Pre- and postnatal developmental exposure to the polychlorinated biphenyl mixture aroclor 1221 alters female rat pituitary gonadotropins and estrogen receptor alpha levels.

Polychlorinated-biphenyls (PCBs) are industrial compounds, which were widely used in manufacturing of electrical parts and transformers. Despite being banned in 1979 due to human health concerns, they persist in the environment. In humans and experimental model systems, PCBs elicit toxicity in part by acting as endocrine-disrupting chemicals (EDCs). Aroclor 1221 (A1221) is a weakly estrogenic PCB mixture known to alter reproductive function in rodents. EDCs can impact hormone signaling at any level of the hypothalamic-pituitary-gonadal (HPG) axis, and we investigated the effects of A1221 exposure during the prenatal and postnatal developmental periods on pituitary hormone and steroid receptor expression in female rats. Examining offspring at 3 ages, postnatal day 8 (P8), P32 and P60, we found that prenatal exposure to A1221 increased P8 neonate pituitary luteinizing hormone beta (Lhb) mRNA and LHß gonadotrope cell number while decreasing LH serum hormone concentration. No changes in pituitary hormone or hormone receptor gene expression were observed peri-puberty at P32. In reproductively mature rats at P60, we found pituitary follicle stimulating hormone beta (Fshb) mRNA levels increased by prenatal A1221 exposure with no corresponding alterations in FSH hormone or FSHß expressing cell number. Estrogen receptor alpha (ERα) mRNA and protein levels were also increased at P60, but only following postnatal A1221 dosing. Together, these data illustrate that exposure to the PCB A1221, during critical developmental windows, alters pituitary gonadotropin hormone subunits and ERα levels in offspring at different phases of maturation, potentially impacting reproductive function in concert with other components of the HPG axis.

Melanoma risk after in vitro fertilization: A review of the literature.

BACKGROUND: The role of female sex hormones in the pathogenesis of malignant melanoma (MM) remains controversial. Although melanocytes appear to be hormonally responsive, the effect of estrogen on MM cells is less clear. Available clinical data does not consistently demonstrate that increased endogenous hormones from pregnancy or increased exogenous hormones from oral contraceptive pills and hormone replacement affect MM prevalence and outcome. OBJECTIVE: We sought to examine potential associations between in vitro fertilization (IVF) and melanoma. METHODS: A literature review was conducted. Primary outcomes were reported as associations between IVF and melanoma risk compared with the general population. Secondary outcomes included associations stratified by type of IVF regimen and subgroup, such as parous versus nulliparous patients. RESULTS: Eleven studies met our inclusion criteria. Five studies found no increased risk for MM among IVF users compared with the general population. Two studies found an increase in MM in clomiphene users, and 4 studies found an increase in MM among patients who were gravid or parous either before or after IVF. CONCLUSION: The reviewed studies do not reveal consistent patterns of association between IVF and MM among all infertile women. However, the data indicates a potential increased risk for MM in ever-parous patients treated with IVF. High-quality studies including a large number of MM cases that control for well-established MM risk factors are needed to adequately assess the relationship between IVF and MM, particularly among ever-parous women.

Novel evidence that pituitary sex hormones regulate migration, adhesion, and proliferation of embryonic stem cells and teratocarcinoma cells.

The pituitary sex hormones (SexHs): follicle­stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) regulate several functions crucial for reproduction, including oogenesis, spermatogenesis, and lactation. An important source of prolactin-like hormones, known as lactogens, is the placenta, and lactogens bind to the PRL receptor (PRLR) with high affinity and thereby mimic the actions of PRL. Recently, it has been demonstrated that pituitary SexHs were involved in metastatic lung cancer, certain sarcomas, and leukemia. In the present study we aimed to investigate whether FSH, LH, and PRL were able to stimulate stem cells involved in early development. To address this issue we employed a murine embryonic stem cell line (ES-D3) as well as two teratocarcinoma cell lines, P19 (murine) and NTera2 (human). We determined that all these cells expressed SexH receptors at the mRNA and protein levels and that stimulation of these receptors induced phosphorylation of p42/44 MAPK, p38 MAPK, and AKT. Moreover, ES-D3, P19, and NTera2 cells responded with increased migration and adhesion to physiological concentrations of pituitary SexHs. In view of these findings we proposed that maternal-derived pituitary SexHs regulate the biology of stem cells involved in early development.

