Enhanced antitumor effects of follicle-stimulating hormone receptor-mediated hexokinase-2 depletion on ovarian cancer mediated by a shift in glucose metabolism.

BACKGROUND: Most cancers favor glycolytic-based glucose metabolism. Hexokinase-2 (HK2), the first glycolytic rate-limiting enzyme, shows limited expression in normal adult tissues but is overexpressed in many tumor tissues, including ovarian cancer. HK2 has been shown to be correlated with the progression and chemoresistance of ovarian cancer and could be a therapeutic target. However, the systemic toxicity of HK2 inhibitors has limited their clinical use. Since follicle-stimulating hormone (FSH) receptor (FSHR) is overexpressed in ovarian cancer but not in nonovarian healthy tissues, we designed FSHR-mediated nanocarriers for HK2 shRNA delivery to increase tumor specificity and decrease toxicity. RESULTS: HK2 shRNA was encapsulated in a polyethylene glycol-polyethylenimine copolymer modified with the FSH ß 33-53 or retro-inverso FSH ß 33-53 peptide. The nanoparticle complex with FSH peptides modification effectively depleted HK2 expression and facilitated a shift towards oxidative glucose metabolism, with evidence of increased oxygen consumption rates, decreased extracellular acidification rates, and decreased extracellular lactate and glucose consumption in A2780 ovarian cancer cells and cisplatin-resistant A2780CP counterpart cells. Consequently, cell proliferation, invasion and migration were significantly inhibited, and tumor growth was suppressed even in cisplatin-resistant ovarian cancer. No obvious systemic toxicity was observed in mice. Moreover, the nanoparticle complex modified with retro-inverso FSH peptides exhibited the strongest antitumor effects and effectively improved cisplatin sensitivity by regulating cisplatin transport proteins and increasing apoptosis through the mitochondrial pathway. CONCLUSIONS: These results established HK2 as an effective therapeutic target even for cisplatin-resistant ovarian cancer and suggested a promising targeted therapeutic approach.

Acute effects of lead on porcine neonatal Sertoli cells in vitro.

Environmental pollution is one of the main factors responsible for reducing fertility in males. Lead is one of the major heavy metal contaminants that impairs several organs; it preferentially accumulates in male reproductive organs and alters sperm quality both in vivo and in vitro. However, the underlying mechanisms remain unclear. Sertoli cells (SCs) provide structural and physiological support to spermatogenic cells within seminiferous tubules. Therefore, changes in SCs affect the developing germ cells and alter spermatogenesis. This study aimed to assess whether exposure to subtoxic doses of adversely affects SC functioning in higher mammals. Purified and functional porcine neonatal SCs were exposed to lead acetate at three different concentrations. Lead exposure decreased the mRNA expression and protein levels of inhibin B and anti-Mullerian hormone (AMH) compared to control, indicating loss of FSH-r integrity in terms of 17-ß-oestradiol production under FSH stimulation. In addition, we observed an increase in the mRNA levels of Akt and mTOR, and the phosphorylation of p38 and Akt in SCs exposed to lead at all concentrations compared to unexposed control SCs. In conclusion, lead is toxic to SCs, even at low concentrations, and is expected to alter spermatogenesis.

An in vitro prototype of a porcine biomimetic testis-like cell culture system: a novel tool for the study of reassembled Sertoli and Leydig cells.

At present, there is no reliable in vitro assembled prepubertal testis-like biomimetic organ culture system designed to assess the functional effects of human gonadotropins on Sertoli and Leydig cells. Spermatogenesis is regulated by endocrine, paracrine, and juxtacrine factors (testicular cross-talk), mainly orchestrated by gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that play a pivotal role by stimulating Leydig and Sertoli cells, respectively. The aim of our study was to set up an in vitro prepubertal porcine bioengineered construct as a new model for experimental studies on reassembled Sertoli and Leydig cells. We have evaluated Sertoli and Leydig cells obtained from 15- to 20-day-old neonatal pig testes in terms of purity and function. Subsequently, purified Sertoli and enriched Leydig cells were subjected to coincubation to obtain an in vitro prepubertal porcine testis-like culture system. We performed enzyme-linked immunosorbent assay (ELISA) for anti-Müllerian hormone (AMH), inhibin B, and testosterone secretion in the medium, and Real-Time PCR analysis of AMH, inhibin B, FSH-r, aromatase, LHr, and 3ß-HSD mRNA expression levels. This in vitro testis-like system was highly responsive to the effects of human gonadotropins and testosterone. AMH mRNA expression and secretion declined, and inhibin-B increased, while FSH-receptor expression was downregulated upon FSH/LH exposure/treatment. Finally, the production of testosterone was increased selectively upon LH treatment. In summary, our proposed model could help to better determine the action of human gonadotropins on Sertoli and Leydig cells. The potential usefulness of the system for shedding light into male infertility-related issues is evident.

