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1.
Gen Comp Endocrinol ; 285: 113241, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31400434

RESUMO

Pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), play central roles in the control of gonadal development of vertebrates. In mammals, Fsh and Lh exclusively activate their respective cognate receptors: Fsh receptor (Fshr) in the Sertoli cell and Lh/choriogonadotropin receptor (Lhcgr) in the Leydig cell. In teleosts, the distinct functions of Fsh and Lh and information on cellular localization of their receptors are still poorly understood. Recently we established FreeStyle 293-F cell lines producing recombinant Japanese eel Fsh and Lh (reFsh and reLh), which form a single chain consisting of a common α-subunit and ß-subunits. In this study, we conducted functional analyses of reFsh and reLh, focusing on the binding specificities to their receptors and effects on testicular steroidogenesis in vitro. Assays with gonadotropin receptors-expressing COS-7 cells indicated reFsh stimulated its cognate receptor, meanwhile reLh activated both receptors. Although results of in vitro incubations showed that reFsh and reLh induced testicular 11-ketotestosterone production in a dose and time-dependent manner by upregulating expression of steroidogenic enzymes, the effective doses of reLh were apparently lower and the effects of reLh emerged faster in comparison with reFsh. Results of quantitative real-time PCR using testicular cell fractions showed that fshr and lhcgr1 mRNA were detected both in Sertoli and Leydig cells. These analyses revealed that reFsh and reLh were biologically active and hence will be useful for future studies. Moreover, our data showed that both eel Fsh and Lh acted as steroidogenic hormones through their receptors in testicular somatic cells; however, Lh was more potent on androgen production, implying differential functions on spermatogenesis.


Assuntos
Anguilla/metabolismo , Hormônio Foliculoestimulante/metabolismo , Hormônio Luteinizante/metabolismo , Receptores do FSH/metabolismo , Receptores do LH/metabolismo , Proteínas Recombinantes/metabolismo , Esteroides/metabolismo , Testículo/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Japão , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do FSH/genética , Receptores do LH/genética , Testosterona/análogos & derivados , Testosterona/metabolismo
2.
Gen Comp Endocrinol ; 285: 113276, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31536722

RESUMO

Reproduction in vertebrates is controlled by the brain-pituitary-gonad axis, where the two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play vital parts by activating their cognate receptors in the gonads. The main purpose of this work was to study intra- and interspecies ligand promiscuity of teleost gonadotropin receptors, since teleost receptor specificity is unclear, in contrast to mammalian receptors. Receptor activation was investigated by transfecting COS-7 cells with either Fsh receptor (mdFshr, tiFshr) or Lh receptor (mdLhr, tiLhr), and tested for activation by recombinant homologous and heterologous ligands (mdFshßα, mdLhßα, tiFshßα, tiLhßα) from two representative fish orders, Japanese medaka (Oryzias latipes, Beloniformes) and Nile tilapia (Oreochromis niloticus, Cichliformes). Results showed that each gonadotropin preferentially activates its own cognate receptor. Cross-reactivity was detected to some extent as mdFshßα was able to activate the mdLhr, and mdLhßα the mdFshr. Medaka pituitary extract (MPE) stimulated CRE-LUC activity in COS-7 cells expressing mdlhr, but could not stimulate cells expressing mdfshr. Recombinant tiLhßα, tiFshßα and tilapia pituitary extract (TPE) could activate the mdLhr, suggesting cross-species reactivity for mdLhr. Cross-species reactivity was also detected for mdFshr due to activation by tiFshßα, tiLhßα, and TPE, as well as for tiFshr and tiLhr due to stimulation by mdFshßα, mdLhßα, and MPE. Tissue distribution analysis of gene expression revealed that medaka receptors, fshr and lhr, are highly expressed in both ovary and testis. High expression levels were found for lhr also in brain, while fshr was expressed at low levels. Both fshr and lhr mRNA levels increased significantly during testis development. Amino acid sequence alignment and three-dimensional modelling of ligands and receptors highlighted conserved beta sheet domains of both Fsh and Lh between Japanese medaka and Nile tilapia. It also showed a higher structural homology and similarity of transmembrane regions of Lhr between both species, in contrast to Fshr, possibly related to the substitution of the conserved cysteine residue in the transmembrane domain 6 in medaka Fshr with glycine. Taken together, this is the first characterization of medaka Fshr and Lhr using homologous ligands, enabling to better understand teleost hormone-receptor interactions and specificities. The data suggest partial ligand promiscuity and cross-species reactivity between gonadotropins and their receptors in medaka and tilapia.