GnRH Neuron-Specific Ablation of Gαq/11 Results in Only Partial Inactivation of the Neuroendocrine-Reproductive Axis in Both Male and Female Mice: In Vivo Evidence for Kiss1r-Coupled Gαq/11-Independent GnRH Secretion.

The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility and kisspeptin (KP) is a potent trigger of GnRH secretion from GnRH neurons. KP signals via KISS1R, a Gαq/11-coupled receptor, and mice bearing a global deletion of Kiss1r (Kiss1r(-/-)) or a GnRH neuron-specific deletion of Kiss1r (Kiss1r(d/d)) display hypogonadotropic hypogonadism and infertility. KISS1R also signals via ß-arrestin, and in mice lacking ß-arrestin-1 or -2, KP-triggered GnRH secretion is significantly diminished. Based on these findings, we hypothesized that ablation of Gαq/11 in GnRH neurons would diminish but not completely block KP-triggered GnRH secretion and that Gαq/11-independent GnRH secretion would be sufficient to maintain fertility. To test this, Gnaq (encodes Gαq) was selectively inactivated in the GnRH neurons of global Gna11 (encodes Gα11)-null mice by crossing Gnrh-Cre and Gnaq(fl/fl);Gna11(-/-) mice. Experimental Gnaq(fl/fl);Gna11(-/-);Gnrh-Cre (Gnaq(d/d)) and control Gnaq(fl/fl);Gna11(-/-) (Gnaq(fl/fl)) littermate mice were generated and subjected to reproductive profiling. This process revealed that testicular development and spermatogenesis, preputial separation, and anogenital distance in males and day of vaginal opening and of first estrus in females were significantly less affected in Gnaq(d/d) mice than in previously characterized Kiss1r(-/-) or Kiss1r(d/d) mice. Additionally, Gnaq(d/d) males were subfertile, and although Gnaq(d/d) females did not ovulate spontaneously, they responded efficiently to a single dose of gonadotropins. Finally, KP stimulation triggered a significant increase in gonadotropins and testosterone levels in Gnaq(d/d) mice. We therefore conclude that the milder reproductive phenotypes and maintained responsiveness to KP and gonadotropins reflect Gαq/11-independent GnRH secretion and activation of the neuroendocrine-reproductive axis in Gnaq(d/d) mice. SIGNIFICANCE STATEMENT: The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility. Over the last decade, several studies have established that the KISS1 receptor, KISS1R, is a potent trigger of GnRH secretion and inactivation of KISS1R on the GnRH neuron results in infertility. While KISS1R is best understood as a Gαq/11-coupled receptor, we previously demonstrated that it could couple to and signal via non-Gαq/11-coupled pathways. The present study confirms these findings and, more importantly, while it establishes Gαq/11-coupled signaling as a major conduit of GnRH secretion, it also uncovers a significant role for non-Gαq/11-coupled signaling in potentiating reproductive development and function. This study further suggests that by augmenting signaling via these pathways, GnRH secretion can be enhanced to treat some forms of infertility.

Luteal phase support with progesterone in intrauterine insemination: a prospective randomized study.

OBJECTIVE: To determine the effect of vaginal progesterone as luteal support on pregnancy outcomes in infertile patients who undergo ovulation induction with gonadotropins and intrauterine insemination (IUI). DESIGN: Prospective randomized trial. SETTING: Tertiary referral center. PATIENT(S): About 398 patients with primary infertility were treated during 893 ovarian stimulation and IUI cycles from February 2010 to September 2012. METHODS: All patients underwent ovarian stimulation with gonadotropins combined with IUI. Patients in the supported group received vaginal micronized progesterone capsules 200 mg once daily from the day after insemination until next menstruation or continuing for up to 8 weeks of pregnancy. Women allocated in the control group did not receive luteal phase support. MAIN OUTCOME MEASURE(S): Livebirth rate, clinical pregnancy rate and early miscarriage rate per cycle. RESULT(S): Of the 893 cycles, a total of 111 clinical pregnancies occurred. There were no significant differences between supported with progesterone and unsupported cycle in terms of livebirth rate (10.2% versus 8.3%, respectively, with a p value = 0.874) and clinical pregnancy rate (13.8% compared with 11.0% in unsupported cycle with a p value = 0.248). An early miscarriage rate of 3.6% was observed in the supported cycles and 2.7% in the unsupported cycles, with no significant differences between the groups (p value = 0.874). CONCLUSION(S): In infertile patients treated with mildly ovarian stimulation with recombinant gonadotropins and IUI, luteal phase support with vaginal progesterone is not associated with higher livebirth rate or clinical pregnancy rate compared with patients who did not receive any luteal phase support.

