FSH Receptor Levels Research Articles

Differential Regulation of Human and Mouse Myometrial Contractile Activity by FSH as a Function of FSH Receptor Density.

Previous studies from our laboratory revealed that the follicle-stimulating hormone receptor (FSHR) is expressed at low levels in nonpregnant human myometrium and that it is up-regulated in pregnant term nonlaboring myometrium; however, the physiological relevance of these findings was unknown. Herein, we examined signaling pathways stimulated by FSH in immortalized uterine myocytes expressing recombinant FSHR at different densities and showed that cAMP accumulation is stimulated in all cases but that inositol phosphate accumulation is stimulated only at high FSHR densities. Because an increase in cAMP quiets myometrial contractile activity but an increase in 1,4,5-triphosphoinositol stimulates contractile activity, we hypothesized that FSHR density dictates whether FSH quiets or stimulates myometrial contractility. Indeed, in human and mouse nonpregnant myometrium, which express low levels of FSHR, application of FSH resulted in a quieting of contractile activity. In contrast, in pregnant term nonlaboring myometrium, which expresses higher levels of FSHR, application of FSH resulted in increased contractile activity. Examination of pregnant mouse myometrium from different stages of gestation revealed that FSHR levels remained low throughout most of pregnancy. Accordingly, through mid-gestation, the application of FSH resulted in a quieting of contractile activity. At Pregnancy Day (PD) 16.5, FSHR was up-regulated, although not yet sufficiently to mediate stimulation of contractility in response to FSH. This outcome was not observed until PD 19.5, when FSHR was further up-regulated. Our studies describe a novel FSHR signaling pathway that regulates myometrial contractility, and suggest that myometrial FSHR levels dictate the quieting vs. stimulation of uterine contractility in response to FSH.

Downregulation of LH and FSH receptors after hCG and eCG treatments in the porcine oviduct

The influence of induction of ovulation and superovulation with eCG and hCG on LH and FSH receptor levels in porcine oviducts on day 3 postcoitum was studied. In experiment I, gilts were assigned into cyclic (control; n = 5) and inseminated (n = 5) groups. In experiment II, there were 3 groups of animals: inseminated (n = 5), induced ovulation/inseminated (750 IU eCG, 500 IU hCG; n = 5) and superovulated/inseminated (1500 IU eCG, 1000 IU hCG; n = 5) gilts. Oviduct tissues were collected 3 d after insemination or PBS infusion. The messenger RNA (mRNA) expression of FSH receptor (FSHR) and luteinizing hormone/chorionic gonadotropin receptor (LH/CGR) was measured by real-time reverse transcription PCR and protein levels using Western blots. Localization of LH/CGR and FSHR-positive cells was studied by immunohistochemical staining. Insemination by itself did not influence mRNA and protein levels of LH/CGR. However, FSHR mRNA expression in the isthmus and ampulla of the oviduct was affected by insemination (P < 0.05). Similarly, insemination decreased FSHR protein level in the isthmus (P < 0.05). Stimulation with hCG and eCG did not affect LH/CGR and FSHR mRNA expression, either in the isthmus or in the ampulla. Nevertheless, superovulation decreased LH/CGR protein level in the oviductal ampulla (P < 0.05) in comparison with inseminated gilts. Similarly, protein levels of FSHR in the oviductal ampulla decreased after superovulation (P < 0.05). LH/CGR-positive cells were observed in the mucosa as well as in smooth muscle cells of both parts of the oviduct. Follicle-stimulating hormone receptor–positive cells were observed in smooth muscle cells and blood vessels of the isthmus. In the ampulla, FSHR-positive cells were observed in the smooth muscle as well as in the mucosa. Summarizing, the present study revealed for the first time that stimulation with eCG and hCG, especially in high doses, can change LH/CGR and FSHR levels in porcine oviducts. This may in turn alter many signaling pathways, eg, PGs or vascular endothelial growth factor synthesis, and consequently disturb the oviductal environment, with possible detrimental effects on fertilization and/or embryonic development.

Zearalenone impairs the male reproductive system functions via inducing structural and functional alterations of sertoli cells.

