Expression Of FSHR Research Articles

Which follicles make the most anti-Mullerian hormone in humans? Evidence for an abrupt decline in AMH production at the time of follicle selection

Anti-Müllerian hormone (AMH) is exclusively produced by granulosa cells (GC) of the developing pre-antral and antral follicles, and AMH is increasingly used to assess ovarian function. It is unclear which size follicles make the most AMH (total content) and are the main contributors to circulating AMH concentrations. To determine AMH gene expression in GC (q-RT-PCR) and follicular AMH production (Elisa and RIA) in relation to follicular development, 87 follicles (3-13 mm diameter) including both GC and the corresponding follicular fluid (FF) were collected in connection with fertility preservation of human ovaries. Further, follicle number and diameter, graded in 1 mm increments, were determined by 3D ultrasound in 113 women in their natural menstrual cycle to determine follicle number and diameter in relation to circulating AMH levels. This study demonstrates for the first time a positive association between AMH gene expression in human and both total follicular fluid AMH (P < 0.02) and follicular fluid AMH concentration (P < 0.01). AMH gene expression and total AMH protein increased until a follicular diameter of 8 mm, after which a sharp decline occurred. In vivo modelling confirmed that 5-8 mm follicles make the greatest contribution to serum AMH, estimated for the first time in human to be 60% of the circulating concentration. Significant positive associations between gene expression of AMH and FSHR, AR and AMHR2 expression (P < 0.00001 for all three) and significant negative association between follicular fluid AMH concentration and CYP19a1 expression were found (P < 0.0001). Both AMH gene expression (P < 0.02) and follicular fluid concentration of AMH (P < 0.00001) correlated negatively with estradiol concentration.

Sexual Dimorphism of Steroidogenesis Regulated by GnIH in the Goldfish, Carassius auratus1

Gonadotropin-inhibitory hormone (GnIH) has been shown to inhibit reproduction in several species. GnIH suppresses gonadotropin synthesis/release at the hypothalamic and pituitary levels; however, increasing evidence suggests that GnIH has a putative function in the gonad. In this study, we demonstrated that GnIH receptors localize to the ovary and testis in goldfish. In situ hybridization illustrated that goldfish GnIHRs were localized exclusively to the oocytes before the cortical alveolus stage and to the interstitial tissue to the testis. Implantation of goldfish GnIH peptides did not affect the serum estradiol levels in female goldfish, but it did enhance the serum testosterone levels in males. Conversely, injecting goldfish GnIH peptides increased the expression of StAR and 3bHSD mRNA and decreased the expression of CYP19 mRNA significantly in the testis, but these genes remained unchanged in the ovary. In addition, goldfish GnIH peptides not only increased the expression of StAR and 3bHSD and decreased CYP19 mRNA, but they also increased the expression of FSHR and LHR mRNA in testicular cells. However, they did not affect the expression of these genes in ovarian cells in vitro. Thus, we suggest that GnIH may contribute to the sexual dimorphism of steroidogenesis in goldfish.

Effects of p,p′-DDE on the mRNA and protein expressions of vimentin, N-cadherin and FSHR in rats testes: An in vivo and in vitro study

To elucidate the mechanism underlying the testicular effects of 1,1-dichloro-2,2bis(p-chlorophenyl)ethylene (p,p′-DDE), the expressions of vimentin, neural cadherin and follicle-stimulating hormone receptor mRNA and proteins were measured in vivo and in vitro. Sprague-Dawley rats were dosed with p,p′-DDE at 0, 20, 60 and 100mg/kg every other day by intraperitoneal injection for 10 days, and Sertoli cells were treated with p,p′-DDE (0, 10, 30, and 50μM) for 24h. Results indicated that the survival rate of Sertoli cells was decreased with increasing doses of p,p′-DDE. In vitro and in vivo studies, p,p′-DDE could increase the expression of neural cadherin, follicle-stimulating hormone receptor mRNA, while decrease the levels of vimentin, neural cadherin and follicle-stimulating hormone receptor proteins. Moreover, immunohistochemistry analysis revealed that the protein expressions of vimentin, neural cadherin and follicle-stimulating hormone receptor in pubertal rat testes were disrupted by treatment with p,p′-DDE. Taken together, these results suggested that p,p′-DDE exposure could induce testicular toxicity through the changes of the mRNA and protein expressions of vimentin, neural cadherin and follicle-stimulating hormone receptor in vitro and in vivo.

