Heme oxygenase in the rat anterior pituitary: immunohistochemical localization and possible role in gonadotropin and prolactin secretion

The objectives of this study were to determine if heme oxygenase (HO), which catalyzes the degradation of heme and the formation of carbon monoxide (CO), is localized in the rat anterior pituitary and, if so, to determine if hemin (a substrate for HO) or chromium mesoporphyrin (CrMP) (an inhibitor of HO), alter pituitary gonadotropin and prolactin secretion. For localization of HO, sections of anterior pituitaries obtained from mature Holtzman Sprague-Dawley rats in different stages of the estrous cycle were immunostained for two of the HO isoforms, HO-1 and HO-2. The immunostaining for the inducible HO isoform (HO-1) was limited to discrete populations of pituitary cells, whereas the constitutive isoform (HO-2) had a more widespread distribution. The afternoon surge of leutinizing hormone (LH) in the plasma of ovariectomized, estradiol-treated rats was advanced by 2 hr after 7 days of treatment with CrMP (4 micro M/kg), and this effect was reversed when hemin (30 micro M/kg) was co-administered with CrMP. The afternoon follicle-stimulating hormone (FSH) surge was not affected by either treatment. In contrast, the afternoon prolactin (PRL) surge was completely blocked or delayed by CrMP treatment, and this effect was not reversed by hemin. In vitro perifusion of pituitary explants with CrMP also significantly reduced PRL release compared with secretion from untreated explants. In vitro gonadotropin-releasing hormone (GnRH)-stimulated FSH secretion was significantly increased from pituitary explants of ovariectomized, estradiol-treated rats treated in vivo with hemin but was unaffected by CrMP treatment, whereas GnRH-stimulated LH release was not affected by hemin but was increased by CrMP treatment. In conclusion, this study demonstrates that HO exists in the rat anterior pituitary gland, and that a substrate and an inhibitor of this enzyme alter the secretion of gonadotropins and PRL.     

Effects of metformin on bovine granulosa cells steroidogenesis: possible involvement of adenosine 5' monophosphate-activated protein kinase (AMPK)

In mammals, IGFs are important for the proliferation and steroidogenesis of ovarian cells. Metformin is an insulin sensitizer molecule used for the treatment of the infertility of women with polycystic ovary syndrome. It is, however, unclear whether metformin acts on ovarian cells. Adenosine 5' monophosphate-activated protein kinase (AMPK) is involved in metformin action in various cell types. We investigated the effects of metformin on bovine granulosa cell steroidogenesis in response to IGF1 and FSH, and studied AMPK in bovine ovaries. In granulosa cells from small follicles, metformin (10 mM) reduced production of both progesterone and estradiol and decreased the abundance of HSD3B, CYP11A1, and STAR proteins in presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). In cows, the different subunits of AMPK are expressed in various ovarian cells including granulosa and theca cells, corpus luteum, and oocytes. In bovine granulosa cells from small follicles, metformin, like AICAR (1 mM) a pharmaceutical activator of AMPK, increased phosphorylation of both Thr172 of AMPK alpha and Ser 79 of ACACA (Acetyl-CoA Carboxylase). Both metformin and AICAR treatment reduced progesterone and estradiol secretion in presence or absence of FSH and IGF1. Metformin decreased phosphorylation levels of MAPK3/MAPK1 and MAPK14 in a dose- and time-dependent manner. The adenovirus-mediated production of dominant negative AMPK abolished the effects of metformin on secretion of progesterone and estradiol and on MAPK3/MAPK1 phosphorylation but not on MAPK14 phosphorylation. Thus, in bovine granulosa cells, metformin decreases steroidogenesis and MAPK3/MAPK1 phosphorylation through AMPK activation.     