The aim of this study was to investigate the effects of ZEA on the cytoskeletal structure, and factors specifically expressed by Sertoli cells. Primary Sertoli cells from rats aged 18-21 days were exposed to increasing ZEA concentrations (0, 5, 10, 20 μg mL(-1)) for 24 h. The results of immunofluorescence showed disruption of α-tubulin filaments and F-actin bundles, and damage to the nucleus of Sertoli cells on exposure to ZEA. In the control group, the protein level expression of androgen-binding protein (ABP), transferrin, vimentin, N-cadherin, and follicle-stimulating hormone receptor (FSHR) were decreased significantly (p<0.05, p<0.01). The mRNA levels of ABP, transferrin, vimentin, N-cadherin, and FSHR varied significantly in the experimental group (p<0.05). The results of enzyme-linked immunosorbent assay indicated a significant decrease in the levels of inhibin-β and transferrin in the cultural supernatants (p<0.05). Additionally, the ultrastructural analysis indicated the absence of mitochondria and Golgi apparatus, and presence of vacuoles in the cytoplasm. These findings showed that ZEA treatment can damage the cytoskeletal structure and affect specific secretory functions of Sertoli cells, which may be an underlying cause of ZEA-induced reproductive toxicity.

Equine chorionic gonadotropin influence on sheep oocyte in vitro maturation, apoptosis, and follicle-stimulating hormone receptor and luteinizing hormone receptor expression.

We assessed the effects of equine chorionic gonadotropin (eCG) on oocyte in vitro maturation (IVM), apoptosis, and follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and gonadotropin-releasing hormone receptor (GnRHR) expression and mRNA levels. Cumulus-oocyte complexes (COCs) were recovered from sheep ovaries and pooled in groups, before being cultured in IVM media containing varying eCG concentrations. Maturation and apoptosis rates were then calculated. Expression of FSHR, LHR, and GnRHR mRNA in oocytes was measured using quantitative reverse transcription polymerase chain reaction. Protein levels were ascertained by western blotting. Matured oocytes displayed and released an intact first polar body. Sheep oocyte maturation rates gradually increased as eCG concentration was raised from 0 to 20 µg/mL. Apoptosis rates of eCG-treated oocytes were lower than those of the control group, and were lowest using 20 µg/mL eCG. FSHR, LHR, and GnRHR mRNA expression increased (P < 0.01, P < 0.05, and P < 0.05, respectively, compared to 0 µg/mL eCG) with eCG concentration, being highest following exposure to 20 µg/mL. FSHR and GnRHR protein levels were significantly higher in oocytes administered 20 µg/mL eCG compared with those matured in the absence of eCG. eCG dose positively correlated with FSHR, LHR, and GnRHR mRNA and protein expression. In conclusion, eCG enhances maturation and decreases apoptosis of oocytes undergoing IVM, and heightens FSHR, LHR, and GnRHR expression. Such increased expression may facilitate oocyte IVM. These findings contribute to our understanding of the mechanisms of underlying hormonal control of sheep oocyte IVM, advancing ovine reproductive methods.

High levels of testosterone inhibit ovarian follicle development by repressing the FSH signaling pathway.

The effect of high concentrations of testosterone on ovarian follicle development was investigated. Primary follicles and granulosa cells were cultured in vitro in media supplemented with a testosterone concentration gradient. The combined effects of testosterone and follicle-stimulating hormone (FSH) on follicular growth and granulosa cell gonadotropin receptor mRNA expression were also investigated. Follicle growth in the presence of high testosterone concentrations was promoted at early stages (days 1-7), but inhibited at later stage (days 7-14) of in vitro culture. Interestingly, testosterone-induced follicle development arrest was rescued by treatment with high concentrations of FSH (400 mIU/mL). In addition, in cultured granulosa cells, high testosterone concentrations induced cell proliferation, and increased the mRNA expression level of FSH receptor (FSHR), and luteinized hormone/choriogonadotropin receptor. It was concluded that high concentrations of testosterone inhibited follicle development, most likely through regulation of the FSH signaling pathway, although independently from FSHR downregulation. These findings are an important step in further understanding the pathogenesis of polycystic ovary syndrome.