The compensatory expression of reproductive hormone receptors in the thymus of the male rat following active immunization against GnRH

To determine whether hormone-receptor signaling pathways in the thymus are altered by active immunization against gonadotrophin-releasing hormone I (GnRH), 3-week-old Sprague–Dawley male rats received GnRH-tandem-OVA peptides (200μg/ml), and the effects were compared to a control group. Serum testosterone, LH and FSH concentrations were markedly reduced, with severe testicular atrophy, compared to controls, demonstrating effective blockade of the pituitary–gonadal axis. The reduction in LH and FSH concentrations in the thymus of immunized animals was lower than that observed in the serum, where a significant difference (P<0.001) in concentration was observed between both groups. Concentrations of GnRH were increased in the thymus of immunized rats. In thymic tissue, GnRHR, FSHR and LHR demonstrated stronger immunostaining, and AR weaker staining, in the immunized group compared to controls. Reproductive hormone receptor mRNA expression was consistent with protein variations in the immunized thymus. Compared to controls, GnRHR gene levels were significantly increased (P<0.05), however, AR mRNA expression were greatly decreased with immune week-age (P<0.05). Both FSHR and LHR mRNA expression levels were significantly higher in the treated group than in controls in the first three samples (P<0.05). When GnRHR was blocked by an antagonist in thymocytes, all reproductive hormone receptor gene expressions were significantly increased (P<0.001). In summary, these findings suggest that active immunization against GnRH can up-regulate GnRH receptor and gonadotropin receptor signaling, by stimulating thymic autocrine and paracrine function, whereas the androgen receptor is down-regulated due to a lack of testosterone secretion in the thymus.

The effects of epidermal growth factor (EGF) on the in vitro development of isolated goat secondary follicles and the relative mRNA expression of EGF, EGF-R, FSH-R and P450 aromatase in cultured follicles

The effects of varying concentrations of EGF were evaluated in terms of in vitro follicular development and the mRNA expression levels of EGF, EGF-R, FSH-R and P450 aromatase. After 6days, the addition of 50ng/mL of EGF to the culture medium increased the antrum formation rates in comparison to cultured control and after 18days of culture produced oocytes with higher rates of meiosis resumption when compared to the other treatments (P<0.05). The daily follicular growth rates in presence of EGF (50 or 100) were increased in comparison to the cultured control (P<0.05). Treatment with EGF 50 stimulated the expression of EGF mRNA but reduced EGF-R mRNA expression and estradiol secretion as compared to the cultured control (P<0.05). After 18days of culture, the mRNA levels for FSH-R and P450 aromatase were greater than those of the non-cultured controls (P<0.05). In conclusion, the effects of EGF treatment on the mRNA levels for EGF, EGF-R, FSH-R, and P450 aromatase varied according to the stage of follicle development.

214 EFFECT OF OVARIAN SUPERSTIMULATION ON EXPRESSION OF GENES ASSOCIATED WITH THE OOCYTE DEVELOPMENTAL COMPETENCE OF NELORE COWS