Active immunization with GnRH-tandem-dimer peptide in young male rats reduces serum reproductive hormone concentrations, testicular development and spermatogenesis.

GnRH sterilization vaccines have been developed for various practical and clinical reasons. However, conjugation of GnRH peptide to carrier protein has many drawbacks, hampering the further commercialization of GnRH vaccines. In this study, a new nonconjugated GnRH vaccine, D-Lys6-GnRH-tandem-dimer peptide (TDK), emulsified in Specol adjuvant was investigated for its immunocastration efficacy in young male rats. Prepubertal male rats were randomly allocated into three groups (n = 12): control (no treatment), surgically castrated or immunized against 100 µg TDK in Specol adjuvant at 6 weeks of age (with a booster 8 weeks later). Blood samples (for antibody titers and hormone concentrations) were collected at 2-week intervals until rats were killed (18 weeks of age). Compared to intact controls, active immunization against TDK reduced (P < 0.05) serum concentrations of testosterone, inhibin B, LH and FSH, prevented the onset of spermatogenesis at puberty. Furthermore, mRNA expressions of GnRH receptor, LH-ß and FSH-ß in the pituitary, LH receptor, FSH receptor, inhibin α, ßA and ßB subunit in the testes were decreased in immunocastrated rats compared to intact controls (P < 0.05). These results demonstrate for the first time that GnRH-tandem-dimer peptide emulsified in Specol is a promising veterinary sterilization medicine.

Toxicity of cadmium on Sertoli cell functional competence: an in vitro study.

Cadmium (Cd), an ubiquitous environmental metal, mainly used for industrial purposes, may be toxic at level of the reproductive system. Testis tubular-based Sertoli cells (SC), play a major role in constituting the blood-testis barrier and provide a unique microenvironment for the genesis and differentiation of germ cells. Hence SC strictly control sperm qualitative and quantitative parameters. We aimed to assess whether exposure to Cd would adversely affect superior mammal SC viability and function. We isolated and purified SC from pre-pubertal pig testes according to our method and incubated the retrieved cells with three different Cadmium chloride concentrations (5-10-15 microM). Parameters of SC function such as inhibin B and anti-Mullerian hormone (AMH) were depressed by Cd exposure, contrary to what observed in untreated controls. No impairment of the FSH receptor integrity on the SC, as assessed by 17-beta-estradiol production, upon stimulation with FSH, was observed in either 5 microM Cd-treated or untreated controls. Differences, on the contrary, were observed for higher Cd concentrations (10 and 15 mM), in terms of FSH receptor integrity, that was altered, as compared to untreated controls, in terms of lower production of 17-beta-estradiol. In addition, the apoptotic test showed a significant increase of early (ANNEXIN V-/Propidium Iodide+) (AV-/PI+) and late apoptotic cells (AV+/ PI+) in all Cd -treated SC conditions as compared to controls. In conclusion, the Cd -related toxicity on SC, clearly demonstrated by our study, even at low concentrations, is expected to damage spermatogenesis that directly is dependent upon retention of SC viability and function.

Treatment with anticancer agents induces dysregulation of specific Wnt signaling pathways in human ovarian luteinized granulosa cells in vitro.

Chemotherapy has been associated with premature ovarian failure and infertility in women with cancer. It is well known that anticancer drugs reduce the primordial follicle pool and harm the ovarian blood vascularization leading to ovarian atrophy. However, their mechanism of injury still remains unclear. The aim of this study was to identify the cellular mechanisms involved in the toxicity of chemotherapy drugs belonging to different classes on human ovarian luteinized granulosa cells (LGCs). Treatment with doxorubicin (DXR), paclitaxel (PC), and cisplatin (CP) affected LGCs viability by inducing apoptosis and downregulating both estrogen receptor ß and follicle-stimulating hormone receptor in a dose-dependent manner. Several members of the WNT signaling pathway are expressed in granulosa cells where they regulate follicle development, ovulation, and luteinization. Here we show that treatment with DXR, PC, and CP induced upregulation of WNT4 expression, whereas WNT3 expression was downregulated by DXR and PC and upregulated by CP. Analysis of the WNT3 downstream signaling pathway showed that total ß-catenin protein levels were reduced upon treatment with DXR and PC. Additionally, restoration of ß-catenin signaling by lithium chloride protected LGCs from the injury induced by chemotherapy. The in vitro LGC toxicity model described might represent a tool to identify components of specific signaling pathways, such as the Wnt pathway, that can be targeted in order to limit the follicular damage caused by chemotherapy.

