Follicle-stimulating Hormone Expression Research Articles

Gene Expression of ACTH, Glucocorticoid Receptors, 11βHSD Enzymes, LH‐, FSH‐, GH Receptors and Aromatase in Equine Epididymal and Testicular Tissue

Glucocorticoids (GCs) are important mediators of the stress response and have been implicated in the function and regulation of testicular functions in different species. In many tissues, intracellular glucocorticoid activity is controlled by either or both of the two known isoforms of 11β-hydroxysteroid dehydrogenase (11βHSD) type 1 and 2, which interconvert active and inactive GCs. Little is known about the effects of stress on fertility in the equine species. The main objective of the present study was to investigate the expression of receptors for GCs and adrenocorticotropic hormone [ACTH, melanocortin 2 receptor (MC2R)] as well 11βHSD1 and 11βHSD2 in male equine epididymal and testicular tissue. In addition, expression of aromatase P-450 and receptors for luteinizing hormone (LHR), follicle stimulating hormone (FSHR) and growth hormone (GHR) was studied. Reverse transcriptase PCR and quantitative real-time PCR were performed in tissue from the epididymis (caput and cauda) and testes collected from nine healthy mature stallions (age 4-10 years). mRNA for ACTH and GC receptors as well as 11βHSD1 and -2 were found in epididymal and testicular tissue. Expression of the genes studied was always positive in testicular tissue, while it was inconsistent in epididymal tissue. Quantitative gene expression in relation to β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was significantly correlated (R = 0.403, p < 0.001). Quantitative PCR in relation to β-actin revealed significant differences in the gene expression of 11βHSD1, 11βHSD2, LHR, FSHR, MC2R and aromatase between tissue collected from caput epididymidis, cauda epididymidis and testicular parenchyma (p < 0.05). With GAPDH, differences between tissues were significant for 11βHSD1, 11βHSD2 and MC2R (p < 0.05) In addition, high concentrations of mRNA of aromatase and receptors of LH and FSH were found in testicular tissue, while a pronounced expression of GH receptor was present in epididymal tissue. The results support the hypothesis of an interaction between the pituitary-adrenal axis and testicular function in the stallion.

Follicle-Stimulating Hormone influences biliary epithelium growth in Autosomal Dominant Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder characterized by the development of renal cysts and various extrarenal manifestations, such as hepatic cysts that bud from biliary epithelium (Alvaro et al., 2008; Onori et al., 2010). We previously showed that Follicle-Stimulating Hormone (FSH) is a trophic factor for the biliary cells in normal rat and in experimental model of bile duct ligation. (Mancinelli et al, 2009). From these data, we aimed to investigate the role of FSH on biliary epithelium in ADPKD. In vivo evaluation of FSH receptor, FSH, p-ERK and c-myc expression in liver fragments from normal and ADPKD patients and in vitro PCNA and cAMP levels in normal human cholangiocytes (H69) and in a cell line obtained from the epithelium lining the hepatic cysts (LCDE) was performed. We found that FSH induces the proliferation of the cystic epithelium and co-localize with p-ERK and c-myc, proteins activated in cAMP signalling mechanism. In vitro FSH sustains cellular growth by the activation of cAMP/ERK signalling pathway with or without the transient silencing of the FSH gene in LCDE by siRNA. These results indicate that FSH has an important role in cystic growth via the cAMP pathway. FSH candidates as a possible target for medical therapy of hepatic cysts during ADPKD.

The Synthetic Gestagen Levonorgestrel Disrupts Sexual Development in Xenopus laevis by Affecting Gene Expression of Pituitary Gonadotropins and Gonadal Steroidogenic Enzymes

