM medroxyprogesterone acetate (17 alpha-acetoxy-6 alpha-methyl-4-pregnene-3,20-dione), specific binding of [125I]human GH ([125I]hGH) and [125I]human PRL was increased to 205 +/- 22% (+/- SE) (P less than 0.01) and 175 +/- 32% (P less than 0.05), respectively, of that in control cultures. There was no significant effect on cell number and no significant enhancement of specific binding of [125I]porcine insulin, [125I]salmon calcitonin, [125I]human transferrin, or [3H] Concanavalin A. Lactogenic receptor number was increased from 6,490 +/- 500 (n = 12) to 13,180 +/- 3,270 (n = 7; P less than 0.01) sites/cell, with no significant change in affinity for hGH. Progesterone, which is readily metabolized by these cells, was less potent than the synthetic progestins (medroxyprogesterone acetate, R 5020 (17 alpha, 21-dimethyl-19-norpregn-4,9-diene-3,20-dione), and ORG 2058 (16 alpha-ethyl-21-hydroxy-19-norpregn-4-en-3,20-dione), but physiological concentrations of progesterone (1 nM) significantly enhanced specific binding of [125I]hGH to 153 +/- 23% of the control value (n = 6; P less than 0.05). Physiological concentrations of androgens, estrogens, and glucocorticoids had no significant effect. MCF-7 cells were considerably less sensitive to these effects of progestins than T-47D cells, probably due to the lower PgR concentration in MCF-7 cells. These observations, which indicate that lactogenic receptor expression is controlled, at least in part, by progestins in these mammary carcinoma cell lines, may have important implications in the management of human breast cancer, where high levels of this receptor may reflect a functional PgR and a highly hormone-dependent phenotype.

In vitro modulation of prolactin binding to human mammary carcinoma cells by steroid hormones and prolactin.

This study demonstrates the specific binding of human (h) PRL to mammary carcinoma cells of the newly established line EFM-19. Under saturating conditions, [125I]hPRL bound to these cells with high affinity (Ka = 4.3 X 10(10) M-1) and low capacity (4080 binding sites/cell). In serum-free medium, stimulation of cell proliferation by PRL was found, suggesting a biological role in the growth of human breast cancer. hPRL was more effective in binding to and promoting the growth of EFM-19 cells than ovine PRL or other lactogenic hormones. Up- and down-regulation of [125I]hPRL binding occurred after pretreatment of EFM-19 cell cultures with subphysiological or higher hPRL concentrations, respectively. Physiological concentrations of 17 beta-estradiol or dihydrotestosterone increased the cellular capacity of hPRL binding. Pharmacological concentrations of dihydrotestosterone or physiological concentrations of progesterone reduced the binding of [125I]hPRL. The results provide evidence for complex regulatory mechanisms of PRL binding by human mammary carcinoma cells involving steroid hormones.

Hydrocortisone--a possible physiological regulator of human granulopoiesis.

The effect of hydrocortisone (HC) on the formation of granulocyte-macrophage (GM) colonies was assessed in vitro in cultures of normal human bone marrow. Subphysiological concentrations (less than or equal to 10(-8) mol/l) of HC had no impact on GM colony formation, but at physiological and therapeutic levels (10(-7) mol/l-10(-5) mol/l) the hormone stimulated the production of an increased number of colonies. By contrast, physiological concentrations of progesterone (10(-9) mol/l-10(-7) mol/l) were without effect and even at 10(-6) mol/l there was no influence of progesterone alone on GM colony formation. However, progesterone at 10(-6) mol/l inhibited the stimulant effect of HC to a degree which was related inversely to the concentration of HC and the duration of the culture interval. The data from this study suggest that the neutrophilia which accompanies glucocorticosteroid therapy is matched by an increase in the production of granulocytes and that HC may play a rôle, which is mediated by steroid receptors, in the physiological regulation of human granulopoiesis.

Evidence that changes in tonic luteinizing hormone secretion determine the growth of preovulatory follicles in the rat.