Heme oxygenase-1 protects HepG2 cells against cytochrome P450 2E1-dependent toxicity

The inducible form of heme oxygenase (HO-1) is increased during oxidative injury and HO-1 is believed to be an important defense mechanism against such injury. Arachidonic acid (AA) and l-buthionine-(S,R)-sulfoximine (BSO), which lowers GSH levels, cause cytochrome P450 2E1 (CYP2E1)-dependent oxidative injuries in HepG2 cells (E47 cells). Treatment of E47 cells with 50 microM AA or 100 microM BSO for 48 h was recently shown to increase HO-1 mRNA, protein, and activity. The possible functional significance of this increase in protecting against CYP2E1-dependent toxicity was evaluated in the current study. The treatment with AA and BSO caused loss of cell viability (40 and 50%, respectively) in E47 cells. Chromium mesoporphyrin (CrMP), an inhibitor of HO activity, significantly potentiated this cytotoxicity. ROS production, lipid peroxidation, and the decline in mitochondrial membrane potential produced by AA and BSO were also enhanced in the presence of CrMP in E47 cells. Infection with an adenovirus expressing rat HO-1 protected E47 cells from AA toxicity, increasing cell viability and reducing LDH release. HO catalyzes formation of CO, bilirubin, and iron from the oxidation of heme. Bilirubin was not protective whereas iron catalyzed the AA toxicity. The carbon monoxide (CO) scavenger hemoglobin enhanced AA toxicity in E47 cells analogous to CrMP, whereas exposure to exogenous CO partially reduced AA toxicity and the enhanced AA toxicity by CrMP. Addition of exogenous CO to the cells inhibited CYP2E1 catalytic activity, as did overexpression of the rat HO-1 adenovirus. These results suggest that induction of HO-1 protects against CYP2E1-dependent toxicity and this protection may be mediated in part via production of CO and CO inhibition of CYP2E1 activity.     

Actions of epidermal growth factor receptor/mitogen-activated protein kinase and protein kinase C signaling in granulosa cells from Gallus gallus are dependent upon stage of differentiation

Studies in both mammalian and nonmammalian ovarian model systems have demonstrated that activation of the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways modulates steroid biosynthesis during follicle development, yet the collective evidence for facilitory versus inhibitory roles of these pathways is inconsistent. The present studies in the hen ovary describe the changing role of MAPK and PKC signaling in the regulation of steroidogenic acute regulatory protein (STAR) expression and progesterone production in undifferentiated granulosa cells collected from prehierarchal follicles prior to follicle selection versus differentiated granulosa from preovulatory follicles subsequent to selection. Treatment of undifferentiated granulosa cells with a selective epidermal growth factor receptor (EGFR) and ERBB4 receptor tyrosine kinase inhibitor (AG1478) both augments FSH receptor (Fshr) mRNA expression and initiates progesterone production. Conversely, selective inhibitors of both EGFR/ERBB4 and MAPK activity attenuate steroidogenesis in differentiated granulosa cells subsequent to follicle selection. In addition, inhibition of PKC signaling with GF109203X augments FSH-induced Fshr mRNA plus STAR protein expression and initiates progesterone synthesis in undifferentiated granulosa cells, but inhibits both gonadotropin-induced STAR expression and progesterone production in differentiated granulosa. Granulosa cells from the most recently selected (9- to 12-mm) follicle represent a stage of transition as inhibition of MAPK signaling promotes, while inhibition of PKC signaling blocks gonadotropin-induced progesterone production. Collectively, these data describe stage-of-development-related changes in cell signaling whereby the differentiation-inhibiting actions of MAPK and PKC signaling in prehierarchal follicle granulosa cells undergo a transition at the time of follicle selection to become obligatory for gonadotropin-stimulated progesterone production in differentiated granulosa from preovulatory follicles.     

Testosterone environment of splenocytes modifies the steroidogenesis of polycystic ovary in rats.