Assuntos
Oryzias/metabolismo , Receptores do FSH/metabolismo , Receptores do LH/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Feminino , Hormônio Foliculoestimulante/química , Hormônio Foliculoestimulante/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hormônio Luteinizante/química , Hormônio Luteinizante/metabolismo , Masculino , Modelos Moleculares , Receptores do FSH/genética , Receptores da Gonadotropina/metabolismo , Receptores do LH/genética , Transdução de Sinais
3.
Genes (Basel) ; 10(12)2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842416

RESUMO

Anti-Mullerian hormone (AMH) is an important reproductive marker of ovarian reserve produced by granulosa cells (GCs) of pre-antral and early-antral ovarian follicles in several species, including cattle. This hormone plays a vital role during the recruitment of primordial follicles and follicle stimulating hormone (FSH)-dependent follicular growth. However, the regulatory mechanism of AMH expression in follicles is still unclear. In this study, we compared the expression of AMH, AMHR-II, BMP2, BMP6, FSHR, and LHCGR genes during follicular development. In-vitro expression study was performed with and without FSH for AMH, AMHR-II, BMP2, and BMP6 genes in bovine GCs which were isolated from 3-8 mm follicles. Association among the mRNA expression and hormone level was estimated. GCs were collected from small (3-8 mm), medium (9-12 mm) and large size (13 to 24 mm) follicles before, during onset, and after deviation, respectively. Further, mRNA expression, hormones (AMH, FSH, and LH), apoptosis of GCs, and cell viability were detected by qRT-PCR, ELISA, flow cytometry, and spectrophotometry. AMH, AMHR-II, BMP2, and FSHR genes were highly expressed in small and medium follicles as compared to large ones. In addition, the highest level of AMH protein (84.14 ± 5.41 ng/mL) was found in medium-size follicles. Lower doses of FSH increased the viability of bovine GCs while higher doses repressed them. In-vitro cultured GCs treated with FSH significantly increased the AMH, AMHR-II, and BMP2 expression levels at lower doses, while expression levels decreased at higher doses. We found an optimum level of FSH (25 ng/mL) which can significantly enhance AMH and BMP2 abundance (p < 0.05). In summary, AMH, AMHR-II, and BMP2 genes showed a higher expression in follicles developed in the presence of FSH. However, lower doses of FSH demonstrated a stimulatory effect on AMH and BMP2 expression, while expression started to decline at the maximum dose. In this study, we have provided a better understanding of the mechanisms regulating AMH, AMHR II, and BMP2 signaling in GCs during folliculogenesis, which would improve the outcomes of conventional assisted reproductive technologies (ARTs), such as superovulation and oestrus synchronization in bovines.


Assuntos
Hormônio Antimülleriano/genética , Bovinos/genética , Células da Granulosa/metabolismo , Animais , Hormônio Antimülleriano/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/genética , Líquido Folicular/metabolismo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Folículo Ovariano/metabolismo , RNA Mensageiro/genética , Receptores do FSH/genética , Receptores do FSH/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo
4.
Chem Biol Interact ; 312: 108817, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499053

RESUMO

Aconitine might have reproductive toxicity and the effects of aconitine on androgen synthesis in Leydig cells remain unclear. Here, we explore how aconitine affects androgen synthesis and metabolism in rat immature Leydig cells in vitro. Immature Leydig cells were isolated from 35-day-old male Sprague Dawley rats and cultured with 0-50 µM aconitine for 3 h in combination with LH, 8Br-cAMP, 22R-hydroxycholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively. Medium androgens were measured. The levels of Leydig cell mRNAs, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14, were measured by qPCR. ROS and apoptosis were determined after 24-h aconitine treatment. Aconitine inhibited basal androgen production in Leydig cells at 0.05 µM and the higher concentrations. Aconitine blocked pregnenolone, progesterone, and androstenedione mediated androgen outputs without affecting 22R-hydroxycholesterol-mediated androgen production at 5 µM. Aconitine also inhibited LH and 8Br-cAMP stimulated androgen outputs at 5 µM. Further investigation showed that aconitine blocked androgen synthesis via down-regulating the expression of Scarb1, Hsd3b1, Cyp17a1, and Hsd17b3. At 50 µM, aconitine also induced ROS generation and increased apoptotic rate of Leydig cells. Aconitine lowered serum testosterone levels at 1.5 mg/kg after 7 days of oral exposure from postnatal day 35. In conclusion, aconitine inhibits androgen synthesis.


