LHR mRNA Research Articles

Gonadal and nongonadal FSHR and LHR dysfunction during lipopolysaccharide induced failure of blastocyst implantation in mouse

The purpose of the present study was to investigate the impact of lipopolysaccharide (LPS) on follicle-stimulating hormone (FSH), luteinizing hormone (LH) and their receptors during preimplantation days of pregnancy. The PBS or lipopolysaccharide (LPS) was injected intraperitoneally in the pregnant females on day 0.5 of pregnancy and serum, embryos, ovaries and uterine horns were collected on days 1.5, 2.5, 3.5, 4.0, 4.125, 4.33 and 4.42 of pregnancy. In the LPS-treated pregnant females, the secretion of FSH and LH is disturbed with respect to normal pregnancy. Furthermore, the expression of FSHR mRNA in embryos and ovaries, LHR mRNA in embryos and uterus get modulated in response to LPS during preimplantation days of pregnancy. The disturbance in the serum level of FSH and LH in response to LPS leads implantation failure in mouse which suggests that these gonadotropins plays an integral role in the process of the successful implantation. This study also suggests a possible nongonadal role of FSHR and LHR in LPS-induced implantation failure in the mouse.

Prolactin acts on the hypothalamic–pituitary axis to modulate follicle-stimulating hormone gene expression in the female brushtail possum ( Trichosurus vulpecula)

Brushtail possums exhibit a distinct preovulatory pattern of prolactin (Prl) secretion suggesting that Prl is involved in normal reproductive function. In some mammals, Prl is essential for corpus luteum (CL) function and/or modulation of steroidal effects on hypothalamic–pituitary activity. The aim of this study was to test the effects of biologically active recombinant possum Prl (recPosPrl) on both pituitary gland and CL function in possums. To confirm biological activity, administration of recPosPrl-N2C1 (10 μg) resulted in an 18-fold stimulation ( P < 0.05) of progesterone (P 4) production by possum granulosa cells in vitro. Based on these findings, minipumps containing either recPosPrl-N2C1 ( n = 10) or saline ( n = 8) were inserted into lactating female possums. The expression levels of pituitary-derived PRL, LHB, FSHB and GNRHR and CL-derived LHR mRNA were quantified. Following a resumption of reproductive activity, no differences in ovulation incidence or plasma Prl concentrations were observed. Plasma Prl levels were less variable ( P < 0.001) in Prl-treated possums, confirming a self-regulatory role for Prl in this species. There was a marked down-regulation ( P < 0.001) of FSHB mRNA at the mid-luteal stage in Prl-treated possums, whereas mean PRL, LHB, GNRHR and LHR mRNA expression levels were not different between experimental groups. Plasma P 4 concentrations were not different ( P = 0.05) in Prl-treated possums, although tended to be higher in the peri-ovulatory and early-luteal phase. We conclude in the brushtail possum that Prl is self-regulated via a short-feedback loop common to all mammals studied and is able to modulate FSHB expression probably at the level of the hypothalamus and/or pituitary gland.

Gonadotropins and Their Receptors in the Hermaphroditic Ricefield Eel Monopterus albus: cDNA Cloning and mRNA Expression.