Cetrorelix and Triptorelin active immunization influences follicle development and receptor expressions of ovaries in mice

The study investigated comparatively the effects of active immunization of Cetrorelix and Triptorelin on the follicle development, histological structures, and expressions of FSHR and LHR proteins in the ovary. One hundred and five female mice, 35 days old, and body weight of 26.56±2.89g, were randomly assigned into Cetrorelix (CET), Triptorelin (TRI) and control groups (CG). Mice in CET-1, CET-2 and CET-3 (n=15) were subcutaneously injected with 0.1, 0.2 and 0.4mL Cetrorelix antigen (100μg/mL) for seven days, respectively. Mice in TRI-1, TRI-2 and TRI-3 were injected with 0.1, 0.2 and 0.4mL Triptorelin antigen (100μg/mL) for seven days. Mice in CG (n=15) were injected with 0.2mL saline for seven days. Western blotting was utilized to determine the expressions of follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) proteins. ELISA was used to measure the serum FSH and LH concentrations. The ovarian slices were observed under optical microscopy. The results showed that the ovarian weights of CET and TRI groups increased slightly with a maximum increment of 27.02% of CET-1 mice on day 35. Ovarian weights in TRI group increased dose-dependently with a maximum increment of 45.77% in TRI-3 on day 35. The numbers of the primordial follicle (POF), primary follicle (PF), secondary follicle (SF) and mature follicle (MF) of CET and TRI groups increased at different degrees when compared to CG. The granular layer in SF was arranged tightly and zona pellucida (ZP) was thickened. Follicles developed fully. Follicle longitudinal diameter (FLD), follicle transverse diameter (FTD) and follicle wall thickness (FWT) in CET and TRI groups increased as compared to CG. FLD, FTD and FWT of TRI-3 had a larger increment than CG and CET-1 on days 21 and 35. Expression levels of FSHR and LHR proteins in TRI group increased when compared to CG. Expression levels of FSHR and LHR proteins of CET group decreased. Serum FSH of TRI group was slightly higher than the CG and CET group on day 21. On day 35, serum FSH levels of CET-1 and TRI-1 were greater than CG and the rest of the subgroups. Serum FSH levels had no significant differences among all groups. Expression levels of LHR and FSHR proteins in CET group had obvious negative correlations to FLD, FTD and FWT. However, in the TRI group, these correlations were positive. In conclusion, Cetrorelix and Triptorelin immunization could improve ovarian growth, increase the follicle numbers, enhance dose-dependently the FLD, FTD and FWT, and eventually promote the ovary and follicle development. Cetrorelix decreased expression levels of FSHR and LHR proteins in the ovaries of mice. Triptorelin enhanced expression levels of FSHR and LHR proteins. Triptorelin treatment had more obvious effects than Cetrorelix treatment.

Fluoride exposure changed the structure and the expressions of reproductive related genes in the hypothalamus–pituitary–testicular axis of male mice

Numerous studies have shown that fluoride exposure adversely affected the male reproductive function, while the molecular mechanism is not clear. The present study was to investigate the effects of fluoride exposure (60days) on the expressions of reproductive related genes, serum sex hormone levels and structures of the hypothalamus–pituitary–testicular axis (HPTA), which plays a vital role in regulating the spermatogenesis in male mice. In this study, 48 male mice were administrated with 0, 25, 50, and 100mg/L NaF through drinking water. Results showed that the malformation ratio of sperm was significantly increased (P<0.05). At transcriptional level, the expression levels of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), inhibin alpha (INHα), inhibin beta-B (INHβB), and sex hormone binding globulin (SHBG) mRNA in testis were significantly decreased (P<0.05). Moreover, histological lesions in testis and ultrastructural alterations in hypothalamus, pituitary and testis were obvious. However, the same fluoride exposure did not lead to significant changes of related mRNA expressions in hypothalamus and pituitary (P>0.05). Also, there were no marked changes in serum hormones. Taken together, we conclude that the mechanism of HPTA dysfunction is mainly elucidated through affecting testes, and its effect on hypothalamus and pituitary was secondary at exposure for 60days.

PET of follicle-stimulating hormone receptor: broad applicability to cancer imaging.