Effects of flavonoids from semen cuscutae on the hippocampal-hypothalamic-pituitary-ovarian sex hormone receptors in female rats exposed to psychological stress.

OBJECTIVE: To investigate the effects of flavonoids from semen cuscutae (FSCs) on the hippocampal-hypothalamic-pituitary-ovarian sex hormone receptors in female rats exposed to psychological stress and to explore the related mechanism. MATERIALS AND METHODS: Flavonoids were obtained from semen cuscutae using solvent extraction and polyamide column chromatography. Sound, light, and electricity were combined into psychological stress for endocrine dysfunction model establishment in female rats. The effects of FSCs on estrogen receptor (ER) in the hippocampus, hypothalamus, and pituitaries, as well as on follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in the ovaries of the psychologically stressed rats were quantitatively analyzed using immunohistochemistry and image analysis. RESULTS: FSCs increased ER expression in the hippocampus, hypothalamus, and pituitaries, as well as LHR expression in the ovaries, but had no effect on FSHR expression in the ovaries. CONCLUSION: FSCs are an effective medicine in the treatment of ovarian endocrine dysfunction in psychologically stressed rats.

Pharmacogenetics of follicle-stimulating hormone action.

PURPOSE OF REVIEW: To review the current knowledge of genetic variants in the two genes affecting the individual responsiveness to follicle-stimulating hormone (FSH) action-the FSH beta-subunit (FSHB) and the FSH receptor (FSHR), as well as the pharmacogenetic ramifications of the findings. RECENT FINDINGS: Four common variants in the FSHB and the FSHR genes were shown to exhibit significant effect on FSH action: linked FSHR variants Thr307Ala and Asn680Ser determining common receptor isoforms, and gene expression affecting polymorphisms FSHR -29G/A and FSHB -211G/T. In women, the FSHR Thr307Ala/Asn680Ser polymorphisms show consistent predictive value for estimating the most optimal recombinant FSH dosage in controlled ovarian hyperstimulation (COH). The same variants exhibit a potential for the pharmacogenetic assessment of the treatment of polycystic ovarian syndrome. The FSHR -29G/A variant was also shown to contribute to ovarian response to COH. Pilot studies have suggested the FSHB -211 TT homozygous oligozoospermic men with genetically determined low concentration of FSH, as potentially the best responders to FSH treatment; furthermore, modulation of this response by FSHR polymorphisms is possible. SUMMARY: Genetic variants in FSHB and FSHR exhibit a potential for pharmacogenetic applications in selecting appropriate treatment options (timing and dosage) in male and female conditions requiring or benefiting from FSH therapy.

Clomiphene citrate resistance in relation to follicle-stimulating hormone receptor Ser680Ser-polymorphism in polycystic ovary syndrome.

BACKGROUND: Clomiphene citrate (CC) response in anovulatory women is difficult to predict and patient-tailored treatment would benefit patient care and time-management. The objective of this study was to evaluate the role of the follicle-stimulating hormone receptor (FSHR) Ser680Ser-polymorphism as a predictor for CC response. METHODS: In this retrospective study, 193 patients, diagnosed with polycystic ovary syndrome (PCOS) according to Rotterdam criteria and treated with ovulation induction, were included over a 5-year period in a university hospital in the Netherlands. Data on demographics, BMI, menstrual cycle, laboratory screening (including FSHR genotyping), transvaginal ultrasonography of ovaries and ovulation parameters were collected. Main outcome measures were response to CC and FSHR genotype. RESULTS: The frequency distribution of the 680-polymorphism was 26% (Asn/Asn), 50% (Asn/Ser) and 24% (Ser/Ser). No significant differences in basal characteristics were found. Significantly more patients with Ser/Ser-polymorphism were resistant to CC (28%) compared with Asn/Ser (14%) and Asn/Asn group (15%), with an odds ratio for ovulation of 0.44 (95% CI, 0.21-0.97). Patients with higher FSH levels, higher age and lower BMI were significantly more likely to ovulate in univariate analysis. In a multivariate logistic regression model, corrected for age, BMI, mean ovarian, volume, hyperandrogenism, and amenorrhoea, only FSHR and basal FSH levels were predictive for ovulation. CONCLUSIONS: Chance of resistance to CC is almost double in women with PCOS harbouring the Ser/Ser genotype.