The ricefield eel is a protogynous hermaphroditic teleost fish that changes sex from functional female, through an intersexual stage, to the functional male phase during its life cycle, and a good model organism to study sex determination and differentiation of vertebrates. Previous studies suggested that HPG axis may play important roles in the initiation of sex change in ricefield eel; however, the gonadotropins and their cognate receptors remains to be elucidated in this fish. By using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), cDNAs encoding Cgalpha, Lhbeta, Fshbeta, Fsh receptor (Fshr), and Lh receptor (Lhr) of ricefield eel were cloned in the present study. Homology analysis showed that ricefield eel Cgalpha, Lhbeta, Fshbeta, Fshr, and Lhr shared highest identities with their counterparts in perciform fish,which conforms to the lineage relationship. A lhr pseudogene was identified from the genome of ricefield eel, and what is interesting is that phylogenetic analysis categorized teleost lhrs into two clusters, and ricefield eel lhr gene and pseudogene are grouped into one of the two clusters, respectively, which suggests that either one of the duplicated lhr genes became a pseudogene during evolution of different teleost lineages. During ovarian development, realtime PCR analysis showed an increasing trend of fshbeta mRNA levels in the pituitary from previtellogenesis to vitellogenesis, but then a decreasing trend toward maturation. As for lhbeta mRNA levels in the pituitary, a continuous increasing trend was observed from previtellogenesis to maturation. The ovarian fshr mRNA levels were significantly increased from previtellogenesis to early vitellogenesis, but then decreased significantly thereafter. However, the ovarian lhr mRNA levels were increased significantly from vitellogenesis to the mature stage. The above results may suggest that Fsh signal pathway is more important for vitellogenesis, whereas Lh signal pathway more important for oocyte maturation in ricefield eel. During sex change from female through an intersexual stage to male, fshr and lhr showed lower expression levels in transitional gonads than either ovary or testis. In the pituitary, fshbeta and lhbeta mRNA levels were significantly lower during the intersexual and male stages than the female stage. However, lhbeta /fshbeta mRNA ratios were increased significantly during sex change, suggesting that the changes in the ratio of LHbeta to FSHbeta may be important for the gonadal differentiation of ricefield eel. The present study provided a foundation for further elucidation of the roles of HPG axis in the gonadal development and sex change of ricefield eel. This research was supported by Natural Science Foundation of China (30970359, 30771651) and National High Technology Research and Development Program of China (863 Program) (No.2008AA09Z406). (poster)

Bone morphogenetic protein 2 inhibits FSH responsiveness in hen granulosa cells

Prior to follicle selection into the preovulatory hierarchy, hen granulosa cells from prehierarchal follicles remain undifferentiated, as defined in part by the virtual absence of LHR mRNA expression and inability to produce progesterone. It has previously been proposed that prior to follicle selection, granulosa cells are actively maintained in an undifferentiated state by epidermal growth factor receptor ligands (EGFRL) signaling via the MAP kinase/extracellular regulated kinase pathway. Moreover, there is recent evidence that EGFRL/MAP kinase signaling modulates FSH receptor (FSHR) transcription, in part, via inhibitor of differentiation/DNA-binding (ID) proteins. In the present studies with undifferentiated granulosa, recombinant human (rh) bone morphogenetic protein 2 (BMP2) induced the phosphorylation of SMAD1/5/8, and blocked transforming growth factor β and FSH-induced FSHR expression and progesterone production. Significantly, BMP2 rapidly induced mRNAs encoding betacellulin and EGF, plus ID proteins (ID1, ID3, and ID4). Alternatively, the bioactivity of BMPs can be modulated by one or more BMP antagonists, including noggin (NOG). NOG mRNA is expressed by both hen granulosa and theca tissues from prehierarchal follicles. Pretreatment of cultured granulosa with rh NOG reversed both the stimulatory effects of BMP2 on ID1, ID3, and ID4 expression and the inhibitory effects of BMP2 on FSHR mRNA levels and progesterone production. Collectively, these data provide evidence that prior to follicle selection, BMP2 signaling contributes toward maintaining granulosa cells in an undifferentiated state. The actions of BMP2 are, at least in part, mediated indirectly via enhanced EGFRL expression and ERBB receptor-mediated MAP kinase signaling, and can be modulated by the autocrine/paracrine production of NOG.