Selective overexpression of follicle-stimulating hormone receptor (FSHR) inside the vascular endothelium of tumors has been confirmed to play critical roles in angiogenesis, tumor invasion, and metastases. The expression level of FSHR correlates strongly with the response of tumors to antiangiogenic therapies. In this study, an immunoPET tracer was developed for imaging of FSHR in different cancer types. A monoclonal antibody (FSHR-mAb) against FSHR was conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and used for subsequent (64)Cu-labeling. NOTA-FSHR-mAb preserved FSHR specificity/affinity, confirmed by flow cytometry measurements. (64)Cu-labeling was successfully conducted with decent yields (∼25%) and high specific activity (0.93 GBq/mg). The uptake of (64)Cu-NOTA-FSHR-mAb was 3.6 ± 0.8, 13.2 ± 0.7, and 14.6 ± 0.4 %ID/g in FSHR-positive CAOV-3 tumors at 4, 24, and 48 h postinjection, respectively (n = 3), significantly higher (p < 0.05) than that in FSHR-negative SKOV-3 tumors (2.3 ± 1.2, 8.0 ± 0.9, and 9.1 ± 1.3 %ID/g at 4, 24, and 48 h postinjection, respectively (n = 3)) except at 4 h p.i. FSHR-relevant uptake of (64)Cu-NOTA-FSHR-mAb was also readily observed in other tumor types (e.g., triple-negative breast tumor MDA-MB-231 or prostate tumor PC-3). Histology studies showed universal FSHR expression in microvasculature of these four tumor types and also prominent expression in tumor cells of CAOV-3, PC-3, and MDA-MB-231. Correlations between tumor FSHR level and uptake of (64)Cu-NOTA-FSHR-mAb were witnessed in this study. FSHR-specific uptake of (64)Cu-NOTA-FSHR mAb in different tumors enables its applicability for future cancer theranostic applications and simultaneously establishes FSHR as a promising clinical target for cancer.

Follicle-stimulating hormone induced epithelial-mesenchymal transition of epithelial ovarian cancer cells through follicle-stimulating hormone receptor PI3K/Akt-Snail signaling pathway.

It has previously been shown that follicle-stimulating hormone (FSH) and its receptor contribute to epithelial ovarian cancer (EOC) development. Epithelial-mesenchymal transition (EMT) is the early event of metastasis in cancer. Therefore, the aim of this study was to investigate the roles of FSH and the FSH receptor (FSHR) in EMT of EOC. Ovarian cancer cells treated with various doses of FSH were used to investigate the effect of FSH on EMT. Small interfering RNA-mediated FSHR depletion or reexpression of FSHR by acute transfecting pcDNA-hFSHR plasmid was performed to determine the role of FSHR in FSH-induced EMT. Moreover, LY294002, a potent and specific cell-permeable inhibitor of phosphatidylinositol 3-kinases (PI3K), was selected to pretreat ovarian cancer cells to confirm whether PI3K/Akt signaling is involved in this event. In the current study, FSH was found to induce the phenotypes of EMT including migration and invasion in EOC cells. Elevated FSHR levels promoted EMT, migration, and invasion, whereas small interfering RNA-mediated FSHR knockdown inhibited these processes. Moreover, the inhibition of FSH-induced PI3K/Akt signaling pathway attenuated Snail expression and the EMT process. Collectively, the findings of the current study indicate that FSH induced the EMT of ovarian cancer cells through the FSHR-PI3K/Akt-Snail signaling pathway.

The extracts of Pacific oyster (Crassostrea gigas) alleviate ovarian functional disorders of female rats with exposure to bisphenol a through decreasing FSHR expression in ovarian tissues.

Bisphenol-A (BPA) is one of the widespread industrial compounds, which has adverse effects on animal and human health. The study was aimed to explore the effects of Crassostrea gigas extracts (CGE) in alleviating ovarian functional disorders of female rats with exposure to BPA and the underlying possible mechanism. Eighteen four-week-old female Sprague-Dawley (SD) rats were randomly divided into BPA group (50mg/kg BPA), BPA+CGE group (50mg/kg BPA+50mg/kg CGE), and control group (equivalent dosage of vehicle) with 6 rats in each group. After a 6-week treatment ended, the serum levels of estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH) were measured by using commercial standard assay kits. The expression levels of FSH receptor (FSHR) in the rat ovarian tissues were respectively detected by immunohistochemistry and Real-time PCR. CGE treatment markedly increased E2 levels and decreased FSH levels in the serum (P<0.05), however, the alterations of serum LH levels were not significant (P>0.05). The protein and mRNA expression levels of FSHR were the lowest in the ovaries of control rats and the highest in BPA rats (P<0.05). CGE treatment markedly decreased the expression levels of FSHR in the ovarian tissues (P<0.05). Crassostrea gigas successfully alleviates ovarian functional disorders of female rats with exposure to BPA partly through decreasing FSHR expression levels in the ovarian tissues.