Physiological concentrations of progesterone (20-100 ng/ml), maintained by the insertion of implants into 30-day-old rats, delayed first ovulation, and withdrawal of progesterone on day 47 of age synchronized first ovulation in rats. Inhibition of ovulation involved negative feedback regulation of tonic LH and FSH secretion, blockage of gonadotropin surges, and suppression of preovulatory, but not antral, follicular growth. Removal of implants resulted in a rapid decline in serum progestrone from 100 to 5 ng/ml within 0-12 h. Between 0-36 h there were progressive increases in serum concentrations of LH and FSH, enhanced accumulation of estradiol by individual follicles incubated in vitro with or without exogenous substrate, and marked progressive increases in the content of LH (but not FSH) receptors in both thecal and granulosa cells. These events were followed by gonadotropin surges at 48 h (1800 h on day 49), ovulation, and morphological and biochemical signs of luteinization, including decreases in follicular gonadotropin receptor content and estradiol accumulation, evident by 60 h. With the exception of changes in basal LH, this sequence of events is remarkably similar in time and pattern to that after the decline of progesterone on diestrous day 2 and ovulation on proestrus of a 5-day cycle. Although a direct effect of progesterone on ovarian follicular cell function cannot be excluded, the data suggest that subtle but sustained increases in LH (and possibly FSH) are required for the enhanced follicular accumulation of estradiol and LH-binding activity occurring between diestrus and proestrus of the rat estrous cycle. Thus, perhaps some of the mystery surrounding the endocrine events between diestrus and proestrus can be ascribed to changes in serum LH that have been too small and/or variable for current nonserial sampling methods and RIAs to detect reliably.

In vitro actin of progesterone on myometrium: I. Reversible modulation of the resistance of rabbit uterus to excitation-contraction uncoupling.

Active isometric tension provoked in isolated uterine strips by optimal electric field stimulation is an index of sarcoplasmic Ca++ concentration or the degree of excitation-contraction coupling in the myometrium. Removal of Ca++ from the bathing medium, while maintaining electric stimulation, results in a progressive uncoupling and a consequent loss of tension. The rate at which tension is lost by the uterine strips can be influenced by progesterone. When uterine strips from rabbits at Day 26 of pregnancy are incubated in tissue culture medium containing no added progesterone, there is a time dependent reduction in resistance to uncoupling. This response can be prevented in a log-dose manner by incubation in medium containing near physiological concentrations of progesterone while nonprogestagenic C21 steroids are ineffective. Incubation without steroid for 6 h followed by addition of progesterone results in a reversal of the initial loss of resistance to uncoupling. This reversal occurs after a latent period of ∼ 4 h. Because resistance to uncoupling is related to the availability of Ca++ in the vicinity of the sarcolemma, this study demonstrates that the association between the sarcolemma and Ca++ can be altered by progesterone in vitro.

Uterine excitability and distensibility influenced by treatment [formula omitted] with progesterone

Myometrial strips excised from rabbits at day 25 or 26 of pregnancy develop the same level of active isometric tension as those obtained at parturition when tetanized under optimal conditions in Ca ++-replete medium. When Ca ++ is selectively removed from the medium, active tension decays and the rate of this uncoupling reflects the progestational state of the myometrium. Strips from pregnancy which are under the influence of progesterone display a resistance to uncoupling relative to the rapid rate of uncoupling of the uterine strips obtained at term. It has been proposed that the progesterone-influenced uterus possesses a pool of Ca ++-binding sites in the sarcolemma and that this reserve of Ca ++ protects the strip from the uncoupling effects of removing the ion from the bathing medium. This expression of the action of the steroid can be predictably altered by incubating uterine strips in culture medium with or without added progesterone. In the absence of added steroid, the strips metabolize the endogenous progesterone and the rate of uncoupling is accelerated over the course of a few hours. Resistance to uncoupling is maintained by near physiological concentrations of progesterone in the medium. The effect of incubation without added progesterone can subsequently be reversed by delayed exposure to the steroid. The reversal process displays a 2–4 hour latency but is complete by 6 hours. This action of progesterone can be mimicked by exposure of the strips to cycloheximide or actinomycin D. The rapidity of action of these agents in establishing high resistance to uncoupling is cycloheximide > actinomycin D > progesterone. Progesterone appears to maintain high resistance to uncoupling by a tonic inhibition of transcriptional events that would otherwise lead to the sarcolemmal changes normally associated with the inhibition of labor. Steroid treatments imposed in vitro also alter resting length-tension relationships in myometrial strips. During mid to late pregnancy, the strips are very distensible and contrast with the relatively indistensible strip collected at parturition. Strips obtained during days 21 – 29 of pregnancy then incubated without progesterone acquire the indistensible properties of strips taken at parturition. Exposure to progesterone in vitro maintains distensibility but this action is progressively overcome in a dose-related fashion by incubation with bacterial collagenase. These findings indicate that changes in the organization of the collagen in the uterine strips markedly affect distensibility and suggest that progesterone may act to inhibit collagenolysis. Distensibility can be maintained by exposure to porcine relaxin in vitro , despite the absence of progesterone. The results suggest that hormonally-controlled changes in the myometrial connective tissue may contribute to the characteristic changes in the properties of the uterus attending the initiation of labor.