In the present study, Xenopus laevis tadpoles were chronically exposed to four concentrations of the synthetic gestagen Levonorgestrel (LNG; 10(-11), 10(-10), 10(-9), and 10(-8)M) starting at Nieuwkoop and Faber (NF) stage 48 until completion of metamorphosis. At NF 58 and 66, brain-pituitary and gonad samples were taken for gene expression analyses of gonadotropins and gonadal steroidogenic enzymes. Exposure to 10(-9) and 10(-8)M LNG until NF 58 repressed messenger RNA (mRNA) expression of luteinizing hormone (LH) β in both genders. This decrease was persistent after further treatment until NF 66 in the 10(-8)M LNG treatment. Expression of follicle-stimulating hormone (FSH) β was affected sex-specifically. No effect was present in NF 58 females, whereas LNG at 10(-9) and 10(-8)M significantly increased FSHβ mRNA levels in males. In NF 66 females, 10(-9)M LNG treatment increased FSHβ gene expression, whereas a decrease was observed in NF 66 males exposed to 10(-8)M LNG. In gonads, expression of steroid-5-alpha-reductase was affected sex-specifically with increased mRNA levels in females but repressed levels in males. Gene expression of further gonadal steroidogenic factors was decreased by 10(-8)M LNG in both genders at NF 66. Assessment of gonad gross morphology and histology revealed poorly developed testes in the 10(-8)M LNG treatment. Our results reveal considerable effects of chronic LNG exposure on sexual development of amphibians. The persistent inhibition of LHβ expression concomitant with decreased mRNA levels of gonadal steroidogenic enzymes is suggested to result in the disruption of reproduction in adult amphibians.

A study on expression of FSH and its effects on the secretion of insulin and glucagon in rat pancreas

Studies indicate that many tissues could express follicle-stimulating hormone (FSH) besides pituitary. New functions of FSH are also been recognized beyond reproduction regulation. However, no report has been made about the expression and function of FSH in rat pancreas yet. Dual-labeled immunofluorescence stain, in situ hybridization and dual-labeled immunohistochemistry stain in adjacent sections were used to study the expression of FSH and its receptor, and co-localization of FSH with gonadotropin-releasing hormone (GnRH) receptor in rat pancreas. Tissue incubation and enzyme-linked immunosorbant assay (ELISA) were used to study the effects of FSH on the secretion of insulin and glucagon in rat pancreas in vitro. The results showed that rat pancreas could express FSH and its receptor, some of islet cells co-expressed FSH and its receptor, some of islet cells co-expressed FSH and GnRH receptor. FSH has the same bidirectional regulation effects on insulin and glucagon in vitro. These data suggested that rat pancreas is a target organ of FSH, and GnRH might regulate FSH through GnRH receptor in rat pancreas. FSH might regulate the endocrine function of rat pancreas through FSH receptor.

Effects of Orexin A on mRNA Expression of Various Neuropeptides in the Hypothalamus and Pituitary, and on Serum LH Levels in Ovariectomized Gilts

Orexin has several biological functions, including the regulation of reproductive endocrine signaling, which has received much attention. However, little is known about the mechanism through which orexin regulates the levels of neuroendocrine hormones and peptides. We injected orexin A or physiological saline into the lateral ventricle of 10 ovariectomized (OVX) gilts, and determined the subsequent changes in serum luteinizing hormone (LH) concentration by using radioimmunoassay (RIA). We also examined the expression of GnRH, NPY, and POMC mRNAs in the hypothalamus and that of LH, follicle-stimulating hormone (FSH), POMC, and ghrelin mRNAs in the pituitary by using semi-quantitative reverse transcription polymerase chain reaction. We found the following results: (1) Orexin A transiently promoted LH secretion; serum LH concentration started to increase at 10 min after the orexin injection, peaked at 30 min, and returned to its initial level at 1.5 h; (2) orexin A upregulated GnRH mRNA expression and downregulated NPY and POMC mRNAs expression in the hypothalamus; (3) orexin A upregulated LH and FSH mRNAs expression (FSH, P > 0.05), but downregulated ghrelin mRNA expression in the pituitary. No significant effects were observed on the pituitary expression of FSH and POMC mRNAs. Our data suggest that orexin A regulates reproductive function by stimulating GnRH and LH release directly and indirectly via its effects on NPY, POMC and ghrelin expression.