[Stimulation in vitro of oocytes of acipenserids with progesterone and homologous gonadotropic hormone of hypophysis].

We showed that the percentage of oocytes of acipenserids ovulating in vitro in Ringer solution modified for sturgeons (RMS) considerably depends on the concentration of sodium bicarbonate and the concentration of progesterone. Under optimal conditions (0.5 g/L of sodium bicarbonate and 30 ng/mL of progesterone), it can be higher than 80. Oocytes that matured and ovulated under such conditions are capable of normal development. In the best case, approximately 70% of developing embryos (of the number of ovulated oocytes) reach the stage of hatching (dead-line of observation). This method of producing offspring based on the insemination of oocytes that have matured and ovulated in vitro can be used in work with single females of rare and disappearing species of acipenserids.

Neonatal gonadotropin therapy in male congenital hypogonadotropic hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) causes pubertal failure and infertility in both women and men due to partial or total secretory failure of the two pituitary gonadotropins lutropin (LH) and follitropin (FSH) during periods of physiological activation of the gonadotropic axis. Men and women with CHH frequently seek treatment for infertility after hypogonadism therapy. Some etiologies, such as autosomal dominant or X-linked Kallmann syndrome, raise the question of hereditary transmission, leading to increasing demands for genetic counseling and monitoring of medically assisted pregnancies. Diagnosis and treatment of newborn boys is, therefore, becoming an increasingly important issue. In male individuals with complete forms of CHH, the antenatal and neonatal gonadotropin deficit leads to formation of a micropenis and cryptorchidism, which could undermine future sexual and reproductive functions. Standard treatments, usually started after the age of puberty, often only partially correct the genital abnormalities and spermatogenesis. The aim of this Review is to examine the possible additional benefits of neonatal gonadotropin therapy in male patients with CHH. Encouraging results of neonatal therapy, together with a few reports of prepubertal treatment, support the use of this novel therapeutic strategy aimed at improving sexual and reproductive functions in adulthood.

Bone morphogenetic protein-6 enhances gonadotrophin-dependent progesterone and inhibin secretion and expression of mRNA transcripts encoding gonadotrophin receptors and inhibin/activin subunits in chicken granulosa cells.

The aims were to examine ovarian expression of bone morphogenetic protein (BMP) ligands/receptor mRNAs in the chicken and to test the hypothesis that theca-derived BMP(s) modulates granulosa cell function in a paracrine manner. RT-PCR revealed expression of multiple BMPs in granulosa and theca cells from pre hierarchical and preovulatory follicles with greater expression in theca cells; both cell types expressed BMP receptors-IA, -IB and -II consistent with tissue responsiveness. Preovulatory granulosa cells (F1, F2 and F3/4) were cultured with BMP-6 (expressed by theca but not granulosa) in the presence/absence of LH, FSH or 8-Br-cAMP. BMP-6 increased 'basal' and gonadotrophin-induced inhibin-A and progesterone secretion by each cell type but did not enhance the effect of 8-Br-cAMP. This indicates that the observed synergism between BMP-6 and gonadotrophin might involve BMP-induced up-regulation of gonadotrophin receptors. In support of this, BMP-6 alone increased LH-receptor (LHR) mRNA in F1 cells and FSH-receptor (FSHR) mRNA in F1, F2 and F3/4 cells. BMP-6 also enhanced LH/FSH-induced LHR transcript amount in each cell type but did not raise FSHR transcript amounts above those induced by BMP-6 alone. To further explore BMP-6 action on inhibin-A secretion, we quantified inhibin/activin subunits (alpha, beta(A), beta(B)) mRNAs. Consistent with its effect on inhibin-A secretion, BMP-6 enhanced 'basal' expression of alpha- and beta(A)-subunit mRNA in F1, F2 and F3/4 cells, and beta(B)-subunit mRNA in F3/4 cells. BMP-6 markedly enhanced FSH/LH-induced expression of alpha-subunit in all follicles and FSH-induced beta(A)-subunit in F2 and F3/4 follicles but not in F1 follicles. Neither BMP-6 alone, nor FSH/LH alone, affected 'basal' beta(B) mRNA abundance. However, co-treatment with gonadotrophin and BMP-6 greatly increased beta(B)-subunit expression, the response being lowest in F1 follicles and greatest in F3/4 follicles. Collectively, these results support the hypothesis that intraovarian BMPs of thecal origin have a paracrine role in modulating granulosa cell function in the chicken in a preovulatory stage-dependent manner.