Lentivirus vector-mediated mitofusin-2 overexpression in rat ovary changes endocrine function and promotes follicular development in vivo.

The aim of the present study was to evaluate the expression and effect of rat mitofusin-2 (rMfn2) in the ovaries and other organs in rats. Rat models were developed by the intraovarian microinjection of an rMfn2-overexpressing lentiviral vector. Lenti-green fluorescent protein (GFP)-rMfn2 was microinjected into rat ovaries at a dosage of 2×106 tuberculin units virosome (n=25) and lenti-GFP was microinjected as a control (n=25). The expression of rMfn2 in the ovaries and other tissues was observed by fluorescence microscopy on days 7, 15, 30, 45 and 60 after the microinjection (n=5/day from each group). The serum levels of estradiol (E2), progesterone (P), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined by radioimmunoassay. Western blotting was used for the quantitative analysis of the expression of rMfn2 and the progesterone receptor (PR), estradiol receptor (ER), luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). The expression of rMfn2 was detected on day 7 after infection, increased with time and was maintained efficiently until day 60. In addition, rMfn2 was highly expressed in the fallopian tubes, uterus, cardiac muscle, liver and kidney, but expressed at a low level in adipose tissue. The serum levels of E2 and P in the model group were significantly increased compared with those in the control group, whereas the FSH and LH levels showed no significant difference between groups. The expression levels of the ER and PR in the model group were higher than those in the control group; however, no significant difference was observed between groups for the expression levels of LHR and FSHR. These findings suggest that the intraovarian microinjection of lenti-GFP-rMfn2 resulted in a significant time-dependent overexpression of rMfn2 in various organs, and that rMfn2 overexpression in rat ovaries changed the endocrine function and promoted follicular development.

Effects of Smad3 on the proliferation and steroidogenesis in human ovarian luteinized granulosa cells.

Granulosa cells (GCs) are essential for proper oocyte, follicular development, and steroidogenesis in the ovary. Transforming growth factor β (TGF-β) superfamily members are critical in regulating GCs growth and differentiation. Smad3 is known to serve as a signaling intermediate for the TGF-β; however, the functions of Smad3 in the human GCs remain unidentified. In this study, the luteinized GCs collected from follicular aspirates from patients undergoing in vitro fertilization were cultured and engineered to overexpress and knockdown Smad3, which were validated by RT-PCR and Western blotting. Immunocytochemistry showed that Smad3 protein was strongly expressed in human ovarian luteinized GCs. EdU incorporation demonstrated that Smad3 promoted the proliferation of GCs, and the expression of PCNA was also enhanced by Smad3. ELISA analysis indicated that the secretion of both estradiol and progesterone was stimulated by Smad3. In addition, Smad3 upregulated the level of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and protein kinase A (PKA) proteins. We subsequently added special PKA inhibitor H89 into the GCs and found that the stimulating effect on the growth of GCs by Smad3 was blocked partly. The morphology of cultured GCs was changed by Smad3, and the expression level of integrin β1 was enhanced by Smad3. Kindlin-2, an important cellular mediating molecule of integrin β signaling, was expressed in human ovarian luteinized GCs and was upregulated by Smad3. Our results indicated that Smad3 promoted the proliferation and steroidogenesis of human ovarian luteinized GCs, and these effects may be mediated by the FSHR/LHR-PKA signaling pathway.