The functional relationship between the ovary and immune cells is well known. The modulation of ovarian steroidogenesis in adult rats with polycystic ovary (PCO) by secretions of cultured splenocytes treated with 10 (-6) M testosterone or 10 (-6) M testosterone plus 10 (-4) M flutamide, an androgen receptor antagonist, was investigated. Polycystic ovary was induced by estradiol valerate (2 mg/rat). Polycystic ovary splenocyte secretions decreased the release of androstenedione from PCO ovaries in contrast to the effect of non-PCO splenocyte secretions. This decrease was associated with a significant decrease in androgen receptor and IL-12 mRNA expression in PCO splenocytes. When splenocytes were treated with testosterone, their conditioned media further decreased androstenedione release from the ovary and had a greater inhibitory effect on PCO ovary compared with non-PCO ovary. This effect was reversed by flutamide. Polycystic ovary splenocytes showed a decrease in IL-1 beta mRNA expression. Their secretions scarcely affected progesterone release from non-PCO ovaries but significantly stimulated progesterone release from PCO ovary by an androgen-independent mechanism. The differential steroidogenic ability of splenocyte secretions from PCO rats is associated with the IN VITRO testosterone environment. Polycystic ovary splenocytes might exert a protective action against PCO effects through their secretions by inducing a low androstenedione response from the ovary.     

心房钠尿肽（ANP）对大鼠卵巢细胞生成雌激...

Isolated ovarian corpus luteal cells and granulosa cells of rat were employed to investigate the effect of alpha-ANP on the secretion of progesterone and estradiol. The contents of the steroid hormones are determined by RIA. The results showed that 0.1-10 ng/ml ANP promoted progesterone production in a dose dependent manner. alpha-ANP also enhanced progesterone production by granulosa cells, but not estradiol. It seems that the effect of alpha-ANP on ovarian steroidogenesis is a direct one.     

Fibroblast growth factor 18 regulates steroidogenesis in fetal bovine ovarian tissue in vitro

In cattle and other species, the fetal ovary is steroidogenically active before follicular development commences, and there is evidence that estradiol and progesterone inhibit follicle formation and activation. Estradiol levels decline sharply around the time of follicle formation. In the present study, we hypothesized that FGF10 and FGF18, which inhibit estradiol secretion from granulosa cells of antral follicles, also regulate fetal ovarian steroid production. Fetuses were collected at local abattoirs, and age determined by crown‐rump length measurements. Real‐time polymerase chain reaction assays with RNA extracted from whole ovaries revealed that the abundance of CYP19A1 messenger RNA (mRNA) decreased from 60 to 90 days of gestation, which is consistent with the decline in estradiol secretion previously observed. Immunohistochemistry revealed the presence of FGF18 in ovigerous cords in early gestation and in oocytes later in fetal age (≥150 days). The abundance of FGF18 mRNA increased after Day 90 gestation. Addition of recombinant FGF18 to fetal ovarian pieces inhibited estradiol and progesterone secretion in vitro, whereas FGF10 was without effect. Consistent with these results, FGF18 decreased levels of mRNA for CYP19A1 and CYP11A1 in ovarian pieces in vitro. These data suggest that FGF18 may be an intraovarian factor that regulates steroidogenesis in fetal ovaries.     

Effects of chronic administration of a heme oxygenase substrate or inhibitor on progression of the estrous cycle,pregnancy and lactation of Sprague-Dawley rats

The purpose of this study was to investigate whether a heme oxygenase substrate (hemin) or an inhibitor (chromium mesoporphyrin IX; CrMP) had any effect on the normal course of the estrous cycle, pregnancy, parturition or lactation in rats. The hypothesis was that these agents, acting on HO to increase or decrease endogenous production of carbon monoxide (CO) respectively, would disrupt these reproductive processes. The results showed that hemin administered s.c. at 30 micromoles/kg for 10 or 11 days, did not markedly influence the estrous cycle; whereas CrMP blocked the estrous cycle in a dose dependant fashion. At 2 and 4 micromoles/kg for 11 days CrMP significantly reduced the occurrence of estrus phase of the estrous cycle and the effect continued after the treatments were discontinued, while a dose of 1 micromole/kg produced no significant effects. CrMP, administered at 4 micromoles/kg during days 5-14 of pregnancy, led to massive fetal resorption with no live births from 14 successfully mated rats. Administration of hemin at 30 micromoles/kg for 10 days during lactation did not have any effect on milk production, whereas administration of CrMP at 4 micromoles/kg significantly decreased lactational performance which was attributed to milk production and not to suckling intensity of the pups. From these observations we conclude that heme oxygenase, and presumably endogenous CO, play positive roles in female reproductive processes and lactation in the rat.     