The P36 protocol has contributed to the genetic improvement of Brazilian herd through its successful use in embryo transfer programs. We aimed to investigate the effect of P36 protocol on embryo yield and mRNA expression of genes correlated with the competence of cumulus–oocyte complex (COC): receptors of FSH (FSHR), EGF (EGFR), and pentraxin 3 (PTX3) in cumulus cells; receptors of LH (LHR) and angiotensin 2 (AT2) in granulosa cells; and GDF9, BMP15, and histone H2A (H2A) in oocytes. Multiparous Nelore cows were allocated in control and P36 groups. Control group (non-superovulated, n = 15) received a progesterone intravaginal device (P4, 1.0 g, Primer®, Tecnopec, Sao Paulo, Brazil) and 2.5 mg of oestradiol benzoate (EB, IM, BER-BE®, Syntex, Buenos Aires, Argentina) at a random day of the oestrous cycle (Day 0). A PGF2α analogue (150 mg d-cloprostenol, IM, Prolise®, RARS SRL) was administered (Day 8) and Primer® was removed. The P36 group (n = 10) received a Primer® and 2.0 mg of EB (Day 0). The FSH treatment (160 mg Folltropin®, Bioniche Animal Health, Ontario, Canada) was initiated at decreasing doses: 40, 30, 20, and 10% of the total dose twice daily for 4 days (Day 5). The PGF2α analogue was administered (Day 8) and after 36 h primer was removed. Animal slaughter to ovary collection was performed 12 h after Primer® removal (Day 9). Some of the oocytes were matured (TCM199), fertilized with Nelore semen (n = 6), and cultured (SOF-synthetic oviduct fluid) to the blastocyst stage. Embryos were removed from culture (Day 6), allocated in 5 pools with 5 embryos in each group, and subjected to RNA extraction. Remaining oocytes were denuded from cumulus and zona pellucida (vortex and Protease®, Sigma-Aldrich, St. Louis, MO, USA). Pools of 20 oocytes and of their respective cumulus cells (n = 6 pools; control group and n = 4 pools, P36 group) were subjected to RNA extraction (RNeasy kit, Qiagen, Valencia, CA, USA). Gene expression was performed by real-time RT-PCR using oligo-dT in reverse transcription and bovine-specific primers. Expression of cyclophilin A was used as endogenous control. Change to developmental rates to the blastocyst stage and transcript abundance were compared by t-test and significance was considered when P &lt; 0.05. Blastocyst rates were also similar (P &gt; 0.05) in groups P36 (40/99; 40%) and control (16/43; 37%). Expression of H2A, EGFR, FSHR, and PTX3 in cumulus cells did not differ (P &gt; 0.05) among treatment groups. The expression of GDF9 and BMP15 in cumulus cells was higher (P &lt; 0.05) in the P36 group, but in oocytes these transcripts were more expressed in the control group (P &lt; 0.05). Although important genes (GDF9 and BMP15) were less expressed in oocytes from superstimulated cows, the maintenance of H2A in oocytes, as well as PTX3, EGFR, and FSHR, and the increases in GDF9 and BMP15 expression in cumulus cells do not seem to affect oocyte competence due to the similar embryo yield of both groups. Supported by FAPESP.

Long-term in vitro culture of ovarian cortical tissue in goats: effects of FSH and IGF-I on preantral follicular development and FSH and IGF-I receptor mRNA expression

Long-term in vitro culture (16 days) of caprine ovarian cortical tissue was performed to test the effect of FSH and IGF-I on the viability and development of preantral follicles and mRNA expression for FSH and IGF-I receptors. Fragments were cultured in α-MEM(+) alone or supplemented with different combinations of FSH and IGF-I (sequential medium). The culture period was divided into two parts. Follicles were isolated and classified as normal or abnormal and primordial, primary or secondary. Viability of isolated follicles was determined by staining with Trypan Blue dye. Expression of FSHR and IGFR-1 mRNA was evaluated by qPCR. At day 8 of culture, more (P < 0.05) follicles in treatments containing IGF-I alone or associated with FSH were normal and viable (overall mean, 81 % and 79 % respectively) than the treatments cultured with FSH or α-MEM(+) alone (68 % and 63 %). At day 16 of culture, treatments with FSH and/or IGF-I had more (P < 0.05) viable follicles (69 %) than α-MEM(+) (38 %). The percentages of follicular development observed in the IGF-I/FSH, FSH+IGF-I/FSH+IGF-I and FSH/IGF-I treatments were similar but higher (P < 0.05) than the other treatments. FSH and IGF-I during the entire culture period maximized (P < 0.05) follicular and oocyte diameters and the percentage of secondary follicles (28 %). FSHR mRNA expression in the non-cultured control was similar to the treatment supplemented with FSH and IGF-I but higher (P < 0.05) than α-MEM(+). IGFR-1 expression did not differ among treatments. Association of FSH and IGF-I in long-term in vitro culture promoted follicular development, maintaining FSHR mRNA expression.