Assuntos
Aconitina/farmacologia , Androgênios/metabolismo , Regulação para Baixo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Testosterona/sangue , Testosterona/farmacologia
5.
J Steroid Biochem Mol Biol ; 194: 105460, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31470110

RESUMO

The bile acid receptor Farnesoid-X-Receptor alpha (FXRα), a member of the nuclear receptor superfamily, is well known for its roles in the enterohepatic tract. In addition, FXRα regulates testicular physiology through the control of both endocrine and exocrine functions. The endocrine function of the Leydig cells is mainly controlled by the hypothalamo-pituitary axis viaLH/chorionic gonadotropin (CG). If FXRα was demonstrated to control the expression of the Lhcgr gene, encoding the LH receptor; the impact of the LH/CG signaling on the Fxrα expression has not been defined so far. Here, we demonstrate that hCG increases the Fxrα gene expression through the protein kinase-A signaling pathway. Fxrα is then involved in a negative feedback of steroid synthesis. These data improve our knowledge of the local control of the testicular steroidogenesis with the identification of the link between the hypothalamo-pituitary axis and the FXRα signaling pathway.


Assuntos
Gonadotropina Coriônica/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Testículo/efeitos dos fármacos , Animais , Linhagem Celular , Masculino , Camundongos Endogâmicos C57BL , Fosfoproteínas/genética , Progesterona/metabolismo , Receptores do LH/genética , Transdução de Sinais/efeitos dos fármacos , Testículo/metabolismo , Testosterona/sangue , Testosterona/metabolismo
6.
Life Sci ; 233: 116694, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31351970

RESUMO

AIMS: The hypoxia-stimulated response of the endocrine system depends on the kind and duration of hypoxia. Hypoxia has been reported to stimulate testosterone (T) production in rats, but the mechanisms remain to be investigated. MATERIALS AND METHODS: Male rats were divided into two groups. The rats exposed to chronic intermittent hypoxia (CIH) at 8 h/day were housed in a hypoxic chamber (12% O2) for 14 days. Normoxic rats were used as control animals. T was measured after challenging the rat Leydig cells (LCs) with different stimulators, including hCG (0.01 IU/ml), forskolin (10-5 M), 8-bromo-cAMP (10-4 M), A23187 (10-5 M), cyclopiazonic acid (10-4 M), and androstenedione (10-8 M). Meanwhile, the LCs were incubated with trilostane (10-5 M) and/or 25-OH-hydroxycholesterol (10-5 M); thereafter the media were collected for pregnenolone assay. KEY FINDINGS: In the CIH group, plasma T levels were increased, but the serum luteinizing hormone (LH) was decreased. Furthermore, at several time intervals after hCG injection, plasma T levels were higher in the CIH group. The evoked-release of T and pregnenolone were significantly increased in the CIH group. Compared with the normoxic group, the CIH group had higher mRNA and protein expression levels of the LH receptor and CYP11A1 but not StAR. The plasma and testicular microvasculature VEGF levels were increased in the CIH group. The testicular vessel distribution was more obvious in CIH rats. SIGNIFICANCE: CIH-induced T secretion might be partially mediated by mechanisms involving the induction of LH receptor expression, testicular angiogenesis, CYP11A1 activity, 17ß-HSD activity, and calcium-related pathway.


Assuntos
Hipóxia Celular/fisiologia , Colforsina/farmacologia , Células Intersticiais do Testículo/metabolismo , Testosterona/metabolismo , Animais , Células Cultivadas , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores do LH/genética , Receptores do LH/metabolismo , Vasodilatadores/farmacologia
7.
Eur J Endocrinol ; 181(2): K11-K20, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31167162

RESUMO

Testosterone production by the fetal testis depends on a functional relationship between hCG and the LH/chorionic gonadotropin receptor (LHCGR). Failure of the receptor to correctly respond to its ligand leads to impaired sexual differentiation in males. A phenotypically female patient with pubertal delay had a 46,XY karyotype and was diagnosed with 46,XY disorder of sex development (DSD). Novel compound heterozygous LHCGR mutations were found in the signal peptide: a duplication p.L10_Q17dup of maternal origin, and a deletion (p.K12_L15del) and a p.L16Q missense mutation of paternal origin. cAMP production was very low for both the deletion and duplication mutations and was halved for the missense mutant. The duplication and missense mutations were both expressed intracellularly, but at very low levels at the cell membrane; they were most likely retained in the endoplasmic reticulum. The deletion mutant had a very limited intracellular expression, indicating impaired biosynthesis. There was reduced expression of all three mutants, which was most marked for the deletion mutation. There was also decreased protein expression of all three mutant receptors. In the deletion mutation, the presence of a lower-molecular-weight band corresponding to LHCGR monomer, probably due to lack of glycosylation, and a lack of bands corresponding to dimers/oligomers suggests absent ER entry. This novel case of 46,XY DSD illustrates how different LHCGR signal peptide mutations led to complete receptor inactivation by separate mechanisms. The study underlines the importance of specific regions of signal peptides and expands the spectrum of LHCGR mutations.