Stage of ovarian follicular development associated with the initiation of steroidogenic competence in avian granulosa cells.

Previously described models for avian ovarian steroidogenesis, using mature, 25-40-mm preovulatory follicles as the source of tissues, were based on the assumption that interaction of the granulosa layer, as the predominant source of progesterone, with adjacent theca cells is required for maximal production of C21, C19, and C18 steroids. In the present study, we evaluated the steroidogenic capacity of ovarian cells isolated from less mature, 6-8-mm and 9-12-mm follicles in the chicken ovary (representative of a stage of development 2-3 wk prior to ovulation) to determine at which stage of follicular development granulosa and/or theca cells become steroidogenically competent. Granulosa cells collected from 6-8-mm follicles were found to be virtually incompetent to produce steroids, containing extremely low basal levels of progesterone (12 pg/5 x 10(5) cells) and failing to respond with increased steroid output following a 3-h exposure to ovine LH (oLH; 0.1 and 100 ng/0.5 ml), ovine FSH (oFSH; 100, 500, and 1,000 ng/0.5 ml), 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP; 0.33 and 3.33 mM) or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, addition of pregnenolone (20 and 200 ng/0.5 ml) to granulosa incubations resulted in significantly increased progesterone levels. Granulosa cells of 6-8-mm follicles also failed to increase cAMP formation in the presence of oLH (10, 100, and 1,000 ng/0.5 ml) and 3-isobutyl-1-methylxanthine (IBMX; 10 microM), but responded to stimulation with 1,000 ng oFSH (4.4-fold increase over basal) or 10 microM forskolin (32-fold increase over basal) in the presence of IBMX. In contrast, granulosa cells isolated from 9-12-mm follicles and incubated for 3 h in vitro were found to contain basal progesterone levels 200-fold higher than those found in granulosa cells of 6-8-mm follicles. Furthermore, granulosa cells of 9-12-mm follicles markedly increased progesterone production following incubation in the presence of oFSH (100-1,000 ng/0.5 ml), 8-bromo-cAMP (0.33 and 3.33 mM), or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, these granulosa cells remained unresponsive to oLH (0.1, 10, and 100 ng/0.5 ml), failing to increase cAMP accumulation (in the presence of IBMX) and progesterone output. Theca cells of small yellow follicles were found to produce measurable basal levels of progesterone, androstenedione, and estradiol, and levels of each steroid were significantly increased following a 3-h challenge with oLH, 8-bromo-cAMP, 25-hydroxycholesterol, and pregnenolone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.     

Effects of angiogenin on granulosa and theca cell function in cattle

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P<0.05) granulosa cell progesterone production and theca cell androstenedione production but did not affect (P>0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P<0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P<0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.     

Androgen inhibition of FSH-stimulated progesterone production by granulosa cells of prepubertal pigs

Androgens have been reported to stimulate progesterone production by granulosa cells of several species, and to act synergistically with FSH in stimulation of progesterone accumulation by rat granulosa cells. Studies were undertaken to examine the effect of androgens on FSH-stimulated progesterone production in culture by granulosa cells derived from prepubertal pig ovaries. When included in 24-h culture with FSH, both androstenedione and testosterone caused a reduction in progesterone accumulation, but dihydrotestosterone and androsterone did not. Granulosa cells were cultured for 24 h with FSH and [14C]progesterone with or without testosterone; testosterone did not affect the rate of overall metabolism of [14C]progesterone and it was therefore concluded that testosterone inhibited progesterone synthesis, rather than enhancing its catabolism. 17 beta-Estradiol also inhibited FSH-stimulated progesterone accumulation. To determine whether the action of testosterone was mediated by conversion to estradiol, granulosa cells were cultured with FSH and testosterone with or without an aromatase inhibitor (4-acetoxy-androstenedione). The aromatase inhibitor failed to prevent the testosterone-induced reduction in progesterone accumulation, although it markedly inhibited estradiol accumulation. These results indicate that theca-derived androgens can inhibit FSH-stimulated progesterone production by granulosa cells in the prepubertal pig, independently of estradiol.     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