Differential regulation of gonadotropin receptors (fshr and lhcgr) by epidermal growth factor (EGF) in the zebrafish ovary

Epidermal growth factor (egf) is expressed in the zebrafish oocyte whereas its receptor EGF receptor (egfr) is expressed in the somatic follicle layer, strongly suggesting a role for Egf in the intrafollicular paracrine communication that mediates an oocyte-to-follicle cell signaling pathway. However, the exact function of Egf in the follicle remains largely unknown. The present study aimed to explore the possible role of Egf in regulating gonadotropin receptors (fshr and lhcgr) in cultured zebrafish follicle cells. EGF down-regulated lhcgr expression dose-dependently in a biphasic manner with significant effect observed at 1.5 and 24h. The effect was mediated via Egfr on the follicle cells. On the contrary, EGF also tended to decrease fshr expression at 1.5h but it appeared to up-regulate fshr at 24h. The EGF suppression of lhcgr expression was functionally relevant as pre-exposure to EGF reduced the follicle cell responsiveness to LH/hCG. We have recently reported that estradiol (E2) strongly stimulated lhcgr expression in the zebrafish ovary. In the current study, we further demonstrated that EGF and other EGF family members, heparin-binding EGF-like growth factor (HBEGF), transforming growth factor α (TGFα) and betacellulin (BTC), all reduced basal and E2-induced lhcgr expression. This study provides evidence for a potential paracrine role of Egf and its related peptides in the zebrafish follicle. The oocyte-derived EGF family ligands may actively control the process of follicle growth and maturation by differentially controlling the expression of fshr and lhcgr in the follicle cells in a paracrine manner.

Dietary Carbohydrate Source Alters Gene Expression in the Uterine and Ovarian Tissues of Pre-Pubertal Female Pigs.

The objective of this study was to assess the effects of dietary carbohydrate source and high fat intake on mRNA concentrations within the reproductive tissues of pre-pubertal female pigs. Gilts (28 d-old) were fed either control or high-fat diets for 8 weeks. High-fat diets contained either complex (corn) or simple (glucose or fructose) carbohydrates. Control pigs were pair-fed with high-fat pigs, resulting in similar final body weight across treatments. Ultrasound back-fat depth (indicative of body adiposity) was 84% higher in gilts consuming high-fat diets, and simple carbohydrate diets tended to increase back-fat. Reproductive tissues were collected after exsanguination for analysis of gene expression by RT-qPCR. Concentrations of ESR1, ESR2, GDF9, IGF1 and IGF1R mRNA were measured in whole ovarian and uterine tissues while expression of LHCGR, FSHR and CYP19A1 was measured in whole ovarian tissue only. Dietary treatment effects on reproductive tissues were almost exclusively based on carbohydrate source. Simple carbohydrate diets resulted in heavier uteri and ovaries and more antral follicles compared to control or complex carbohydrate diets. Gilts fed simple carbohydrate diets tended to have higher uterine ESR1 concentrations than those fed control or complex carbohydrate diets. And while ovarian ESR1 did not differ amongst dietary treatments, ovarian ESR2 was lower in gilts fed the simple carbohydrate diets compared to control or complex carbohydrate diets. Opposing trends in expression of GDF9 were also observed, with higher uterine GDF9 and lower ovarian GDF9 in gilts fed simple carbohydrate diets compared to control or complex carbohydrate treatments. Gilts consuming the simple carbohydrate diet also had a greater than two-fold increase in uterine IGF1 concentrations. Furthermore, ovarian IGF1R and FSHR concentrations were greater in the simple carbohydrate group. There were no observed differences in LHCGR or CYP19A1 gene expression. Total tract weights of the gilts consuming control and complex carbohydrate diets were positively correlated with ovarian expression of IGF1R and FSHR, and negatively correlated with ovarian expression of ESR2 and GDF9. None of these correlations existed for the gilts fed the simple carbohydrate diets. Despite similar body weights, the reproductive tracts of gilts fed diets differing in carbohydrate source were strikingly different. Gilts consuming simple carbohydrate diets appeared to be closer to ovulation and puberty than those consuming control or complex carbohydrate diets. The competency of their follicles and the oocytes contained within those follicles remains to be determined.