Potential detrimental effect of soy isoflavones on testis sertoli cells

To determine the effect of soy isoflavones on cell proliferation and the transcription levels of follicle-stimulating hormone receptor (FSHR), inhibin α (INHα), INHβB, androgen binding protein (ABP), transferrin (Tf) and vimentin in testis sertoli cells in SD rats. Sertoli cells were cultured in vitro, exposed to daidzein at 0.03, 0.3, 3, and 30 μmol/L and genistein at 0.05, 0.5, 5 and 50 μmol/L, respectively. MTT was used to detect the proliferation of sertoli cells. Real-time PCR was used to detect the relative mRNA expressions of FSHR, INHα, INHβB, ABP, Tf and vimentin. Compared with control groups, cell proliferation and the relative mRNA expression levels of INHβB and ABP in the treated cells showed no significant alternation. The INHα mRNA expression levels were increased in 0.3 and 3 μmol/L Dai and 0.05 μmol/L Gen, while the mRNA expression levels of FSHR were downregulated in 30 μmol/L Dai and Gen at all concentrations. Tf mRNA expression levels were downregulated in 30 μmol/L Dai and 5 μmol/L and 50 μmol/L Gen, and the mRNA expression levels of vimentin were downregulated in 3 and 30 μmol/L Dai and 50 μmol/L Gen. Soy Isoflavones may have potential detrimental effect on the male reproductive system, as they may impact the function of sertoli cells by downregulating the transcription levels of some important proteins.

The effect of the intracervical application of follicle-stimulating hormone or luteinizing hormone on the pattern of expression of gonadotrophin receptors in the cervix of non-pregnant ewes.

During the periovulatory period, the cervix relaxes in response to changes in circulating concentrations of reproductive hormones. The present study investigated the role of gonadotrophins in cervical function by examining the expression of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) and their mRNAs following intracervical treatment with either FSH or LH. Eighteen ewes were assigned to four groups, and they were then treated with progestagen sponges and PMSG to synchronize their oestrous cycles. Intracervical treatments were given 24h after sponge removal as follows: Group 1: FSH 2mg; Group 2: LH 2mg; Group 3: Vehicle and Group 4: Control. Cervices were collected 54h after sponge removal and then divided into three regions. The expression of FSHR and LHR was determined by immunohistochemistry and FSHR mRNA and LH mRNA by in situ hybridization. The expression of LHR, FSHR and their respective mRNAs was compared in six tissue layers (luminal epithelium, subepithelial stroma, circular, longitudinal and transverse muscle and serosa) and in three cervical regions (vaginal, mid and uterine). The results showed that FSH increased transcription of the FSHR gene and the levels of its receptor, but only in subepithelial stroma of the cervix. FSH also increased the levels of LHR in the cervix, but only in the muscle layers. LH had no effect on the levels of FSHR despite the fact that it did increase the level of transcription of the FSHR gene and LH also increased the levels of its own receptor in the cervix, but only in the muscle layers, and this action was independent of increased levels of transcription of the LHR gene. These findings suggest multiple levels of regulation of cervical LH and FSH receptors and that the gonadotrophins may have a role in relaxation of the cervix during oestrus by regulating their own receptors.

The effects of 11-ketotestosterone on ovarian physiology of previtellogenic captive hapuku (Polyprion oxygeneios)

The present study investigated, for the first time in a perciform teleost, the effects of in vivo 11-ketotestosterone (11-KT) treatment using slow-release implants on ovarian development and gonadotropin receptor mRNA levels in captive previtellogenic females of hapuku (Polyprion oxygeneios). At the cellular/functional level, ovarian development and ovarian and hepatic total lipid levels were examined. At the molecular level, transcript abundance of ovarian follicle-stimulating hormone receptor (FSH-R) and luteinizing hormone receptor (LH-R) was measured. Additionally, cyclic adenosine monophosphate (cAMP) levels in ovarian fragments from placebo and 11-KT implanted fish incubated with or without human chorionic gonadotropin (hCG) in vitro were compared between groups. There were no significant differences between treatments with regard to oocyte size and lipid contents of liver and ovary. Messenger RNA levels of FSH-R and LH-R were significantly lower in the treated females. Similarly, cAMP levels were significantly lower in the ovarian fragments of the 11-KT implanted females. These results suggest that 11-KT specifically, but possibly androgens in general, may not have an important function in regulating gonadal development of previtellogenic female hapuku; indeed, if anything, 11-KT appeared to have a detrimental effect and its use will not be beneficial in advancing sexual maturity of hapuku in aquaculture.