125. ELEVATED FSH INCREASES PRIMORDIAL FOLLICLE RESERVE WITHOUT INCREASING PRIMORDIAL FOLLICLE FORMATION OR DECREASING OOCYTE QUALITY

Declining ovarian follicle reserve and oocyte quality with age can dictate the mammalian female reproductive lifespan. Elevated circulating levels of follicle-stimulating hormone (FSH) coincides with reproductive ageing in women, and is predicted to accelerate the depletion of ovarian follicle reserve by increasing the recruitment of remaining follicles. Unexpectedly, we found that transgenic expression of human FSH (TgFSH) increased primordial follicle reserve in mature GnRH-deficient hypogonadal (hpg) female mice functionally lacking endogenous gonadotrophin secretion1. Advancing our finding of increased primordial reserve in TgFSH-hpg mice, we find that total primordial follicle numbers is significantly increased (60%) in mature 26 week old non-hpg TgFSH females compared to WT controls. Thus, the FSH-induced increase in primordial reserve is maintained despite increased ovulation rate2 in mature TgFSH females. Embryo transfer experiments showed that embryos derived from 26 week old TgFSH females developed normally in recipient WT females, indicating no significant reduction in the viability of oocytes or early embryos produced by TgFSH mice. In addition, equal primordial numbers in 5 day old TgFSH versus WT ovaries suggested that FSH-induced changes to ovarian reserve was not due to an increased initial primordial follicle pool. Initial analysis of postnatal gene expression (qPCR) found elevated expression of granulosa cell markers (Kit-L, inhibin β) in TgFSH-hpg vs non-Tg hpg ovaries, indicating FSH actions during early follicle development. Therefore, contrary to a classical view that preantral follicles are gonadotrophin-independent, we provide in vivo evidence demonstrating FSH-induced changes to early follicle populations, as well as elevated FSH activity increasing or maintaining primordial reserve without compromising oocyte quality. (1) Allan CM et al. J Endocrinol. 2006; 188(3): 549–57.(2) McTavish KJ et al. Endocrinology. 2007; 148(9): 4432–9.

Identification of Ovarian Genes Regulated by Follicle-Stimulating Hormone During Early Secondary Oocyte Growth in Salmon.

It is well established in mammals that follicle-stimulating hormone (FSH) plays a major role in regulating oocyte growth and development by modulating the expression of follicle cell and oocyte factors. In fishes, however, little is known about the function(s) of FSH and regulation of early oocyte growth, a period of development that may affect later egg quality and fecundity. In species that exhibit synchronous gonad development (e.g., salmonids, catfish, striped bass), plasma FSH and/or pituitary gonadotropin subunit expression increases significantly during early secondary oocyte growth and continues to rise during vitellogenesis, suggesting FSH may play a role in regulating follicle development during this period. The goal of this study was to gain a fundamental understanding of how FSH acts at the level of the fish ovary by identifying FSH regulated ovarian genes. To examine time course effects of FSH, coho salmon (Oncorhynchus kisutch) previtellogenic ovarian follicles were cultured with or without 500 ng salmon FSH for 12-72 h. Culture media was collected for estradiol-17beta (E2) measurement, ovarian RNA was isolated, and abundance of candidate oocyte and follicle cell mRNAs was measured by quantitative PCR. Medium E2 levels were significantly elevated in response to FSH by 12 h and plateaued after 48 h. Messenger RNAs encoding steroidogenic enzymes, such as steroid acute regulatory protein (star) and 3beta-hydroxysteroid (hsd3b) were upregulated by 12 h, while P450 aromatase (cyp19a1a) showed no change in response to FSH. Other putative follicle cell transcripts such as FSH receptor (fshr) and lipoprotein lipase (lpl) were depressed initially by FSH treatment and later recovered to control levels. Putative oocyte transcripts such as zona pellucida glycoproteins, alveolin, and a C-type egg lectin showed no change over the 72-h experiment. Additional FSH regulated ovarian genes were subsequently identified through construction of four suppression subtractive hybridization (SSH) libraries with RNA isolated from FSH-treated and untreated whole ovarian follicles, as well as follicle/interstitial cell enriched ovarian tissues. Many of the genes identified by SSH are involved in signal transduction, cell cycle regulation, cell growth and proliferation, apoptosis, cholesterol metabolism and steroidogenesis. Experiments utilizing steroidogenesis inhibitors are underway to distinguish ovarian genes directly regulated by FSH versus those regulated by sex steroids produced in response to FSH. As evident by the rapid and significant increase in E2 release and changes in steroidogenic enzyme gene expression within the ovary, we can conclude that FSH strongly influences steroidogenesis of the salmon ovary during early secondary oocyte growth and may be a critical signal for puberty onset. This is the first comprehensive assessment of the biological actions of FSH in the fish ovary. Discoveries will aid in developing methods to reduce common reproductive dysfunctions in aquaculture, such as delayed puberty and poor egg quality. This project was supported in part by National Research Initiative Competitive Grant no. 2007-35203-18082 from the USDA Cooperative State Research, Education, and Extension Service to JAL. (platform)