In vitro-growth and Gene Expression of Porcine Preantral Follicles Retrieved by Different Protocols.

This study was conducted to determine how the isolation method of the porcine preantral follicles influenced the following follicular growth in vitro. Mechanical and enzymatical isolations were used for retrieving the follicles from prepubertal porcine ovaries, and in vitro-growth of the follicles and the expression of folliculogenesis-related genes were subsequently monitored. The enzymatic retrieval with collagenase treatment returned more follicles than the mechanical retrieval, while the percentage of morphologically normal follicles was higher with mechanical retrieval than with enzymatic retrieval. After 4 days of culture, mechanically retrieved, preantral follicles yielded more follicles with normal morphology than enzymatically retrieved follicles, which resulted in improved follicular growth. The mRNA expression of FSHR, LHR Cx43, DNMT1 and FGFR2 genes was significantly higher after culture of the follicles retrieved mechanically. These results suggest that mechanical isolation is a better method of isolating porcine preantral follicles that will develop into competent oocytes in in vitro culture.

Expression of functional follicle-stimulating hormone receptor and luteinizing hormone/chorionic gonadotrophin receptor in oviduct and uterus in prepubertal gilts

The presence of the FSHR and LH/CGR in nongonadal tissues of reproductive tracts during estrus has been reported. But localization study for FSHR in the endometrium has not been performed. Moreover, the expression of FSHR and LH/CGR in the nongonadal tissues of prepubertal gilts is also unclear. Our objectives were to demonstrate the presence of active and functional LH/CGR and FSHR and to localize the two receptors in uterus and oviducts in prepubertal gilts. Fifteen prepubertal crossbred (Landrance/Yorkshine) gilts were used in the study. RT-PCR revealed that both uterus and oviducts contained a 145bp LH/CGR transcript and a 103bp FSHR transcript. Western blotting revealed that the two tissues also contained a 75KDa immunoreactive LHR protein and a 75KDa immunoreactive FSHR protein. Immunocytochemistry demonstrated that the LHR and FSHR were expressed both in the uterus and oviducts. In the oviducts, the two receptors were all present in the mucosal epithelium, but not detected in the myosalpinx and the serosa. In the uterus, they were present in the luminal and glandular epithelium of the endometrium. Furthermore, the vascular smooth muscle also expressed the LHR protein. Injection of eCG to ovariectomized gilts and intact gilts increased both the concentrations of FSH and LH in serum and expressions of FSHR and LH/CGR proteins in uterus and oviducts. In summary, active FSHR and LH/CGR are present in the uterus and oviducts of prepubertal gilts.

Expression of luteinizing hormone and follicle-stimulating hormone receptor in the dog prostate

A possible role for gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the prostate physiology has been suggested in humans and rats. This study aimed at investigating the presence of receptors for LH and FSH (LHR and FSHR) in the canine prostate. Prostates were collected at post mortem from 6 clinically healthy, sexually intact beagles free from any prostatic disorder. Tissue was sampled from dorsal, middle and ventral regions of each prostate. Immunohistochemical localization was performed on wax-embedded sections using polyclonal antibodies for LHR or FSHR. The pattern and intensity of staining in the parenchyma (glandular epithelium) and stroma were determined using a semiquantitative histologic assessment. Receptors for LH and FSH were consistently present in both the glandular epithelium and the stroma in all tissue samples examined. Expression for both receptors was higher in the glandular epithelium than the stroma of all prostatic regions (P < 0.001). In the glandular epithelium, LHR (P < 0.01) and FSHR (P < 0.05) expression was lower in the lateral than the other regions, and there was no difference between dorsal and ventral regions. However, variations in the expression for LHR and FSHR among prostatic regions were not found in the stroma. These findings have demonstrated that LHR and FSHR are expressed in the dog prostate, and the variation observed in their levels of expression among its regions and tissue layers suggests a potential role of gonadotrophins LH and FSH in the regulation of the prostate physiology, particularly the glandular epithelium.