Stability of housekeeping genes and expression of locally produced growth factors and hormone receptors in goat preantral follicles

The aim of the present study was to investigate the stability of six housekeeping genes, and the relative expression of growth factors (EGF, GDF-9, BMP-15, VEGF, FGF-2, BMP-6, IGF-1 and KL) and hormone receptors (FSH, LH and GH) in goat preantral follicles. To evaluate to stability of housekeeping genes micro-dissected fresh follicles (150-200 μm) as well as follicles that have been in vitro cultured for 12 days were used. In addition, isolated fresh follicles were used to compare expression of various growth factors and hormone receptors before culture. Both fresh and cultured follicles were subjected to total RNA extraction and synthesis of cDNA. After amplification of cDNA by real-time PCR, the geNorm software program was used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), β-tubulin, β-actin, phosphoglycerokinase (PGK), 18S rRNA, ubiquitin (UBQ) and ribosomal protein 19 (RPL-19). In addition, follicular steady-state levels of mRNA from the various growth factors under study were compared. Results demonstrated that, in goat preantral follicles, UBQ and β-actin were the most suitable reference genes and thus could be used as parameters to normalize data from future in vitro studies. In contrast, 18S RNA appeared the least stable gene among the tested housekeeping genes. Analysis of mRNA for several hypophyseal hormone receptors in fresh preantral follicles showed significantly higher FSH-R mRNA levels than those of LH-R and GH-R, and no difference between GH-R and LH-R mRNA levels. In regard growth factor mRNA expression in goat preantral follicles, EGF mRNA levels appeared significantly lower than those of the other studied growth factors. Increasingly higher relative mRNA levels were observed for GDF-9, BMP-15, BMP-6, FGF-2, VEGF, Kl and IGF-1, successively. In conclusion, UBQ and β-actin are the most stable housekeeping genes in fresh and 12-days cultured caprine preantral follicles. Furthermore, in fresh follicles, high levels of FSH-R mRNA are detected while among eight growth factors, IGF-1 is the most highly expressed and EGF the weakest expressed compound.

Molecular cloning and expression analysis of Fshr and Lhr in relation to Fshb and Lhb subunits during the period of temperature‐dependent sex determination in pejerrey Odontesthes bonariensis

In this study, we cloned and characterized the follicle stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhr) cDNAs of pejerrey Odontesthes bonariensis, a species with temperature-dependent sex determination (TSD), and analyzed their expression in relation to Fshb and Lhb subunits during gonadogenesis at temperatures producing only females (17 degrees C, FPT), both sexes (25 degrees C, MixPT), and only males (29 degrees C, MPT). The pejerrey Fshr cDNA had 3,069 bp for a mature protein of 694 amino acids (aa) and a signal peptide of 22 aa; the Lhr cDNA had 2,936 bp for a mature protein of 676 aa and a signal peptide of 25 aa. With the exception of Lhr in fish at the MPT, all genes showed significant increases and/or peaks of expression before histological differentiation of the gonads regardless of temperature. Larvae at the FPT had lower Fshb and Lhb but higher Lhr expression during the TSD period than those at the MPT; a clear pattern could not be ascertained for Fshr. At the MixPT, Fshb, Lhb, and Lhr mRNA increased in approximately half of the fish during TSD and sex differentiation and the sex ratio was 55.2% male. Based on the above results, it is suggested that animals with high Fshb and Lhb and low Lhr values represent putative males. These evidences, together with other studies, suggest that temperature may signal through the pituitary (differential expression of Fshb and Lhb) down to the gonads (differential expression of Lhr), probably affecting the regulation of steroidogenesis during the TSD process of pejerrey.

Expression profiles of LHβ, FSHβ and their gonadal receptor mRNAs during sexual differentiation of Xenopus laevis tadpoles