Effect of activin-a on goat granulosa cell steroidogenesis

Growth factors are said to play a significant role in the development of ovarian follicles. We wished to measure the content of one growth factor, activin-A in goat ovarian follicles, and study its effect on goat granulosa cells steroidogenesis. The follicular fluid content of activin-A from small, medium and large antral follicles was determined by two-site enzyme immunoassay. The results showed that activin-A concentration in the follicular fluid increased as the size of the follicle increased and, thus, may act as a local regulator of follicle development. To examine this possibility, the effect of increasing concentration of activin-A (0, 1, 10, 100 ng/mL) on differentiated goat granulosa cells steroidogenesis was evaluated in vitro for 48 hours in a chemically defined medium. Activin-A treatment resulted in a significant inhibition of progesterone production concomitant with a significant stimulation of estradiol production. These results were confirmed by time-effect of 50 ng/mL activin-A on goat granulosa cells steroidogenesis for 24, 48 and 72 hours. Granulosa cells displayed differential steroidogenic responses to activin-A, estradiol production becoming enhanced and progesterone production suppressed. Based on these findings, it appears that activin-A is a local regulator of goat granulosa cell steroidogenesis, and may act to promote granulosa cell differentiation and inhibit its luteinization.     

Cell-specific expression and regulation of soluble guanylyl cyclase alpha 1 and beta 1 subunits in the rat ovary

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and carbon monoxide, resulting in cGMP production. Recent studies indicate that NO and cGMP influence ovarian functions. However, little information is available regarding the ovarian expression of sGC. The present study examined sGC alpha(1) and beta(1) subunit protein levels in the ovary during postnatal development, gonadotropin-induced follicle growth, ovulation, and luteinization as well as in cultured rat granulosa cells. In postnatal rats, sGC alpha(1) subunit immunoreactivity was high in granulosa cells of primordial and primary follicles on Day 5 but low in granulosa cells of larger follicles on Days 10 and 19. Theca cells of developing follicles, but not stromal cells, also demonstrated moderate sGC alpha(1) immunoreactivity. In gonadotropin- treated immature rats, intense sGC alpha(1) subunit staining was similarly observed in granulosa cells of primordial and primary follicles, but such staining was low in granulosa cells of small antral follicles and undetectable in granulosa cells of large antral and preovulatory follicles. Following ovulation, corpora lutea expressed moderate sGC alpha(1) immunoreactivity. Similar ovarian localization and expression patterns were seen for sGC beta(1), indicating regulated coexpression of sGC subunits. Immunoblot analysis revealed no change in total ovarian sGC alpha(1) and beta(1) subunit protein levels during gonadotropin treatment. Similarly, no effect of FSH on sGC subunit protein levels was apparent in cultured granulosa cells. These findings indicate regulated, cell- specific patterns of sGC expression in the ovary and are consistent with roles for cGMP in modulating ovarian functions.     

Effect of Epidermal Growth Factor on Follicular Development and Steroidogenesis in Perfused Rat Ovary

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Macrophage secretions modulate the steroidogenesis of polycystic ovary in rats: effect of testosterone on macrophage pro-inflammatory cytokines