Differential regulation of gonadotropin receptors by bone morphogenetic proteins in the zebrafish ovary

Follicle-stimulating hormone receptor (fshr) and luteinizing hormone/choriogonadotropin receptor (lhcgr) exhibit differential temporal expression patterns during zebrafish folliculogenesis with fshr being dominant during vitellogenic growth and lhcgr increasing its expression dramatically before maturation. The dynamic and distinct expression patterns of fshr and lhcgr suggest that they are under tight regulatory control. However, the underlying mechanisms for the differential expression of the two receptors remain unknown. We have recently demonstrated that members of bone morphogenetic protein (BMP) family are largely expressed in the oocyte, while their receptors are exclusively localized on the follicle cells, suggesting a potential paracrine signaling from the oocyte to the follicle cells by BMPs. In this study, we investigated the effects of zebrafish BMP2b (zfBmp2b) and BMP4 (zfBmp4) on the expression of fshr and lhcgr using a novel co-culture approach. The recombinant zfBmp2b or zfBmp4-producing CHO cells were co-cultured with the zebrafish follicle cells followed by real-time qPCR analysis of fshr and lhcgr expression. Our results showed that zfBmp2b and zfBmp4 both down-regulated fshr, while up-regulated lhcgr expression at 24h of co-culturing. This finding, together with the high expression level of BMP receptors in the follicle cells prior to oocyte maturation, strongly suggests a potential role for BMPs in the differential expression of fshr and lhcgr, especially in the full-grown follicles before maturation. As BMPs are largely expressed in the oocyte, this also implies an important role for the oocyte in orchestrating the differentiation and function of the follicle cells.

Human chorionic gonadotropin and its relation to grade, stage and patient survival in ovarian cancer

BackgroundAn influence of gonadotropins (hCG) on the development of ovarian cancer has been discussed. Therefore, we quantified serum hCG levels in patients with benign and malignant ovarian tumors and the hCG expression in ovarian cancer tissue in order to analyze its relation to grade, stage, gonadotropin receptor (LH-R, FSH-R) expression and survival in ovarian cancer patients.MethodsPatients diagnosed and treated for ovarian tumors from 1990 to 2002 were included. Patient characteristics, histology including histological subtype, tumor stage, grading and follow-up data were available. Serum hCG concentration measurement was performed with ELISA technology, hCG tissue expression determined by immunohistochemistry.ResultsHCG-positive sera were found in 26.7% of patients with benign and 67% of patients with malignant ovarian tumors. In addition, significantly higher hCG serum concentrations were observed in patients with malignant compared to benign ovarian tumors (p = 0.000). Ovarian cancer tissue was positive for hCG expression in 68%. We identified significant differences in hCG tissue expression related to tumor grade (p = 0.022) but no differences with regard to the histological subtype. In addition, mucinous ovarian carcinomas showed a significantly increased hCG expression at FIGO stage III compared to stage I (p = 0.018). We also found a positive correlation of hCG expression to LH-R expression, but not to FSH-R expression. There was no significant correlation between tissue hCG expression and overall ovarian cancer patient survival, but subgroup analysis revealed an increased 5-year survival in LH-R positive/FSH-R negative and hCG positive tumors (hCG positive 75.0% vs. hCG negative 50.5%).ConclusionsSerum human gonadotropin levels differ in patients with benign and malignant ovarian tumors. HCG is often expressed in ovarian cancer tissue with a certain variable relation to grade and stage. HCG expression correlates with LH-R expression in ovarian cancer tissue, which has previously been shown to be of prognostic value. Both, the hormone and its receptor, may therefore serve as targets for new cancer therapies.