The gonadotropins, luteinising hormone (LH) and follicle stimulating hormone (FSH), are important hormones regulating reproductive biology in vertebrates, especially the processes of steroidogenesis and gamete maturation. Despite the role of gonadotropins during the reproductive cycle in amphibians is well established, much less is known about the functional maturation of the hypothalamus–pituitary–gonad axis during larval development. Therefore, the present study aimed to analyze the expression profiles of hypophyseal LHβ and FSHβ mRNA and of their corresponding gonadal receptors (LH-R, FSH-R) in Xenopus laevis tadpoles during their ontogeny and sexual differentiation. The first significant elevation of LHβ and FSHβ mRNA was observed at late premetamorphosis. A clear raise of LHβ mRNA was present during prometamorphic stages especially in males, while the LH-R only slowly increased during ontogeny with highest levels during metamorphic climax. In contrast, FSHβ mRNA expression only slightly increased during ontogeny, however in both sexes the FSH-R mRNA was considerably elevated at prometamorphosis and further at metamorphic climax. Our results suggest that LHβ and LH-R mRNA expression might be involved in initial maturation events of gametes, at least in males, while the gradually increase of FSH-R mRNA coincided with the advancing process of gamete maturation in both sexes. The present study provides for the first time evidence based on expression of gonadotropins and their corresponding gonadal receptors that the hypothalamus–pituitary–gonad axis evolves already at early stages of ontogeny and sexual differentiation in amphibians.

Effect of FSH and LH hormones on oocyte maturation of buffalo and gene expression analysis of their receptors and Cx43 in maturing oocytes

Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are commonly added to maturation media to improve cumulus expansion known to be a predictor of oocyte maturation. Therefore, effects of various concentrations of FSH (1000 ng/ml), LH (1000 ng/ml) and FSH + LH (1000 ng/ml each) in comparison with control (without FSH + LH) cultured oocytes were investigated. FSH and LH (1000 ng/ml each) induced significantly more cumulus expansion and polar body numbers, as compared with control and treatments of 1000 ng/ml FSH and 1000 ng/ml LH alone. Expression of FSH receptor (r), LHr and Cx43 mRNAs was determined by real-time PCR in cumulus-oocyte complexes (COCs) and denuded oocytes at different maturation times. Expression of all three genes was higher in COCs compared with denuded oocytes, confirming the importance of cumulus cells in oocyte maturation. FSHr and connexin 43 (Cx43) mRNA abundance in both COCs and denuded oocytes was highest at 0-6 h of maturation and decreased subsequently. However, LHr mRNA abundance increased from 6 h up to 24 h of maturation. The study concluded that FSH, LH receptors and Cx43 gene expression regulation is an index related to oocyte maturation.

The expression of pituitary FSHβ and LHβ mRNA and gonadal FSH and LH receptor mRNA in the chicken embryo

In avian species, synthesis of sex steroids by embryonic gonads is regulated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In order to elucidate the role of the two gonadotropins in gonadal axis development during the second half of chicken embryogenesis, pituitary expression of LH beta subunit (LHbeta) and FSH beta subunit (FSHbeta) mRNAs as well as gonadal expression of LH and FSH receptor (LHR and FSHR) mRNAs were determined on days 11 (E11) and 17 (E17) of embryonic development and after hatching (D1). In the pituitary of the female embryo, the gene expression of FSHbeta was the lowest on E11 and increased on E17. In the male pituitary, the expression of FSHbeta did not differ among the studied days. The FSHbeta mRNA expression on E11 was higher in the male than in the female pituitary gland. The expression of LHbeta mRNA in the female pituitary increased on D1 in comparison to E11. In the male pituitary gland, the expression of LHbeta gene was relatively constant. The expression of mRNA encoding FSHR in the ovary increased on E17, while in testes it did not differ among the studied days. There were no significant alterations in LHR gene expression in the ovary or in the testes in the examined period however, the gene expression on E17 was higher in the ovary than in the testes. We observed positive correlations between the pituitary FSHbeta mRNA expression and ovarian expression of FSHR mRNA (r = 0.63; p<0.01) as well as between LHbeta mRNA and LHR mRNA in the testes (r=0.65; p<0.01). The reported alterations in gene expression of FSHbeta, LHbeta and their receptors between sexes and among the stages of embryonic development indicate time- and sex-dependent action of gonadotropins in gonads of chicken embryos.