Alarelin active immunization influences expression levels of GnRHR, FSHR and LHR proteins in the ovary and enhances follicular development in ewes

We investigated the effects of gonadotropin releasing hormone (GnRH) agonist on expressions of GnRH receptor (GnRHR), follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) proteins in the ovaries and follicular development in the ewes. Forty-two pre-pubertal ewes were assigned to experimental groups 1 to 5 (EG-I to EG-V) and control group (CG). Ewes in EG-I, EG-II and EG-III were subcutaneously injected with 200, 300 or 400 μg alarelin antigens twice (on days 0 and 14), respectively. Ewes in EG-IV and EG-V were subcutaneously injected with 200 μg and 300 μg alarelin antigen four times (on days 0, 7, 14 and 21). Ewes in CG were subcutaneously injected with a solvent twice (on days 0 and 14). Serum concentrations of GnRH antibody in the EGs increased and were higher than (P<0.05) that of CG from day 14 to day 60. GnRH antibody concentrations in EG-IV and EG-V were higher than that in EG-I, EG-II and EG-III from days 35 to 45. Expressions of GnRHR protein in EG-IV and EG-V were lower than that in CG (P<0. 01). Expressions of FSHR and LHR proteins in EGs increased. Levels of FSHR and LHR proteins in EG-IV and EG-V (P<0.05) were higher than CG. Ovarian weights in EGs increased. Values of follicle vertical diameter, follicle transverse diameter, follicle wall thickness, follicle externatheca thickness and follicle internatheca thickness in EG-III and EG-V were greater than other groups. Primordial follicles and primary follicles developed quickly in alarelin-immunized animals. Secondary follicles and mature follicles became more abundant. Mitochondria, mitochondrial cristaes and cortical granules increased. Serum FSH concentrations of EGs remained higher than that in CG from days 28 to 70 (P<0.05). Alarelin immunization stimulated GnRH antibody production, suppressed expression of GnRHR protein, enhanced expressions of FSHR and LHR proteins in ovaries, promoted FSH secretion and thereby accelerated the development of ovaries and follicles in ewes.

Manganese-Induced Effects on Testicular Trace Element Levels and Crucial Hormonal Parameters of Hyline Cocks

Manganese (Mn) is an essential element required for normal development and reproduction. However, little is known about the reproductive toxicity of Mn in birds. To investigate the Mn-induced toxicity on testicular trace element levels and crucial hormonal parameters on male reproduction in birds, 50-day-old male Hyline cocks were fed either a commercial diet or a Mn-supplemented diet. The changes in contents of copper (Cu), iron (Fe), zinc (Zn), and calcium (Ca) in testis were detected. Hormonal parameters were evaluated including the levels of testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) in the serum. The mRNA levels of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) were determined in this study. The results showed that Mn was accumulated in testis, and the content of Cu, Fe, Zn, and Ca decreased. Exposure to Mn significantly lowered the content of T, LH, FSH, and the mRNA expression levels of LHR and FSHR. Levels of T3 and T4 appeared with a decreased tendency, and TSH presented no obvious regularity. It indicated that Mn exposure resulted in the disbalance of testicular trace elements and influenced hormone levels in the molecular level, which may be possible underlying reproductive toxicity mechanism induced by Mn.

Cloning of Vasa and expression analysis during the reproductive cycle inCynoglossus semilaevisGünther

The complete cDNA sequence of follicle-stimulating hormone receptor( FSHR) gene was cloned by degenerate primer PCR amplification and RACE cDNA amplification for the first time. The length of the cDNA is 3105 bp,and it encodes a protein of about 704 amino acids,which contains the conserved seven transmembrane helix domains( TM helix) ,and it belongs to glycoprotein hormone receptor( GHR) family. Phosphorylation site predictions identified 19 Ser,4 Thr and 5 Tyr potential phosphorylation sites in tongue sole,and only one protein kinase C phosphorylation site( 441T) was predicted. The Clustal X alignment also revealed the presence of specific signature sequences( e. g. 82CCAF,481ERW,594FTD and 667NPFLY) ,which are highly conserved in GpHRs. And comparison with the halibut FSH-R also allowed the identification of 10 imperfect LRRs in the FSHR of Cynoglossus semilaevis. Tissue expression analysis showed that FSHR mRNA is expressed widely in C. semilaevis. Except the high levels in gonads,strong amplification signals were also detected in extragonadal tissues including the spleen,kidney and head kidney. Plasma 17β- Estradiol( E2) seasonal changes are detected by radio immunoassay( RIA) . The result shows that the lowest level appears in April and the highest peak of a year is in October. And we analyzed the relative expression level of FSHR in reproductive cycle of female. There is the highest level in October and the lowest level in January.