Neonatal Estrogenic Effects upon the Male Rat Pituitary: Early Gonadotrophin Attenuation Precedes Long-term Recovery

Neonatal exposure to potent estrogenic compounds can affect multiple components of the male reproductive system causing impaired development of the epithelium and overgrowth of stromal tissue in the epididymis, vas deferens, seminal vesicles, and prostate. However, very little is known about the direct effects of estrogenic compounds on the anterior pituitary gland. In this study we have investigated the effects of neonatal estrogenic exposure upon the anterior pituitary. Both the early- and late-stage effects of exposure to a synthetic estrogenic agent, diethylstilbestrol (DES), upon pituitary gonadotroph cell function were assessed. We administered either a high dose (10 microg) or a low dose (0.1 microg) of DES to male rats during their neonatal period (P2-12). Gonadotroph function, cell number and morphology shortly after DES treatment (P18) and during adulthood (P90) were assessed. At P18 there was a significant decrease in follicle stimulating hormone (FSH) immunoreactivity in the pituitary gonadotroph cells in the high DES dose treated rats compared to control animals. No significant change in luteinizing hormone (LH) was observed at either DES dose. In adulthood (P90), there was no significant difference in FSH or LH gonadotroph immunoreactivity between control rats and any dose of DES-treated rats. Therefore, despite acute and selective ablation of FSH expression the gonadotrophs were able to recover in adulthood, suggesting that perinatal estrogenic exposure was only temporarily deleterious.

Follicle-stimulating hormone increases cholangiocyte proliferation by an autocrine mechanism via cAMP-dependent phosphorylation of ERK1/2 and Elk-1

Sex hormones regulate cholangiocyte hyperplasia in bile duct-ligated (BDL) rats. We studied whether follicle-stimulating hormone (FSH) regulates cholangiocyte proliferation. FSH receptor (FSHR) and FSH expression was evaluated in liver sections, purified cholangiocytes, and cholangiocyte cultures (NRICC). In vivo, normal female and male rats were treated with FSH or immediately after BDL with antide (a gonadotropin-releasing hormone antagonist blocking FSH secretion) or a neutralizing FSH antibody for 1 wk. We evaluated 1) cholangiocyte proliferation in sections and cholangiocytes and 2) changes in secretin-stimulated cAMP (functional index of cholangiocyte growth) levels, and ERK1/2 and Elk-1 phosphorylation. NRICC were stimulated with FSH before evaluation of proliferation, cAMP/IP(3) levels, and ERK1/2 and Elk-1 phosphorylation. To determine whether FSH regulates cholangiocyte proliferation by an autocrine mechanism, we evaluated the effects of 1) cholangiocyte supernatant (containing FSH) on NRICC proliferation and 2) FSH silencing in NRICC before measuring proliferation and ERK1/2 and Elk-1 phosphorylation. Cholangiocytes and NRICC express FSHR and FSH and secrete FSH. In vivo administration of FSH to normal rats increased, whereas administration of antide and anti-FSH antibody to BDL rats decreased 1) ductal mass and 2) secretin-stimulated cAMP levels, proliferation, and ERK1/2 and Elk-1 phosphorylation in cholangiocytes compared with controls. In NRICC, FSH increased cholangiocyte proliferation, cAMP levels, and ERK1/2 and Elk-1 phosphorylation. The supernatant of cholangiocytes increased NRICC proliferation, inhibited by preincubation with anti-FSH antibody. Silencing of FSH gene decreases cholangiocyte proliferation and ERK1/2 and Elk-1 phosphorylation. Modulation of cholangiocyte FSH expression may be important for the management of cholangiopathies.