Prolactin independent rescue of mouse corpus luteum life span: identification of prolactin and luteinizing hormone target genes

The corpus luteum (CL) plays a central role in the maintenance of pregnancy in rodents, mainly by secreting progesterone. Female mice lacking prolactin (PRL) receptor (R) are sterile due to a failure of embryo implantation, which is a consequence of decreased luteinizing hormone (LH) receptor expression in the CL and inadequate levels of progesterone. We attempted to treat PRLR(-/-) females with human chorionic gonadotropin (hCG) and showed a de novo expression of LHR mRNA in the corpora lutea. Binding analysis confirmed that the LHR in hCG-treated PRLR(-/-) animals was functional. This was accompanied with increased expression of steroidogenic enzymes involved in progesterone synthesis. Despite these effects, no embryo implantation was observed because of high expression of 20alpha-hydroxysteroid dehydrogenase. To better appreciate the molecular mechanisms underlying maintenance of the CL, a series of mRNA expression-profiling experiments was performed on isolated corpora lutea of PRLR(-/-) and hCG-treated PRLR(-/-) mice. This approach revealed several novel candidate genes with potentially pivotal roles in ovarian function, among them, p27, VE-cadherin, Pten, and sFRP-4, a member of the Wnt/frizzled family. This study showed the differential role of PRL and LH in CL function and identified new targets of these hormones in luteal cells.

Pharmacological characterization, localization and quantification of expression of gonadotropin receptors in Atlantic salmon ( Salmo salar L.) ovaries

The gonadotropins Fsh and Lh interact with their receptors (Fshr and Lhr, respectively) in a highly specific manner in mammals with little overlap in biological activities. In fish, the biological activities seem less clearly separated considering, for example, the steroidogenic potency of both Fsh and Lh. Important determinants of the biological activity are the specificity of hormone–receptor interaction and the cellular site of receptor expression. Here, we report the pharmacological characterization of Atlantic salmon Fshr and Lhr, identify receptor-expressing cells in the ovary, and validate receptor mRNA quantification systems. For the pharmacological studies, we used highly purified coho salmon gonadotropins and found that the Fshr preferentially responded to Fsh, but was also activated by ∼6-fold higher levels of Lh. The Lhr was specific for Lh and did not respond to Fsh. Photoperiod manipulation was used to generate ovarian tissue samples with largely differing stages of maturation. Specific real-time, quantitative (rtq) PCR assays revealed up to 40-fold ( fshr) and up to 350-fold ( lhr) changes in ovarian expression levels, which correlated well with the differences in ovarian weight, histology, and circulating oestrogen levels recorded in January and June, respectively. Vitellogenic ovaries were used to localise receptor-expressing cells by in situ hybridization. Granulosa cells of small and large vitellogenic follicles were positive for both receptors. Also theca cells of small and large vitellogenic follicles expressed fshr mRNA, while only in large vitellogenic follicles theca cells were (weakly) positive for lhr mRNA. While only ovulatory Lh levels seem high enough to cross-activate the Fshr, expression by both receptors by granulosa and theca cells suggests that homologous ligand receptor interaction will prevail.