Levels of BMP-6 mRNA in goat ovarian follicles and in vitro effects of BMP-6 on secondary follicle development

Expression of BMP-6 mRNA was quantified by real-time polymerase chain reaction (PCR) and the BMP-6 protein was demonstrated by immunohistochemistry in the primordial, primary, secondary, small and large antral follicles of goat. Furthermore, the influence of BMP-6 on increase in diameter, antrum formation and expression of BMP-6 and FSH-R in in vitro cultured secondary follicles was studied. Therefore, goat primordial, primary and secondary follicles, as well as small and large antral follicles were obtained and the mRNA levels of BMP-6 were quantified by PCR in real time. Expression of BMP-6 protein in goat follicles was demonstrated by immunohistochemistry. The influence of BMP-6 in the presence or absence of follicle-stimulating hormone (FSH) on both the development of secondary follicles and the expression of mRNA for BMP-6 and FSH-R was evaluated after 6 days of culture. Furthermore, the follicular diameter and the formation of the antrum were evaluated before and after 6 days of culture and compared by Kruskal-Wallis and chi-squared tests (P < 0.05), respectively. The results show that the level of mRNA for BMP-6 in primary and secondary follicles was significantly higher than in the primordial follicles (P < 0.05). Similar levels of BMP-6 mRNA were observed in cumulus-oocyte complexes and mural granulosa/theca cells from small and large antral follicles, respectively. BMP-6 protein was expressed in oocytes of all categories of follicles and in granulosa cells from secondary follicles onwards. Addition of BMP-6 to the culture medium increased the diameter of secondary follicles mainly by antrum formation after 6 days' culture, in the presence or absence of FSH (P < 0.05). Furthermore, addition of FSH resulted in increased levels of BMP-6 mRNA in these follicles (P < 0.05). Simultaneous administration of FSH and BMP-6 enhanced the levels of FSH receptor (FSH-R) mRNA (P < 0.05). It is concluded that BMP-6 mRNA is increased during transition from primordial to primary/secondary follicles in the goat ovaries and that BMP-6 enhances the growth of cultured secondary follicles.

Patients with endometriosis and patients with poor ovarian reserve have abnormal follicle-stimulating hormone receptor signaling pathways

To study the integrity of the FSH receptor (FSHR) signaling pathway in granulosa-lutein cells at the time of egg retrieval and its relationship with the infertility diagnosis. In vitro assays. University laboratory and private IVF center. Patients undergoing IVF: 35 controls (no ovarian factor, NOF), 28 poor responders (PR), 32 patients with endometriosis (EM), and 22 patients with polycystic ovary syndrome (PCOS). Quantitative reverse transcriptase-polymerase chain reaction (PCR) in granulosa-lutein cells from pooled follicles. Correlation between expression of FSHR and FSH-regulated genes PAPP/Cyp19A1. Positive correlations among FSHR, PAPP, and Cyp19A1 expression levels are observed in NOF and are lost, with different patterns, in poor responder patients and those with endometriosis. Patients with endometriosis are an heterogeneous group including patients with poor ovarian reserve who behave like other poor responders patients (endometriosis-A) and patients with good response to ovarian stimulation (endometriosis-B) who show a specific alteration of the FSHR signaling pathway. These preliminary data suggest that the different signaling pathways activated through the FSHR in normal ovaries (NOF) are disrupted in poor responders and in patients with endometriosis. A better knowledge of the molecular origin of these errors may guide clinicians in the choice of personalized ovulation induction protocols for each typeof ovarian dysfunction.