Differential regulation of gene expression and release of FSH and prolactin by long day and sulfamethazine in chicks

In several avian species long day exposure results in plasma elevation of gonadotropins and prolactin (PRL). We examined the early (12–72 h) effects of photostimulation on mRNA transcripts and plasma levels of follicle stimulating hormone (FSH) and PRL in three-week old cockerels. In addition, the neuroendocrine influence of the compound, sulfamethazine (SMZ), known to enhance light-induced gonadal development in chicks, was studied when applied with or without long-day photostimulation. Both long day exposure and SMZ intake caused a rapid increase in FSHβ mRNA transcripts at Zeitgeber time 48 (ZT48), while only SMZ stimulated secretion of the hormone into plasma during the course of the study. In contrast to SMZ treatment, photostimulation was more effective at stimulating PRL mRNA transcripts and secretion of PRL. Results demonstrate a differential role of long day exposure and SMZ intake on the regulation of FSH and PRL synthesis and secretion and suggest that some effects of SMZ on gonadal development may be mediated by the pituitary.

Response of cryopreserved ovarian tissue after autologous implantation in mouse stimulated with gonadotrophin

To investigate the response of cryopreserved ovarian tissue after autologous implantation in mouse stimulated with gonadotrophin. Thirty six female mice were randomly divided into three groups, with 12 mice in each group. In group of fresh ovarian tissue, fresh ovarian tissue was implanted into kidney capsule of mice; in group of cryopreserved ovarian tissue, ovarian tissue was implanted into kidney capsule of mice after cryopreserved by vitrification for two weeks. We investigated the response of ovarian tissue two weeks later after autologous implantation stimulated with gonadotrophin. Immunohistochemistry staining method was used to observe the expression of follicle stimulating hormone receptor. Before and after stimulation with gonadotrophin, the mature follicle rate of group of fresh ovarian tissue was 2.3% and 4.2%, that of group of cryopreserved ovarian tissue was 2.3% and 4.0%, and that of group of control was 2.6% and 5.8%. Regarding the percentages of mature follicle, there were significant differences after stimulation with gonadotrophin (P < 0.05). After stimulating with gonadotrophin the percentages of mature follicle were the same in the fresh tissue group, cryopreserved tissue group and control group (P > 0.05). The integrated optical density of follicle stimulating hormone receptor of fresh ovarian tissue in antrofollicle and pre-antrofollicle were 9408 +/- 2777 and 4531 +/- 1903, that of cryopreserved ovarian tissue were 9175 +/- 3093 and 4808 +/- 1386, and that of the control ovarian tissue were 8838 +/- 2064 and 5516 +/- 1136 respectively. There was no significant difference between any two groups (P > 0.05). The follicle stimulating hormone receptor is preserved by cryopreservation and transplantation, small pieces of ovarian tissue response to gonadotropin stimulation is normal.

A Longitudinal study of the expression of Follicle Stimulating Hormone in Prepubertal Mice

Follicle‐stimulating hormone (FSH) is a glycoprotein secreted from the anterior pituitary. Like other glycoproteins, FSH exists as a mixture of isoforms that vary in the number and type of sugar groups. Observed changes in FSH isoforms during critical reproductive events, such as puberty onset, suggest that different combinations of FSH isoforms influence reproduction differently. It has also been suggested that all FSH isoforms may not equally bind to antibody based assays. In order to establish a working model, a study was performed using pre‐pubertal mice to determine at what age FSH mRNA expression begins. Gene expression was determined for the alpha, FSH beta, LH beta, and TSH beta subunits in five‐, ten‐, fifteen‐, twenty‐, thirty‐, and sixty‐day‐old mice. All subunits were expressed in all age groups indicating that FSH expression begins very early in development. However, despite the expression of mRNA, morphological studies do not reflect an active form of FSH is initiating gonad development, hormone production, or gametogenesis. Early expression of FSH may be critical to establish a hormonal milieu that leads to maturation of the gonads. Another possibility is that different isoforms of FSH determine its physiological significance during different metabolic states.