Follicle-stimulating hormone treatment of male infertility.

PURPOSE OF REVIEW: Treatment with gonadotrophins is very effective in patients affected by hypogonadotrophic hypogonadism. The success of follicle-stimulating hormone (FSH) treatment in these men has brought the utilization of the same therapy in infertile oligozoospermic patients, aimed at obtaining a quantitative increase in sperm count. RECENT FINDINGS: FSH plays a crucial role in human reproduction. This physiological role in spermatogenesis has induced various attempts to treat idiopathic oligozoospermic men with FSH, often inducing the restoration of normal spermatogenesis and spontaneous pregnancy. However, the results obtained so far are still controversial. In this research, attention is focused on the possible criteria able to predict a seminal response to the specific hormonal treatment. Moreover, we have correlated different polymorphisms of FSH receptor gene with the outcome of FSH treatment. In this article, the literature is reviewed, and the authors' experience on using FSH treatment in oligozoospermic patients is discussed. SUMMARY: FSH treatment may represent a valid tool for infertile men. However, it should be performed on selected patients utilizing some predictive parameters able to identify a priori responder patients with high probability.

Differing pharmacological activities of thiazolidinone analogs at the FSH receptor.

The follicle-stimulating hormone is critical to reproductive success and is an important target for development of novel reproductive therapies. We have recently reported the development of thiazolidinone positive allosteric modulators of the follicle-stimulating hormone receptor. Here, we demonstrate that discrete modifications in the chemical structure of the thiazolidinone agonists produced compounds with different pharmacological properties. Positive allosteric modulators activated adenylate cyclase signaling (Gs). Using an ADP-ribosylation assay we found that both differing glycosylated variants of human FSH (hFSH) and selected thiazolidinone allosteric modulators of the FSHR induce activation of the Gi signaling pathway. Additionally, we observed that some analogs of this class could activate both pathways. These data suggest that the pharmacological activity of thiazolidinone modulators to the FSHR may be due to the ability of these compounds to induce association of the FSHR with either Gs or Gi signaling pathways in an analog-specific manner.

Effect of octapeptide: FSH binding inhibitor on cyclicity of bonnet monkeys (Macaca radiata).

BACKGROUND: The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone play an important role in the development of ovarian follicles, and a number of autocrine/paracrine factors secreted by the ovary are known to directly or indirectly regulate gonadotropin action. The objective of the present study was to elucidate the effect of octapeptide (OP) on cyclicity and hormonal profile of bonnet monkeys, the menstruating Old World primates. STUDY DESIGN: Our group has purified one such factor from human ovarian follicular fluid, which inhibits the binding of FSH to the granulosa cells. N-terminal eight-amino-acid sequence of this peptide has been deduced, which is referred to as the OP. It has shown an antifertility effect in marmosets, the New World primates. In the present study, the bonnet monkeys were divided into two groups, namely, the treated group (n=5), which was administered with OP (250 mcg/kg body weight/day) intramuscularly during the follicular phase, and the control group (n=6), which was injected with vehicle (saline). Blood was collected every other day, and progesterone levels were estimated by enzyme-linked immunosorbent assay. RESULTS: Animals in the control group demonstrated normal plasma progesterone levels and exhibited normal cyclicity. On the other hand, in the treated group, progesterone levels decreased by 65.8%, as compared with that in pretreatment cycles. This probably disturbed the cyclicity, thus causing amenorrhea (73.0+/-6.7 days).

Functional specificity of the rainbow trout (Oncorhynchus mykiss) gonadotropin receptors as assayed in a mammalian cell line.