Screening for membrane hormone receptor expression in primary aldosteronism

The investigation of expression and functional relevance of G-protein coupled receptors in primary aldosteronism (PA) by molecular and clinical studies. Tissues of 14 aldosterone-producing adenomas (APA), of one unilateral adrenal hyperplasia and of six healthy adult adrenal glands; 12 patients with confirmed PA due to APA; (n=5), idiopathic hyperaldosteronism (n=7) and 8 control subjects (C). The tissues were subjected to a quantitative PCR for determination of mRNA expression levels of AT2R1, GIPR, MC2R, GnRHR, LHR, TRHR, TSHR, glucagon-R, V1aR, V2R, and 5-HT4R. The patients and controls were enrolled in a test protocol consisting of stimulation by posture, mixed meal, ACTH, GnRH, TRH, glucagon, vasopressin, and metoclopramide (MCP). Three patients could be analyzed by both studies. A positive response was defined as an aldosterone increase of more than 50% following stimulation. All the tissues revealed AT2R1, MC2R, AVPR, and 5-HT4R mRNA expression. LHR mRNA was found in normal adrenals and 13 adrenal tumors. By contrast with normal adrenals tumorous adrenal tissue expressed GnRHR mRNA (4/15) and TSHR mRNA (1/15). Both the patients and controls responded to posture, ACTH, glucagon, AVP, and MCP. Specific responses were seen in one patient following TRH and three patients following GnRH stimulation. We provide evidence for peptide hormone responsiveness to various peptide hormones in patients with PA, including GnRH and TRH. A good correlation between clinical and molecular testing could be observed, making an involvement of the receptor expressed in PA possible.

Unilateral testicular tumour associated to congenital adrenal hyperplasia: Failure of specific tumoral molecular markers to discriminate between adrenal rest and leydigioma

Testicular adrenal rest tumours are frequently associated with congenital adrenal hyperplasia (CAH). These ACTH-dependent tumours cannot be easily distinguished histologically from Leydig-cell tumours. We report the case of a 30-year-old man who was explored for infertility, azoospermia and unilateral testicular tumour. High levels of 17-OH progesterone and ACTH, low cortisol and undetectable gonadotropins levels, associated to bilateral adrenal hyperplasia, led to the diagnosis of CAH by 21-OH deficiency with a composite heterozygoty. The testicular tumour was first considered as adrenal rest. However, histological analysis of this unilateral painful tumour showed a steroid-hormone-secreting cell proliferation with atypical and frequent mitosis. To discriminate between a benign adrenal rest tumour and a possible malignant leydigioma, tumoral expression of specific gene products was analyzed by RT-PCR. No 11-beta-hydroxylase nor ACTH receptor mRNAs could be found in the tumour, which did not behave like usual adrenal rest cells. For this unilateral testicular tumour, the lack of adrenal-specific markers associated with a high rate of mitosis and pleiomorphism supported a leydigian origin with malignant potential. However, lack of tumoral LH-R mRNA expression and a tumour-free 3-year follow-up led us to retain the diagnosis of adrenal rest tumour with loss of adrenal gene expression and progressive autonomous behaviour.

Role for Inhibitor of Differentiation/Deoxyribonucleic Acid-Binding (Id) Proteins in Granulosa Cell Differentiation

Recent studies in the hen ovary have linked the initiation of granulosa cell differentiation at follicle selection to the alleviation of inhibitory MAPK signaling. The present studies assessed a role for individual inhibitor of differentiation (Id) protein isoforms as modulators of key transcriptional events occurring within granulosa cells at or immediately subsequent to differentiation. Findings from freshly collected granulosa cells collected at different stages of follicle development demonstrated a negative association between expression levels for Id2 mRNA compared with levels of Id1, Id3, and Id4. Elevated levels of Id2 are related to a differentiating/differentiated phenotype, whereas elevated Id1, Id3, and Id4 are associated with an undifferentiated phenotype. This negative relationship extends to cell signal transduction, because factors that promote inhibitory MAPK signaling (TGF-alpha and betacellulin) block expression of Id2 mRNA but increase levels of Id1, Id3, and Id4. Furthermore, overexpression of Gallus Id2 in cultured granulosa was found to significantly decrease levels of Id1, Id3, and Id4 mRNA but facilitate FSHR mRNA expression and, importantly, initiate LHR mRNA expression plus LH-induced progesterone production. Finally, knockdown studies using small interfering RNA specific for Id2 revealed reduced expression of FSHR and LHR mRNA and attenuated FSH- and LH-induced levels of StAR and p450 cholesterol side-chain cleavage enzyme mRNA plus progesterone production. Collectively, these data demonstrate that Id2 expression is both sufficient and necessary for increasing LHR expression and, as a result, promoting gonadotropin-induced differentiation in hen granulosa cells subsequent to follicle selection.