Expression and Epigenetic Change of the AR and FSHR Genes in the Granulosa Cells of Endometriosis Patients

Background Endometriosis is one of the most common gynecological diseases associated with infertility. Endometriosis may affect the androgen receptor (AR) mRNA expression in human granulosa cells and the methylation of the promoter region of AR. We investigated 28 patients with endometriosis and 47 subjects without endometriosis undertaking IVF treatment. Methods Granulosa cells were obtained from 28 patients with endometriosis and 47 subjects without endometriosis as a control. Expressions of AR and FSHR mRNA were then evaluated by OneStep real-time PCR analysis, and the level of methylation of the promoter region was qualified by methylation-specified PCR (MSP). Results The expression of AR mRNA in the endometriosis group was statistically lower than that in the control group. As well, FSHR mRNA expression in the control group showed a positive correlation with AR mRNA expression; however, there was no such correlation in endometriosis patients. In the control group, AR mRNA expression was statistically higher in pregnant subjects compared with non-pregnant subjects; however, in the endometriosis group, no significant difference was identified. The promoter of AR was heavily methylated in all endometriosis cases; however, only 5 (45.4%) were methylated in the control group. Conclusion Lower AR mRNA expression and methylation of the AR promoter region might affect the expression of AR and FSHR the presence of endometriosis, thus leading to a disturbance in the regulation of AR and FSHR.

Microcystin (-LR) affects hormones level of male mice by damaging hypothalamic-pituitary system

Microcystin (-LR) (MC-LR) is a broad distributed hepatotoxic microcystin produced by common freshwater phytoplankton species. Recently, its toxic effects on male reproductive system drew great attention. To investigate its reproductive toxicity and the underlying mechanism, BALB/c mice were exposed to MC-LR by intraperitoneal injection with different injection duration (1, 4, 7 and 14 days) and injection concentration (3.75, 7.5, 15 and 30 μg kg b.w.−1 day−1) as an in vivo test. For in vitro test, isolated mice Leydig cells were exposed to MC-LR at doses of 1, 10, 100, 250, 500 750 and 1000 nmol/L for 24 h. MC-LR did not induce significant changes in Kiss-1, GPR54, Gnrhr, Fshr or Lhr expression. It decreased the Gnrh expression in a dose- and duration-dependent manner. It increased and subsequently decreased the expressions of FSH, LH and testosterone. In in vivo test, MC-LR was not able to enter Leydig cells and had no cytotoxicity on Leydig cells. The results showed that MC-LR affected male mice serum hormones and mRNA expressions by damaging the hypothalamic-pituitary systems.

GnRH agonist active immunization influences gonadotropin receptor expression in pituitary gland, uterine development and secretion of peripheral reproduction hormones in female mice

Abstract Objective The present study was conducted to assess the effects of the GnRH agonist (alarelin) on the expression of GnRHR, FSHR and LHR mRNAs in the pituitary gland and uterine development in female mice. Methods Sixty pre-estrus female mice were randomly assigned to 4 groups (n = 15). The mice in experiment group 1 (EG-1), experiment group 2 (EG-2), and experiment group 3 (EG-3) were subcutaneously injected with 0.375 μg/kg, 0.75 μg/kg and 1.5 μg/kg alarelin antigen emulsion, respectively, once a day during 7 consecutive days to enhance immune response. Mice in the control group (CG) were injected with 0.l ml solvent once a day on 7 consecutive days. The samples of blood, pituitary and uterine horns were collected aseptically at day 21 from each mouse. Fluorescence quantitative RT-PCR was implemented to detect gene expression of GnRHR, FSHR and LHR in the pituitary glands. ELISA was used to measure the serum LH and FSH concentrations. The uterine slices were observed under an optical microscope;...

Differential Regulation of Gonadotropin Receptors (fshr and lhcgr) by Estradiol in the Zebrafish Ovary Involves Nuclear Estrogen Receptors That Are Likely Located on the Plasma Membrane