In vertebrates, gonadotropins (GTHs) (FSH and LH) are two circulating pituitary glycoprotein hormones that play a major role in the regulation of gonadal functions, including gonadal cell proliferation/differentiation and steroidogenesis. In mammals, it is well known that their biological effects are mediated by highly specific membrane-bound receptors expressed preferentially on the somatic cells of the gonads. However, in fish, binding and functional studies have shown that cross-reactivity may occur in GTH receptors depending on the species. To understand the molecular mechanisms involved in GTH actions, functional characterization of trout GTH receptors and their gonadal gene expression pattern has been carried out. The present study describes the presence of two distinct GTH receptors in trout showing similarities with those of higher vertebrates but also differences in their structural determinants. In vitro functional studies demonstrate that rtLH specifically activates its cognate receptor (EC(50) = 117 ng/ml), whereas purified rainbow trout FSH (rtFSH) activates FSHR but also LHR at supraphysiological doses (EC(50) = 38 vs 598 ng/ml for FSHR and LHR respectively). The high doses of rtFSH required to activate LHR put into question the physiological relevance of this interaction. The use of heterologous chinook GTHs confirms the strong preference of each hormone for its cognate receptor. The gonadal expression pattern of the GTH receptor genes suggests that FSH may play an important role in regulating gonadal functions, not only at the early stages but also at the final stages of the male and female reproductive cycles, in addition to the LH pathway.

Polymorphism of the FSH receptor and ovarian response to FSH.

Follicle-stimulating hormone (FSH) is a key factor in human reproduction. FSH activates its receptor (FSHR) located exclusively on Sertoli cells in the testis and granulosa cells in the ovary. Two common single nucleotide polymorphisms (SNP) within exon 10 of the human FSHR gene result in two almost equally common allelic variants exhibiting threonine (Thr) or alanine (Ala) at position 307 in the hinge region, respectively, asparagine (Asn) or serine (Ser) at codon 680 of the intracellular domain. Clinical studies have demonstrated that p.N680S polymorphism determines the ovarian response to FSH stimulation in patients undergoing IVF-treatment. Patients with the Ser(680) allele need more FSH during the stimulation phase to reach the serum estradiol levels of Asn(680) patients. A study investigating women with normal, mono-ovulatory menstrual cycles revealed that the Ser(680)/Ser(680) genotype leads to higher FSH serum levels and a prolonged cycle. To date, the molecular mechanism underlying the partial "resistance" of the Ser(680)-FSHR to FSH remains unclear. Future experiments should extend our current understanding of FSH action on follicular selection and dominance, thereby permitting novel, patient-tailored therapies for infertility and fertility preservation.

Dichlorodiphenyltrichloroethane impairs follicle-stimulating hormone receptor-mediated signaling in rat Sertoli cells.

Any toxicant that affects Sertoli cell development can potentially disturb male fertility. So far, the effects of organochlorine compounds have been poorly investigated in male. Here, we studied the effects of dichlorodiphenyltrichloroethane (DDT), an organochloride pesticide, on Sertoli cells. DDT inhibited the cAMP response to follicle-stimulating hormone (FSH), the major endocrine control of Sertoli cell development, and to a beta2-agonist, isoproterenol. DDT exposure decreased the level of FSH binding sites. Direct adenylyl cyclase activation by Forskolin was unaltered by DDT, while the activation of Galphas by cholera toxin was decreased by DDT. The DDT inhibitory effect on the FSH response was also observed in Ser W3 cells, a Sertoli cell-derived immortalized cell line. All these effects were reproduced by the lipophilic aromatic bisphenol A but not by structurally unrelated CisPlatin. In conclusion, these results are a first step in understanding the molecular basis of DDT deleterious effects in spermatogenesis.

Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization.

FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized. In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts.A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin.

Follicle-stimulating hormone (FSH) activates extracellular signal-regulated kinase phosphorylation independently of beta-arrestin- and dynamin-mediated FSH receptor internalization.