The gonadotropin receptors FSH-R and LH-R of Atlantic halibut (Hippoglossus hippoglossus)—2. Differential follicle expression and asynchronous oogenesis

The biological activity and spatio–temporal expression patterns of the gonadotropin receptors FSH-R and LH-R were examined in the repetitive spawner Atlantic halibut to elucidate the gonadotropic regulation of the asynchronous follicle development. The cloned receptors were expressed in mammalian COS-7 cells, and stimulation with sea bass FSH and LH increased the cAMP production. The halibut FSH-R and LH-R genes were shown to be highly expressed in the gonads of sexually mature fish, but the transcripts were also found in extra-gonadal tissues such as pituitary and brain. Different expression patterns of FSH-R and LH-R in the developing follicles were documented by semi-quantitative RT-PCR. Abundant FSH-R mRNA was found in the small follicles during primary growth and vitellogenesis, and the signals were localized to the granulosa cells by in situ hybridization. In contrast, follicular LH-R mRNA was hardly detectable during the early stages. Conversely, in follicles during final maturation FSH-R mRNA levels tended to decrease, while the expression of LH-R was highly upregulated. Whereas the pituitary FSH and LH are asynchronously expressed in annual spawners, both gonadotropins were expressed in the female halibut pituitary throughout the reproductive cycle, except in the prespawning females. Hence, the sequential gonadotropic activation of ovarian follicle growth and maturation in repetitive spawners is probably regulated by modulating the temporal expression of FSH-R and LH-R in the follicle membrane.

Effects of Daidzein on mRNA Expression of Gonadotropin Receptors and P450 Aromatase in Ovarian Follicles of White Silky Fowls

Effects of daidzein on expression of mRNAs of gonadotropin receptors (FSHR, LHR) and P450 aromatase (P450arom) were evaluated in ovarian follicles of white silky fowls. The hens were 13 months old in the post-peak period of egg laying and were randomly allocated as control and daidzein-treated groups, with daidzein supplemented to the basal diet at 10 mg/kg for 7 consecutive weeks. The mRNA expression of related genes was measured by semi-quantitative RT-PCR in the granulosa layers of the preovulatory follicle (PRF: Fl, F2 ...) and follicular layers of the small yellow follicle (SYF), large white follicle (LWF) and atretic follicle (ATF). Results showed that daidzein supplementation significantly increased the number of SYF and LWF (p<0.05). The relative abundance of the FSHR mRNA decreased in the granulosa layers from F3 to Fl, but LHR mRNA displayed opposite developmental changes. P450arom mRNA was highest in the SYF, but was very low in the granulosa layers after follicles finished selection. Treatment with daidzein resulted in increased mRNA expression of FSHR in F3 granulosa layer, LHR in granulosa layers of F3 to Fl and P450arom in LWF (p<0.05). These results indicated that dietary supplementation of daidzein up-regulated mRNA expression of gonadotropin receptors and P450arom to improve the development of preovulatory follicles in white silky fowls after the peak-laying period.

Differences in the expression of luteinizing hormone and follicle-stimulating hormone receptors in the lower urinary tract between intact and gonadectomised male and female dogs