Expression of follicle-stimulating hormone and luteinising hormone binding sites in the bitch ovary during the follicular phase

In the female dog, in contrast with most mammals, the growing follicle starts to luteinise several days before ovulation. Little is known about the physiological control of the final follicular growth in this species. In order to better understand the pituitary regulation of follicular growth, specific binding sites for FSH and LH were localised and quantified by autoradiography using [(125)I]-porcine (p) gonadotrophins on ovarian sections (7 microm) from adult Beagle bitches during the follicular phase. Follicles were analysed either before the LH surge (n = 4 bitches; n = 117 follicles) or after the LH surge and before ovulation (n = 5 bitches; n = 110 follicles). FSH binding sites were specifically and homogeneously expressed at high levels on granulosa cells of all healthy follicles from the preantral stage onwards. In contrast, LH binding sites were detected homogeneously and at high levels only on granulosa cells of follicles larger than 1 mm in diameter, including luteinised follicles. Theca binding of LH (but not FSH) was also observed, but only when using high concentrations of [(125)I]-pLH. The overall incidence of atresia was 45.8% and was dependent upon follicular diameter. Quantitative analysis of labelling showed that atretic follicles had reduced levels of both FSH and LH binding sites compared with healthy follicles. In healthy follicles, levels of both FSH and LH binding sites changed with follicle diameter. Compared with other mammals, the acquisition of LH binding on canine granulosa cells occurs in smaller sized follicles relative to the size of ovulation.

Expression of gonadotropin and gonadotropin receptor genes during early sexual maturation in male Atlantic salmon parr

Atlantic salmon males may mature already as small parr in freshwater. Sexual maturation in teleosts as in vertebrates is characterized by the activation of the brain-pituitary-gonad axis. The endocrine regulation of early puberty is still not well understood. In the present study, one-summer-old male Atlantic salmon parr were sampled regularly from December several months prior to the beginning of spermatogenesis until spawning in October. Pituitary expression levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) beta subunit genes were analyzed in parallel with testis expression of FSH receptor (FSHR) and LH receptor (LHR) genes by RT-PCR and plasma 11-ketostestosterone (11-KT) was measured. Expression levels of FSHbeta, low during winter and spring started to increase prior to the onset of gonadal growth at the end of May while LHbeta mRNA levels were hardly detectable. Both gonadotropin receptor genes were expressed in immature testis with FSHR transcripts being more abundant (8-fold). FSHR transcript levels increased in parallel to FSHbeta levels from early spermatogenesis onwards, while LHR mRNA started to increase prior to any large changes in LHbeta expression. Both transcript levels of LHbeta and LHR were highest during spermiation. Plasma 11-KT increased at the beginning of spermatogenesis reaching highest levels at spermiogenesis suggesting a possible role of FSH in inducing 11-KT production during early spermatogenesis while LH stimulates via its specific receptor 11-KT production at spermiogenesis. The commitment into sexual maturation appears to be dependant on both the presence of FSHR in immature testis and the increase of FSH expression.

Expression of follicle stimulating hormone and luteinizing hormone receptors during follicular growth in the domestic cat ovary

In order to better understand the pituitary regulation of follicular growth in the domestic cat, follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors (R) were localized and quantified in relation to follicle diameter and atresia using in situ ligand binding on ovarian sections. Expression of FSHR was homogeneous and restricted to follicle granulosa cells from the early antral stage onwards, whereas expression of LHR was heterogeneous on theca cells of all follicles from the early antral stage onward, and homogeneous on granulosa cells of healthy follicles larger than 800 microm in diameter and in corpora lutea. LHR were also widely expressed as heterogeneous aggregates in the ovarian interstitial tissue. Atretic follicles exhibited significantly reduced levels of both FSHR and LHR on granulosa cells, compared with healthy follicles whatever the follicular diameter, whereas levels of LHR on theca cells were lower only for atretic follicles larger than 1,600 microm in diameter. In healthy follicles, levels of FSHR and LHR in all follicular compartments increased significantly with diameter. Although generally comparable to that observed in other mammals, the expression pattern of gonadotropin receptors in the cat ovary is characterized by an early acquisition of LHR on granulosa cells of growing follicles and islets of LH binding sites in the ovarian interstitial tissue.