AimsThe macrophage secretions' effect on ovarian steroidogenesis is investigated in a polycystic ovary syndrome rat model (PCO rat). The influence of testosterone environment on the expression of macrophage pro-inflammatory cytokines that participate in ovarian steroidogenesis is studied.Main methodsPCO rats were induced by estradiol valerate. Spleen macrophages were cultured with and without testosterone (10^? 6 M) and their secretions were used to stimulate ovaries from PCO and control rats. Ovarian hormones released and ovary mRNA levels of P450 aromatase and 3β-hydroxysteroid dehydrogenase were measured by radioimmunoassay and RT-PCR, respectively. The tumor necrosis factor alpha (TNFα) and nitric oxide (NO) levels in macrophage culture medium, along with the TNFα, interleukin (IL)-6, IL-10 and androgen receptors (AR) mRNA levels in macrophage cells were determined.Key findingsMacrophages from PCO rats released more TNFα and NO, expressed higher TNFα and IL-6, lower AR, and no change in IL-10 mRNA levels than control macrophages. TNFα, IL-6 and AR changes were greater after macrophage testosterone treatment. Macrophage secretions from PCO rats stimulated androstenedione and decreased estradiol release and ovarian mRNA P450 aromatase expression in PCO rats compared to macrophage secretions from control rats.These effects were greater when macrophages from PCO rats were treated with testosterone. Ovarian progesterone response was unchanged.SignificanceThe differential steroidogenic ability of macrophage secretions from PCO rats is associated to the in vitro testosterone environment. Testosterone, probably acting on macrophage AR, induces a greater release of TNFα, modifying ovarian response by increasing androstenedione and slightly decreasing estradiol without affecting progesterone.     

Inhibition of heme oxygenase augments tubular sodium reabsorption

Heme oxygenase (HO) catalyzes the degradation of heme to form iron, biliverdin, and carbon monoxide (CO). The vascular actions of CO include direct vasodilation of vascular smooth muscle and indirect vasoconstriction through inhibition of nitric oxide synthase (NOS). This study was performed to examine the effects in the kidney of inhibition of heme oxygenase alone or combined with NOS inhibition. Chromium mesoporphyrin (CrMP; 45 μmol/kg ip), a photostable HO inhibitor, was given to control rats and N(G)-nitro-l-arginine methyl ester (l-NAME)-treated hypertensive rats (50 mg·kg?1·day?1), 12 h, 4 days). In control animals, CrMP decreased CO levels, renal HO-1 levels, urine volume, and sodium excretion, but had no effect on arterial pressure, renal blood flow (RBF), plasma renin activity (PRA), or glomerular filtration rate (GFR). In l-NAME-treated hypertensive rats, CrMP decreased endogenous CO and renal HO-1 levels and had no effect on arterial pressure, RBF, or GFR but decreased sodium and water excretion in a similar manner to control animals. An increase in PRA was observed in untreated rats but not in l-NAME-infused rats, indicating that this effect is associated with an absent NO system. The results suggest that inhibition of HO promotes water and sodium excretion by a direct tubular action that is independent of renal hemodynamics or the NO system.     

Insulin enhances luteinizing hormone-stimulated steroidogenesis by porcine theca cells

It has been shown recently that insulin enhances differentiation of rat, pig, and human granulosa cells. The present studies were done to determine if insulin also plays a role in the regulation of theca cell steroidogenesis. Theca cells were obtained from prepubertal gilts and cultured under serum-free conditions for 48 h. Theca cell androstenedione production under basal and luteinizing hormone (LH)-stimulated conditions was significantly increased by adding insulin (1 microgram/ml) to the culture medium. Treatment of basal and LH-stimulated cultures with increasing concentrations of insulin (0.001-10 micrograms/ml) caused dose- and time-dependent increments in androstenedione production, but the effect was independent of the dose of LH employed. The ability of insulin to enhance thecal cell androstenedione production was mimicked by somatomedin C, but not by relaxin. Studies to determine the mechanism(s) of action of insulin showed that insulin action is exerted, at least in part, at a site(s) proximal to cyclic adenosine 3'5'-monophosphate (cAMP) generation, since insulin enhanced both basal and LH-stimulated accumulation of extracellular cAMP in addition to increasing androstenedione production. This effect was further enhanced by 3-isobutyl-1-methyl xanthine, an inhibitor of phosphodiesterase activity. Insulin treatment also caused dose-dependent increments in forskolin- and prostaglandin E2-stimulated accumulation of extracellular cAMP and androstenedione. Insulin also increased both the basal and LH-stimulated production of progesterone and its precursor pregnenolone, in addition to the increases in androstenedione.(ABSTRACT TRUNCATED AT 250 WORDS)