Effects of gonadotrophin treatments on meiotic and developmental competence of oocytes in porcine primordial follicles following xenografting to nude mice.

Our objective was to improve the developmental ability of oocytes in porcine primordial follicles xenografted to nude mice, by treating the host mice with gonadotrophins to accelerate follicular growth. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Gonadotrophin treatments were commenced around 60 days after vaginal cornification in the mice. Ovarian grafts were obtained 2 or 3 days after treatment with equine chorionic gonadotrophin (eCG-2 and eCG-3 groups), after porcine FSH infusion for 7 or 14 days, or after infusion of porcine FSH for 14 days with a single injection of estradiol antiserum (FSH-7, FSH-14 and FSH-14EA groups, respectively). Gonadotrophin treatments accelerated follicular growth within the xenografts compared with that in control mice given no gonadotrophins, consistent with higher (P < 0.05) circulating inhibin levels in the gonadotrophin-treated mice. In contrast, circulating mouse FSH levels were significantly (P < 0.05) depressed. We recovered large numbers of full-sized oocytes with meiotic competence to the mature stage from the eCG-3, FSH-7, and FSH-14EA, unlike in the control group. Moreover, 56% of matured oocytes with the first polar body (n = 39) were fertilized in vitro in the FSH-14EA group. After in vitro fertilization and subsequent culture for 7 days, one blastocyst was obtained from each of the eCG-3, FSH-7 and, FSH-14EA groups, whereas no blastocysts appeared in the other groups. Exogenous gonadotrophins--not mouse FSH--stimulated the growing follicles that had developed from the primordial follicles in the xenografts: the effects were incomplete but improved to some extent the meiotic and developmental abilities of the oocytes.

Anti-inflammatory and proliferative responses in human and ovine ovarian surface epithelial cells.

The majority of ovarian cancers (>90%) are believed to derive from the ovarian surface epithelium (OSE); a single layer covering the entire surface of the ovary. At ovulation, the OSE cell layer undergoes an inflammatory response, involving cell death and growth, in order to overcome ovarian surface rupture. Abnormalities during these processes are believed to contribute to the development of tumours. Using primary cultures of OSE cells, we have compared anti-inflammatory and proliferative responses directly between human and ovine OSE cells to further establish the use of ovine OSE cells as a suitable model system for the study of human OSE cells. In order to compare effects of inflammatory stimulation, expression and activity of 11betahydroxysteroid dehydrogenase (11betaHSD) type 1 was measured in OSE cells in response to interleukin (IL)-1alpha. As previously identified in human OSE cells, treatment of ovine OSE cells with IL-1alpha stimulated a concomitant increase of 11betaHSD type 1 mRNA (31-fold; P <0.05) and oxoreductase activity, indicating an increased production of anti-inflammatory cortisol. To compare the growth of human and ovine OSE cells, OSE cell number was measured in response to treatment with gonadotropins or growth factors. In the presence of FSH, LH or human chorionic gonadotropin (hCG), ovine and human OSE cell growth was similarly stimulated >1.2-fold (P <0.05). In the presence of connective tissue growth factor (CTGF) and more significantly insulin growth factor I (IGF-I), human and ovine OSE cell growth was also similarly stimulated >1.2-fold (P <0.05) and >1.5-fold (P <0.01), respectively. The induction of both human and ovine OSE cell growth by IGF-I or hCG was further shown to be dependent on activation of the MAP kinase/extracellular-signal-regulated kinase (ERK) pathway. Stimulation of ovine OSE cell growth by hepatocyte growth factor (HGF) was similarly shown to be ERK-dependent; however, for human OSE cells, HGF only mildly stimulated ERK phosphorylation and failed to stimulate OSE cell growth. The demonstration that human and ovine OSE cells share similarities at the level of cell signalling, gene expression and cellular growth supports the use of ovine OSE cells as a suitable model for the study of human OSE cells.