Altered gene expression: A mechanism for reproductive toxicity in zebrafish exposed to benzo[ a]pyrene

Exposure of fish to polycyclic aromatic hydrocarbons (PAHs) has consistently been shown to have a negative impact on reproduction (e.g. decreased spawning success, decreased ovarian somatic index (OSI) and lower circulating levels of 17β-estradiol and vitellogenin). While an understanding of the mechanism behind these changes has yet to be fully elucidated, it has been proposed that PAHs can alter the expression of genes important in regulating reproduction. The objectives of this study were to: (1) determine the effect of exposure to waterborne benzo[ a]pyrene (B[ a]P) (0, 1.5 and 3.0 μg/L) for 56 days on egg production and OSI in female zebrafish ( Danio rerio) and (2) determine the effect of B[ a]P on transcription of genes involved in reproduction including gonadotropins (follicle stimulating hormone (FSH) and luteinizing hormone (LH)), steroidogenic enzymes (CYP11A1, CYP17, CYP19A1, CYP19A2 and 20β-HSD), estrogen receptor β (ERβ) and vitellogenin. Cytochrome P4501A1 (CYP1A1) was also measured in the liver and heads as an indicator of exposure to B[ a]P. A reduction in total egg output was observed in B[ a]P exposed fish as well as a decrease in OSI in fish exposed to 3.0 μg/L B[ a]P. A significant increase in CYP1A1 expression in the heads as compared to the control was observed whereas no significant difference in CYP1A1 was detected in livers. A significant increase in 20β-HSD mRNA occurred in heads and pre-vitellogenic oocytes from fish exposed to 1.5 and 3.0 μg/L as compared to the controls. CYP19A2 and vitellogenin were significantly increased following exposure to 3.0 μg/L in the heads and liver, respectively. No effects on the expression of FSH, LH, CYP19A1 or ERβ were observed. Results from this study demonstrate that reproduction in zebrafish is affected by waterborne exposure to B[ a]P and that altered transcription of genes important in regulating reproduction (20β-HSD, CYP19A2 and vitellogenin) may be one of the underlying mechanisms of B[ a]P-induced reproductive toxicity.

Differential Regulation of Luteinizing Hormone and Follicle-Stimulating Hormone Expression during Ovarian Development and under Sexual Steroid Feedback in the European Eel

Pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are, in teleosts as in mammals, under the control of hypothalamic factors and steroid feedbacks. In teleosts, feedback regulations largely vary depending on species and physiological stage. In the present study the regulation of FSH and LH expression was investigated in the European eel, a fish of biological and phylogenetical interest as a representative of an early group of teleosts. The eel FSHβ subunit was cloned, sequenced and together with earlier isolated eel LHβ and glycoprotein hormone α (GPα) subunits used to study the differential regulation of LH and FSH. In situ hybridization indicated that FSHβ and LHβ are expressed by separate cells of the proximal pars distalis of the adenohypophysis, differently from the situation in mammals. The profiles of LHβ and FSHβ subunit expression were compared during experimental ovarian maturation, using dot-blot assays. Expression levels for LHβ and GPα increased throughout ovarian development with a positive correlation between these two subunits. Conversely, FSHβ mRNA levels decreased. To understand the role of sex steroids in these opposite variations, immature eels were treated with estradiol (E₂)and testosterone (T), both steroids being produced in eel ovaries during gonadal development. E₂ treatment induced increases in both LHβ and GPα mRNA levels, without any significant effect on FSHβ. In contrast, T treatment induced a decrease in FSHβ mRNA levels, without any significant effect on the other subunits. These data demonstrate that steroids exert a differential feedback on eel gonadotropin expression, with an E₂-specific positive feedback on LH and a T-specific negative feedback on FSH, leading to an opposite regulation of LH and FSH during ovarian development.