BACKGROUND: The follicle-stimulating hormone receptor (FSH-R) is a seven transmembrane spanning receptor (7TMR) which plays a crucial role in male and female reproduction. Upon FSH stimulation, the FSH-R activates the extracellular signal-regulated kinases (ERK). However, the mechanisms whereby the agonist-stimulated FSH-R activates ERK are poorly understood. In order to activate ERK, some 7 TMRs require beta-arrestin-and dynamin-dependent internalization to occur, whereas some others do not. In the present study, we examined the ability of the FSH-activated FSH-R to induce ERK phosphorylation, in conditions where its beta-arrestin- and dynamin-mediated internalization was impaired. METHODS: Human embryonic kidney (HEK) 293 cells were transiently transfected with the rat FSH-R. Internalization of the FSH-R was manipulated by co-expression of either a beta-arrestin (319-418) dominant negative peptide, either an inactive dynamin K44A mutant or of wild-type beta-arrestin 1 or 2. The outcomes on the FSH-R internalization were assayed by measuring 125I-FSH binding at the cell surface when compared to internalized 125I-FSH binding. The resulting ERK phosphorylation level was visualized by Western blot analysis. RESULTS: In HEK 293 cells, FSH stimulated ERK phosphorylation in a dose-dependent manner. Co-transfection of the beta- arrestin (319-418) construct, or of the dynamin K44A mutant reduced FSH-R internalization in response to FSH, without affecting ERK phosphorylation. Likewise, overexpression of wild-type beta-arrestin 1 or 2 significantly increased the FSH-R internalization level in response to FSH, without altering FSH-induced ERK phosphorylation. CONCLUSION: From these results, we conclude that the FSH-R does not require beta-arrestin- nor dynamin-mediated internalization to initiate ERK phosphorylation in response to FSH.

Zebrafish gonadotropins and their receptors: I. Cloning and characterization of zebrafish follicle-stimulating hormone and luteinizing hormone receptors--evidence for their distinct functions in follicle development.

In the present study, we cloned and characterized zebrafish FSH receptor (Fshr) and LH receptor (Lhr). Both fshr and lhr were abundantly expressed in the zebrafish gonads; however, they could also be detected in the kidney and liver, respectively. When overexpressed in mammalian cell lines together with a cAMP-responsive reporter gene, zebrafish Fshr responded to goldfish pituitary extract but not hCG, whereas Lhr could be activated by both. It was further demonstrated that Fshr was specific to bFSH, while Lhr could be stimulated by both bovine FSH and LH. Low level of fshr expression could be detected in the immature ovary, but the level steadily increased during vitellogenesis of the first cohort of developing follicles. In contrast, the expression of lhr could barely be detected in the immature ovary, but it became detectable at the beginning of vitellogenesis and steadily increased afterward with the peak level reached at the full-grown stage. At the follicle level, the expression of fshr was very weak in the follicles of primary growth stage but significantly increased with the follicles entering vitellogenesis. However, after reaching the maximal level in the midvitellogenic follicles, the level of fshr expression dropped slightly but significantly at the full-grown stage. In comparison, the expression of lhr obviously lagged behind that of fshr. Its expression became detectable only when the follicles started to accumulate yolk granules, but the level rose steadily afterward and reached the peak at the full-grown stage before oocyte maturation. These results suggest differential roles for Fshr and Lhr in zebrafish ovarian follicle development.

Mechanisms of action of transforming growth factor beta on the expression of follicle-stimulating hormone receptor messenger ribonucleic acid levels in rat granulosa cells.

The present study was undertaken to identify the mechanisms underlying the effect of transforming growth factor (TGF) beta on FSH receptor (FSH-R) in rat granulosa cells. Compared to the control, the treatment of granulosa cells with TGFbeta (10 ng/ml) increased FSH-R mRNA transcripts (5.5 and 2.4 kilobases) in a time-dependent manner, with a maximum increase of approximately 2-fold at 48 h. We then investigated whether the effect of TGFbeta on FSH-R mRNA levels was the result of increased transcription and/or altered mRNA stability. To determine whether the FSH-R 5'-flanking region plays a role in directing FSH-R mRNA expression, the proximal area of the FSH-R 5'-flanking regions were inserted into an expression vector, pGL-Basic, which contains luciferase as the receptor gene, and the resulting plasmids were transiently transfected into rat granulosa cells. The FSH (30 ng/ml) significantly enhanced the activity of 1862 base pairs of the FSH-R 5'-flanking region, but treatment with TGFbeta did not significantly influence the activity induced by FSH. On the other hand, the decay curves for FSH-R mRNA transcript in primary granulosa cells showed a significant increase in half-life after the addition of TGFbeta. Transforming growth factor beta stimulates the expression of follistatin mRNA accumulation in a dose- and time-dependent manner. Treatment with activin produced a substantial increase in FSH-R mRNA level. Concurrent treatment with follistatin neutralized this activin effect on FSH-R mRNA, as reported, although concurrent treatment with follistatin did not affect TGFbeta-induced FSH-R mRNA. Therefore, the profile of the TGFbeta effect on FSH-R mRNA granulosa cells may be caused by the increased stability of FSH-R mRNA and insensitivity to the follistatin.

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.