Fundulus heteroclitus gonadotropins.5: Small scale chromatographic fractionation of pituitary extracts into components with different steroidogenic activities using homologous bioassays.

Fractionation and characterization of gonadotropins (GtH) from Fundulus heteroclitus pituitary extracts were carried out using a biocompatible liquid chromatographic procedure (Pharmacia FPLC system). Chromatographic fractions were monitored for gonadotropic activities (induction of oocyte maturation and steroid production) using homologous follicle bioassays in vitro. Size-exclusion chromatography eluted gonadotropic activity in one major protein peak (Mr approximately 30,000). Anion-exchange and hydrophobic-interaction chromatography (HIC) yielded two distinct peaks of 17beta-estradiol (E2)- and 17alpha-hydroxy,20beta-dihydroprogesterone (DHP)-promoting activity with associated oocyte maturation. Two-dimensional chromatography (chromatofocusing followed by HIC) resolved pituitary extracts into two active fractions; both induced E2 synthesis, but one was relatively poor in eliciting DHP and testosterone production. Thus, using homologous bioassays, at least two quantitatively different gonadotropic (steroidogenic) activities: an E2-promoting gonadotropin (GtH I-like) and a DHP-promoting gonadotropin (GtH II-like), which has a lower isoelectric point but greater hydrophobicity than the former, can be distinguished from F. heteroclitus pituitaries by a variety of chromatographic procedures. This study complements previous biochemical and molecular data in F. heteroclitus and substantiates the duality of GtH function in a multiple-spawning teleost.

Gonadotropin-releasing hormones modulate electrical activity of vasotocin and isotocin neurons in the brain of rainbow trout.

Gonadotropin-releasing hormone (GnRH) is widely distributed in the vertebrate brains; however, its significance in the brain function is poorly understood. Both GnRH and vasopressin-family hormones are involved in control of reproductive behavior. Anatomical evidence indicated the possible action of GnRH on classical neurosecretory neurons. In the present study, we examined whether GnRH modulates electrical activity of vasotocin (VT) and isotocin (IT) neurons in the brain of rainbow trout (Oncorhynchus mykiss). Two forms of GnRH, salmon GnRH and chicken GnRH II, are present in the rainbow trout brain, and their fibers are localized in the close vicinity of VT and IT neurons. Applications of both GnRH forms elevated the frequency of cell-type-specific synchronous Ca(2+) pulses in VT and IT neurons that are blocked by a GnRH-receptor antagonist. Our results showed facilitatory actions of GnRHs on VT and IT neurons, suggesting that GnRH neurons modulate classical neurosecretory neurons to control reproductive behavior.

The FecB (Booroola) gene acts at the ovary: in vivo evidence.

The aim of this study was to differentiate between pituitary and ovarian actions of the FecB gene by measuring the ovarian response to a standardized treatment with gonadotrophins designed to mimic the changes in FSH and LH that occur in the follicular phase of the ovarian cycle in ewes, with (Fec(B/-), n=6) and without (Fec(+/+), n=9) the gene, that were rendered hypogonadotrophic by pretreatment with a potent antagonist of GnRH. Ewes with ovarian autotransplants were used to facilitate the assessment of follicular function by the collection of ovarian venous blood and ultrasonography. The gonadotrophin regimen resulted in concentrations of FSH and LH that were similar to concentrations found in a normal cycle and did not differ between genotypes. Follicular development and ovulation occurred in all animals, and patterns of secretion of oestradiol, androstenedione and inhibin A were normal. Despite these endocrine similarities, the antral follicle population stimulated by FSH infusion retained the characteristic genotypic difference with the ovaries of Fec(+/+) animals containing a range of follicle sizes with decreasing proportions of small (<3.5 mm in diameter) and medium (3.5-4.5 mm in diameter) follicles as well as large follicles (> or =4.5 mm in diameter), whereas the ovaries of Fec(B/-) ewes contained no follicles of >4.5 mm in diameter. This genotypic difference was retained after ovulation with gene carriers having more preovulatory follicles/corpora lutea (3.8+/-0.3) of a smaller diameter (5.3+/-0.3 mm) than did non-gene carriers (1.7+/-0.3; 11.4+/-0.9 mm; P<0.05). As ewes carrying the FecB gene mutation were able to ovulate more follicles than non-gene carriers, despite identical concentrations and patterns of FSH and LH stimulation, the results of this study support the hypothesis that the FecB gene acts at the ovary to enhance ovarian sensitivity to gonadotrophic stimulation.