Receptors for LH (LHR) and FSH (FSHR) are expressed in the canine lower urinary tract (LUT). As gonadectomy results in an increase in plasma LH and FSH, the objective of this study was to determine whether there are any differences in the expression of LHR and FSHR in the LUT between intact and gonadectomised dogs. Four regions of the LUT, i.e. body and neck of the bladder as well as proximal and distal urethra, were collected from 20 healthy dogs (5 intact males, 5 intact anoestrous females, 4 castrated males and 6 spayed females). The mRNA and protein expression of receptors was determined by in situ hybridization and immunohistochemistry, respectively, and assessed semi-quantitatively incorporating both the distribution and the intensity of specific staining. Expression of LHR and FSHR was present in all tissue layers (epithelium, sub-epithelial stroma and muscle) of each region with different levels of the expression. Overall mRNA and protein expression for both LHR and FSHR was significantly (P<0.001) lower in gonadectomised dogs. Intact dogs had more (P<0.05) LHR and FSHR mRNA and protein in all tissue layers of the four regions, except for LHR mRNA expression in the sub-epithelial stroma where no differences were observed between the two statuses. Decreases in LHR and FSHR mRNA and protein in gonadectomised dogs appeared to be more consistent in spayed bitches compared to castrated males. Lower expression of LHR and FSHR observed in gonadectomised dogs may adversely affect the normal canine LUT function.

Growth Differentiation Factor 9 (GDF9) Stimulates Proliferation and Inhibits Steroidogenesis by Bovine Theca Cells: Influence of Follicle Size on Responses to GDF91

Ovarian follicular development is controlled by numerous paracrine and endocrine regulators, including oocyte-derived growth differentiation factor 9 (GDF9), and a localized increase in bioavailable insulin-like growth factor 1 (IGF1). The effects of GDF9 on function of theca cells collected from small (3-6 mm) and large (8-22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis, as measured by a (3)H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells, but had no effect on IGF1R, STAR, or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated similar to mothers against decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells, but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development.

LHR splicing variants and gene expression in the marmoset monkey

In the marmoset monkey, the LHR type II, lacking exon 10, is the native receptor type. We characterised the LHR splicing pattern in marmoset testes and the adrenals during puberty and in pre- and postpubertal ovaries and quantified mRNA LHR expression in the testis. We detected 11 LHR splicing variants expressed at similar levels and generated by exon skipping and/or usage of cryptic splice sites. No preferred splicing variant during pubertal maturation was observed in both sexes. Testicular and adrenal LHR expression levels did not significantly change with age. However, a significant increase during pubertal maturation for the serum testosterone/ LHR ratio indicated that testosterone secretion increases in the presence of constant LHR mRNA expression levels. We conclude that LHR splicing in the marmoset displays a homogenous pattern and that the main function of the LHR is established in the testis, reaching its highest efficiency during pubertal maturation.

Differential expression and regulation of gonadotropins and their receptors in the Japanese eel, Anguilla japonica

Eel species have a striking life cycle with a blockade of puberty until the oceanic migration. We report the first molecular data on eel gonadotropin receptors. The partial sequences cloned covered two-third of the open reading frame and included most of the extracellular and transmembrane domains. Phylogenetic analysis partitioned the two eel gonadotropin receptors into the two teleost FSHR and LHR clusters, respectively. Real-time quantitative RT-PCR was used to quantify the expression of eel gonadotropins and their receptors. Similar levels of pituitary FSH-β and LH-β transcripts were found in the immature previtellogenic female eels. In contrast, ovarian FSHR mRNA level was at 100- to 185-fold higher than that of LHR. This revealed that FSHR rather LHR would mediate gonadotropin stimulation of the early stages of ovarian growth. Chronic treatment with fish pituitary homogenates, applied to induce eel sexual maturation, stimulated pituitary LH-β but suppressed FSH-β transcripts. In the ovaries, both FSHR and LHR mRNA were significantly increased in experimentally matured eels. Treatments with sexual steroids showed a stimulatory effect of estradiol-17β (E 2) on pituitary LH-β mRNA levels, while FSH-β transcripts were suppressed by E 2 or testosterone (T). In contrast, neither E 2 nor T-treatment had any significant effect on ovarian FSHR nor LHR transcripts. This suggests that steroid feedbacks may be responsible for the opposite regulation of pituitary gonadotropins in experimentally matured eels, but are not involved in the regulation of gonadotropin receptors. In conclusion, these are the first data on the sequence, expression and regulation of gonadotropin receptors in the eel. They provide new foundation for basic and applied research on eel reproduction.