FSH and LH are gonadotropins (GTH) that control all major events of gonadal function. FSH and LH signal through their cognate receptors, FSH receptor and LH/choriogonadotropin receptor, respectively, across vertebrates. Compared with the information in mammals, very little is known about these receptors in fish, especially the regulation of their expression. In female zebrafish, fshr and lhcgr exhibit significant temporal difference in expression, with fshr increasing first when the follicles are activated to enter the vitellogenic growth phase and lhcgr lagging behind. This raises an interesting question on the differential regulation of these two GTH receptors (GTHR) during folliculogenesis. Using a primary follicle cell culture, the present study demonstrated that 17β-estradiol (E2), but not testosterone, was a potent endocrine hormone that differentially regulated the expression of fshr and lhcgr. Although E2 stimulated both receptors, its effect on the steady-state level of lhcgr mRNA was much higher (>8-fold up-regulation) than that of fshr (∼0.5-fold increase). E2 likely acted at the transcription level via its nuclear estrogen receptors (ERα and ERβ), because ICI 182,780 could abolish its effects. However, our evidence suggested that these receptors might be localized on the plasma membrane, because β-estradiol 6-(O-carboxy methyl)oxime:BSA could fully mimic the effects of E2. Demonstrating that E2 is likely one of the differentiating factors for the distinct expression of the two GTHR in the zebrafish ovary, this study sheds important light on the functions of the two GTH and their receptors in fish as well as the conservation and diverse aspects of GTHR regulation across vertebrates.

Upstream Stimulatory Factor Induces Nr5a1 and Shbg Gene Expression During the Onset of Rat Sertoli Cell Differentiation1

Within the testis, each Sertoli cell can support a finite number of developing germ cells. During development, the cessation of Sertoli cell proliferation and the onset of differentiation establish the final number of Sertoli cells and, thus, the total number of sperm that can be produced. The upstream stimulatory factors 1 and 2 (USF1 and USF2, respectively) differentially regulate numerous Sertoli cell genes during differentiation. To identify genes that are activated by USF proteins during differentiation, studies were conducted in Sertoli cells isolated from 5- and 11-day-old rats, representing proliferating and differentiating cells, respectively. Usf1 mRNA and USF1 protein levels were increased between 5 and 11 days after birth. In vitro studies revealed that USF1 and USF2 DNA-binding activity also increased at 11 days for the promoters of four potential target genes, Fshr, Gata4, Nr5a1, and Shbg. Chromatin immunoprecipitation assays confirmed that USF recruitment increased in vivo between 5 and 11 days after birth at the Fshr, Gata4, and Nr5a1 promoters. Expression of Nr5a1 and Shbg, but not of Fshr or Gata4, mRNAs was elevated in 11-day-old Sertoli cells compared with 5-day-old Sertoli cells. Transient transfection of USF1 and USF2 expression vectors up-regulated Nr5a1 and Shbg promoter activity. RNA interference assays demonstrated that USF1 and USF2 contribute to Nr5a1 and Shbg expression in differentiating cells. Together, these data indicate that increased USF levels induce the expression of Nr5a1 and Shbg during the differentiation of Sertoli cells, whereas Fshr and Gata4 expression is not altered by USF proteins during differentiation.

Pituitary gonadotropin and testicular gonadotropin receptor expression in Atlantic cod ( Gadusmorhua L.) during the first reproductive season: Effects of photoperiod modulation

Pituitary mRNA levels of gonadotropin β-subunits and of their cognate receptors in the testis were studied during puberty in Atlantic cod under normal and experimental photoperiod conditions that suppressed, delayed or accelerated testis maturation. Results are discussed in context with changes in testicular histology and plasma androgen levels, considered as end points of gonadotropic regulation. Up-regulation of fshb was closely associated with the onset of puberty, decreased when spermatogenesis was completed and reached minimum levels after spawning. These results demonstrate, for the first time using an experimental approach, that activation of Fsh-dependent signaling is associated with spermatogonial proliferation and formation of spermatogenic cysts. Changes in fshr expression were less prominent and could be explained by changes in the cellular composition and RNA content of cod testis tissue. At more advanced stages of development (spermiogenesis, spermiation and spawning), lhb and, one month later, lhcgr transcript levels increased and reached peak values in spawning fish, in a positive feedback loop involving plasma androgens and Lh/Lhcgr-dependent signaling. This loop was broken by a loss of lhb expression at the end of the spawning season. Continuous light (LL) from summer solstice, ∼8 months prior to spawning, suppressed the start of testis maturation and the changes in gonadotropin and receptor mRNA levels, while LL from winter solstice initially up-regulated lhb and lhcgr expression, before resulting in a precocious termination of the spawning season and low expression of all four genes. Our studies provide experimental evidence for a clear functional discrimination of cod gonadotropins.