Analysis of signal transduction stimulated by gonadotropins in granulosa cells.

Gonadotropins exert their effect on ovarian follicular cells through the activation of the hormone sensitive adenylate cyclase and consequent elevation of intracellular cyclic AMP (cAMP). Desensitization to the hormone in cultured primary granulosa cells can occur within a short period and internalization of the hormone-receptor complex has been observed both in vivo and in vitro. It was recently documented that the gonadotropins as well as cAMP activate MAP kinase (MAPK) in granulosa cells. Moreover we discovered that specific inhibitors of extracellular signal-regulated kinase phosphorylation, 1 and 2, augment steroidogenesis in granulosa cells up-regulating steroidogenic acute regulatory (StAR) protein expression, and that this modulation is blocked by specific inhibitors of protein kinase A. It is therefore suggested that gonadotropins may activate both stimulatory and inhibitory pathways which regulate steroidogenesis. Moreover the ratio between the activity of these two pathways may determine the rate of steroidogenesis, and rapid activation of MAPK may account as part of the mechanism of desensitization to the hormonal action. Steroidogenic factor-1 and DAX-1 may be involved in the regulation of the MAPK-dependent attenuation of steroidogenesis, since they exhibit sites that could be potentially phosphorylated by the MAPK cascade.

Multi-factorial role of GnRH-I and GnRH-II in the human ovary.

Normal ovarian functions are regulated by a wide variety of endocrine hormones, local paracrine and autocrine factors, which functionally interact with each other in a highly coordinated fashion. Recent findings have demonstrated that both forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II) are expressed in various compartments of the human ovary including the granulosa-luteal cells, ovarian surface epithelial cells and ovarian tumors, and their expressions have been shown to be tightly regulated by gonadal steroids and gonadotropins. Functionally, these neuropeptides exert diverse biological effects in the ovary via binding to their cognate receptors, supporting the notion that these peptides act as paracrine and autocrine factors in modulating local ovarian functions. In this review, we will summarize recent literatures regarding the regulation of GnRH-I and GnRH-II gene expressions in the human ovary, and discuss the possible signal transduction mechanisms by which these hormones exert their actions in the gonad. Recent cloning of the second form of the GnRH receptor (GnRH-II receptor) in primates and other vertebrates demonstrated that it was structurally, and thus, functionally distinct from the GnRH-I receptor. Cell proliferation studies showed that GnRH-II inhibited the growth of human ovarian cancer cells that express GnRH-II but not GnRH-I receptor, indicating that the GnRH-II binding sites are functional in these cells. However, it remains unknown if GnRH-II receptor is expressed as a full-length, properly processed and functional gene transcript in humans, and its potential physiological roles such as differential regulation of gonadotropin secretion, neuroendocrine modulation and female sexual behavior await further investigation.

Influence of gonadotropins on insulin- and insulin-like growth factor-I (IGF-I)-induced steroid production by bovine granulosa cells.

To determine the effect of gonadotropins on insulin- and insulin-like growth factor (IGF-I)-induced bovine granulosa cell functions, granulosa cells from bovine ovarian follicles were cultured for 2 days in the presence of 10% fetal calf serum (FCS), and then cultured for an additional 2 days in serum-free medium with added hormones. In the presence of 0 or 1 ng/mL of insulin or IGF-I, FSH had little or no effect (P > 0.05) on estradiol production by granulosa cells from both small (1-5mm) and large (> or = 8mm) follicles. However, in the presence of > or = 3 ng/mL of insulin, FSH increased (P < 0.05) estradiol production by granulosa cells from small and large follicles such that the estimated dose (ED(50)) of insulin necessary to stimulate 50% of the maximum estradiol production was decreased by 2- to 3-fold from 22 to 28 ng/mL in the absence of FSH to 7-14 ng/mL in the presence of FSH. Similarly, in the presence of > or = 3 ng/mL of IGF-I, FSH increased (P< 0.05) estradiol production by granulosa cells from small and large follicles such that the ED(50) of IGF-I for estradiol production was decreased by 4- to 5-fold from 25 to 36 ng/mL in the absence of FSH to 5-6 ng/mL in the presence of FSH. In the presence of FSH, the maximal effect of insulin on estradiol production was much greater than that of IGF-I (137- versus 12-fold increase) and were not additive; when combined, 100 ng/mL of IGF-I completely blocked the stimulatory effect of 100 ng/mL of insulin. In the absence of FSH, the maximal effect of insulin and IGF-I on estradiol production was similar. Concomitant treatment with 30 ng/mL of LH reduced (P<0.05) insulin-stimulated estradiol production by 52% on day 1 and 19% on day 2 of treatment. Insulin, IGF-I and FSH also increased (P<0.05) granulosa cell numbers and progesterone production but their maximal effects were less (i.e., < 4-fold increase) than their effects on estradiol production. In conclusion, insulin and IGF-I synergize with FSH to directly regulate ovarian follicular function in cattle, particularly granulosa cell aromatase activity.