Analysis of the Cys82Arg mutation in follicle-stimulating hormone beta (FSHβ) using a novel FSH expression vector

ObjectiveTo determine the effect of the Cys82Arg FSHβ mutation from a patient with isolated FSH deficiency upon follicle-stimulating hormone (FSH) levels in vitro. DesignIn vitro analysis of the Cys82Arg mutation and comparison with the phenotype. SettingTertiary medical center setting. Patient(s)DNA sequence of the FSHβ gene and clinical description from a patient with isolated FSH deficiency. Intervention(s)Construction of a new vector containing the cDNAs for the α-subunit and β-subunit of FSH (pαFSHβ) followed by mutagenesis and transfection into Chinese hamster ovary cells. Main outcome measure(s)Immunoreactive and bioactive FSH levels from the CHO cellular media. Result(s)Although expression of both subunits was present, both immunoreactive and bioactive FSH levels were unmeasurable from cellular media containing the mutation versus wild type. Conclusion(s)The Cys82Arg mutation in a male with normal puberty and azoospermia results in profound deficiency of FSH in vitro, thereby confirming the molecular basis of hypogonadism in this patient and documenting the importance of the Cys residue at position 82 of the FSHβ subunit.

Contribution of endogenous inhibin to the decline of the secondary surge of follicle-stimulating hormone in the rat.

The involvement of inhibin in the decline of the secondary surge of follicle-stimulating hormone (FSH) was investigated in the rat. After ovariectomy or treatment with inhibin antiserum conducted at 2300 hours during pro-oestrus, plasma concentrations of FSH were maintained at high levels compared with control rats. However, plasma FSH started to decline at 0500 hours during oestrus in both the groups. The same treatments conducted during metoestrus markedly increased plasma FSH after 24 h (twofold compared with the treatments during pro-oestrus), suggesting that the treatments sufficiently depleted circulating inhibin. To examine whether the decline of plasma FSH occurred through a transcriptional mechanism or through a translational mechanism, FSH-beta mRNA expression and the pituitary concentration of FSH were measured. Neither ovariectomy nor inhibin immunization conducted during the night of pro-oestrus, affected the pituitary concentration of FSH after 24 h, whereas a noticeable increase was observed after the treatments conducted during metoestrus. In both stages, both ovariectomy and inhibin immunization significantly increased FSH-beta mRNA expression compared with control rats. In contrast with the pituitary concentration of FSH, the effect of inhibin immunization on FSH-beta mRNA expression was not different between the stages. The present data demonstrate the involvement of inhibin in the decline of the secondary surge of FSH, and suggest that a factor or factors other than inhibin may also be responsible for the fall in FSH. Changes in the pituitary concentration of FSH and FSH-beta mRNA expression suggest that post-transcriptional mechanisms may be involved in the suppression of FSH secretion during oestrus.

LbetaT2 gonadotroph cells secrete follicle stimulating hormone (FSH) in response to active A.

Secretion of luteinizing hormone in response to gonadotropin releasing hormone (GnRH) has been described in the recently developed LbetaT2 gonadotroph cell line. We evaluated the expression of follicle stimulating hormone (FSH)beta mRNA and secretion of FSH from LbetaT2 cells in response to GnRH and activin A. LbetaT2 cells were treated with activin A in doses from 0 to 50 ng/ml, with or without a daily 10 nM GnRH pulse, or with GnRH alone. FSH secretion was stimulated over 6-fold by concomitant GnRH and activin A in a dose-responsive fashion at 72 h of treatment. FSHbeta mRNA was detectable by ribonuclease protection assay only in cells treated with activin A with or without GnRH. The demonstration of FSHbeta gene expression in LbetaT2 cells further validates these cells as mature, differentiated gonadotrophs and as an important tool for the study of gonadotroph physiology.