Biphasic regulation of activin A secretion by gonadotropins in cultured human ovarian granulosa-luteal cells leads to decreasing activin:inhibin ratios during continuing gonadotropin stimulation.

Pituitary gonadotropins mediate part of their effects on ovarian function via local hormones and growth factors produced by granulosa cells. Activins and inhibins are among these factors, and they have often opposite effects on various components of the reproductive system. The purpose of this study was to investigate the regulation of ovarian activin A secretion using cultured human ovarian granulosa-luteal cells as a model. The granulosa-luteal cells, obtained from women taking part in an in vitro fertilization program, were cultured and treated with FSH, LH, 8-bromo cAMP (8-BrcAMP, a protein kinase A activator) and 12-O-tetradecanoyl phorbol-13-acetate (TPA, a protein kinase C activator). Conditioned cell culture media were analyzed for activin A, inhibin A and progesterone concentrations with specific enzyme immunoassays. FSH and LH (1-100 IU/l) increased activin A secretion with 24 h of treatment (to 132% and 253% of control respectively; P<0.05 for both), but their effects were inhibitory in 48-h treatments (26% and 16% decreases respectively; P<0.05 for both). In the same experiments, FSH and LH increased inhibin A and progesterone secretion after both 24 and 48 h of treatment. 8-BrcAMP (0.1-100 muM) increased activin A in 24- and 48-h experiments (to 206% and 148% of control respectively; P<0.01 for both). Inhibin A and progesterone secretion were stimulated by 8-BrcAMP time- and dose-dependently. TPA increased activin A secretion dose-dependently (0.1-100 ng/ml) in both 24- and 48-h experiments. At 100 ng/ml concentration, it increased activin A up to 61-fold and inhibin A up to 16-fold of control in 24-h experiments. We conclude that gonadotropins regulate immunoreactive activin A secretion biphasically in cultured human granulosa-luteal cells: initial stimulation is followed by inhibition. In contrast, gonadotropins increase inhibin A and progesterone secretion continuously. Consequently, continuing gonadotropin stimulation leads to a decreasing activin:inhibin ratio, which may have a significant role in the local fine-tuning of ovarian steroidogenesis.

Hormonal regulation of apoptosis in rabbit granulosa cells in vitro: evaluation by flow cytometric detection of plasma membrane phosphatidylserine externalization.

Annexin V and propidium iodide bivariate analysis and the TUNEL method were used to quantify hormonal regulation of apoptosis in rabbit granulosa cells from preovulatory follicles in vitro. The aim of this study was to analyse comparatively the effects of gonadotrophins and their second messenger in the regulation of granulosa cell apoptosis in (i) cultured isolated granulosa cells and (ii) granulosa cells scraped from cultured follicles. The results showed that increasing doses of FSH had no effect on apoptosis of cultured isolated cells but caused a decrease in the number of apoptotic granulosa cells from preovulatory follicles cultured in serum-free conditions. Unlike FSH, addition of hCG did not modify apoptosis of granulosa cells significantly. In contrast, dibutyryl cAMP had an apoptotic effect in the two cellular models in the presence of serum. Moreover, a biphasic effect of dibutyryl cAMP in isolated granulosa cells was observed with an increase in the incorporation of [(3)H]thymidine into DNA at the lowest dose and an increase in apoptotic cell death at the highest dose. It was concluded that, in rabbits: (i) FSH requires follicle integrity to exert its anti-apoptotic effect in granulosa cells; (ii) dibutyryl cAMP induces a dose-dependent apoptotic effect in granulosa cells cultured alone or obtained from cultured preovulatory follicles; and (iii) cAMP signals induce opposite effects on growth and apoptosis in granulosa cells.

Modulatory effects of gonadotrophins and insulin-like growth factor on the secretion of inhibin A and progesterone by granulosa cells from chicken preovulatory (F1-F3) follicles.

The aim of this study was to compare the actions and interactions of gonadotrophins (LH and FSH) and an analogue of insulin-like growth factor I (LR3-IGF-I) on the secretion of inhibin A, inhibin B and progesterone by cultured chicken granulosa cells derived from the three largest (F1--F3) follicles of the preovulatory hierarchy. Treatment with LH or FSH promoted marked dose-(P < 0.0001) and time- (P < 0.0001) dependent increases in both inhibin A and progesterone secretion, with the magnitude of response (< 15-fold compared with basal) increasing over time in culture. Concentrations of inhibin B were below the detection limit in all samples. Initially, F1 cells were more LH-responsive than were F3 cells in terms of progesterone secretion (P < 0.02) but this difference between follicles decreased over time in culture. In contrast, LH-induced inhibin A secretion tended to be highest from F3 cells, although this was not significant. Cells from F3 follicles were consistently more FSH-responsive than F1 cells in terms of both progesterone (P < 0.01) and inhibin A (P < 0.02) secretion. Initially, F1 cells were more responsive to LR3-IGF-I than were F3 cells in terms of progesterone secretion (P < 0.001) but were less responsive in terms of inhibin A secretion (P < 0.001). Again, these inter-follicle differences decreased over time in culture (not significant on day 3 of treatment). Co-treatment experiments showed that LR3-IGF-I enhanced both LH- and FSH-induced secretion of inhibin A and progesterone in a time- (P < 0.001) and follicle- (P < 0.001) dependent way. Initially, F1 cells showed highest LR3-IGF-I enhancement of LH-induced inhibin A and progesterone secretion; in contrast, F3 cells showed the highest LR3- IGF-1 enhancement of FSH-induced inhibin A and progesterone secretion. These inter-follicle differences persisted over time in the case of FSH-induced hormone responses but not in the case of LH-induced responses, even though the relative degree of LR3-IGF-I enhancement increased markedly over time. Collectively, these data support a positive role for IGF-I, presumably of thecal origin, as an amplifier of gonadotrophin action on granulosa cell inhibin A and progesterone production by preovulatory chicken follicles.

Activin A and gonadotropin regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNA in avian granulosa cells.

Activin A regulation of the expression of mRNA for the LH receptor, FSH receptor, and the inhibin alpha subunit as well as the effect of activin A on the secretion of progesterone were investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F(1) and F(3) + F(4) follicles from five hens, pooled according to size, dispersed, and cultured for 48 h. In experiment 1 (n = 3 replications), granulosa cells were cultured with or without highly purified ovine (o) FSH at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A. Experiment 2 (n = 4 replications) followed the same protocol as experiment 1, except that oFSH was replaced with oLH. Results from these experiments showed that addition of activin A to the granulosa cell cultures had no effect on the expression of mRNA for the inhibin alpha subunit or the FSH receptor, but it did affect the expression of mRNA for the LH receptor. Treatment of F(3) + F(4) granulosa cells with LH stimulated the expression of mRNA for the LH receptor; however, when LH was combined with either dose of activin A, this induction was prevented. The highest dose of activin A with or without LH resulted in decreased expression of the LH receptor compared to the untreated controls in the F(3) + F(4) cell cultures. Progesterone secretion by the granulosa cells from both follicle sizes was not altered by activin A. In experiment 3 (n = 3 replications), the effect of activin A on the growth of granulosa cells was examined with the following treatments: 0, 10, or 50 ng/ml of activin A; 50 ng/ml of either oLH or oFSH; and oLH or oFSH combined with 10 ng/ml of activin A. The highest dose of activin reduced the rate of granulosa cell proliferation in both follicle types. Growth of F(1) and F(3) + F(4) granulosa cells was stimulated by the addition of either gonadotropin, and the presence of 10 ng/ml of activin A with either gonadotropin did not alter this proliferation, except for the LH-treated F(3) + F(4) granulosa cells, in which the increase in proliferation was prevented. The results suggest that activin A could act as a local factor that regulates follicular maturation by preventing excessive or untimely LH receptor expression.