Assuntos
Transtorno 46,XY do Desenvolvimento Sexual/diagnóstico por imagem , Transtorno 46,XY do Desenvolvimento Sexual/genética , Mutação/genética , Receptores do LH/genética , Adolescente , Feminino , Humanos
8.
J Assist Reprod Genet ; 36(7): 1457-1469, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31187330

RESUMO

PURPOSE: To determine whether a selected set of mRNA biomarkers expressed in individual cumulus granulosa cell (CC) masses show association with oocyte developmental competence, embryo ploidy status, and embryo outcomes. METHODS: This prospective observational cohort pilot study assessed levels of mRNA biomarkers in 163 individual CC samples from 15 women stimulated in antagonist cycles. Nineteen mRNA biomarker levels were measured by real-time PCR and related to the development of their corresponding individually cultured oocytes and subsequent embryos, embryo ploidy status, and live birth outcomes. RESULTS: PAPPA mRNA levels were significantly higher in CC from oocytes that led to euploid embryos resulting in live births and aneuploid embryos compared to immature oocytes by ANOVA. LHCGR mRNA levels were significantly higher in CC of oocytes resulting in embryos associated with live birth compared to immature oocytes and oocytes resulting in arrested embryos by ANOVA. Using a general linearized mixed model to assess ploidy status, CC HSD3B mRNA levels in oocytes producing euploid embryos were significantly lower than other oocyte outcomes, collectively. When transferred euploid embryos outcomes were analyzed by ANOVA, AREG mRNA levels were significantly lower and PAPPA mRNA levels significantly higher in CC from oocytes that produced live births compared to transferred embryos that did not form a pregnancy. CONCLUSIONS: Collectively, PAPPA, LHCGR, and AREG mRNA levels in CC may be able to identify oocytes with the best odds of resulting in a live birth, and HSD3B1 mRNA levels may be able to identify oocytes capable of producing euploid embryos.


Assuntos
Anfirregulina/genética , Complexos Multienzimáticos/genética , Oócitos/crescimento & desenvolvimento , Proteína Plasmática A Associada à Gravidez/genética , Progesterona Redutase/genética , Receptores do LH/genética , Esteroide Isomerases/genética , Adulto , Células do Cúmulo/metabolismo , Transferência Embrionária , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Técnicas de Maturação in Vitro de Oócitos , Recuperação de Oócitos , Oócitos/metabolismo , Oogênese/genética , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Ploidias , Gravidez , RNA Mensageiro/genética
9.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30965614

RESUMO

Luteinizing hormone (LH), a pituitary gonadotropin, coupled with LH receptor (LHR) is essential for the regulation of the gonadal maturation in vertebrates. Although LH homolog has been detected by immunocytochemical analysis, and its possible role in ovarian maturation was revealed in decapod crustacean, so far there is no molecular evidence for the existence of LHR. In this study, we cloned a novel LHR homolog (named EsLHR) from the Chinese mitten crab Eriocheir sinensis. The complete sequence of the EsLHR cDNA was 2775bp, encoding a protein of 924 amino acids, sharing 71% amino acids identity with the ant Zootermopsis nevadensis LHR. EsLHR expression was found to be high in the ovary, while low in testis, gill, brain, and heart, and no expression in the thoracic ganglion, eye stalk, muscle, and hepatopancreas. Quantitative PCR revealed that the expression level of EsLHR mRNA was significantly higher in the ovaries in previtellogenic (Pvt), late vitellogenic (Lvt), and germinal vesicle breakdown (GVBD) stages than that in the vitellogenic (Mvt) and early vitellogenic (Evt) stages (P < 0.05), and, the highest and the lowest expression were in Lvt, and Evt, respectively. The strong signal was mainly localized in the ooplasm of Pvt oocyte as detected by in situ hybridization. The crab GnRH homolog can significantly induce the expression of EsLHR mRNA at 36 hours post injection in vivo (P < 0.01), suggesting that EsLHR may be involved in regulating ovarian development through GnRH signaling pathway in the mitten crab.


Assuntos
Braquiúros/metabolismo , Receptores do LH/metabolismo , Animais , Braquiúros/embriologia , DNA Complementar/metabolismo , Feminino , Masculino , Ovário/embriologia , Ovário/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Receptores do LH/genética , Testículo/embriologia , Testículo/metabolismo
10.
Environ Toxicol ; 34(7): 844-852, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30951242

RESUMO

Our goals were to investigate whether environmentally relevant doses of T-2 toxin can affect human ovarian granulosa cells' function and to reveal the potential mechanism of T-2 toxin's action. Results showed that T-2 toxin strongly attenuated luteinizing hormone/choriogonadotropin receptor (LHCGR) mRNA expression in follicle-stimulating hormone (FSH)-stimulated human cumulus granulosa cells. Addition of human chorionic gonadotropin was not able to elicit maximal response of ovulatory genes amphiregulin, epiregulin, and progesterone receptor. T-2 toxin reduced mRNA levels of CYP19A1 and steroidogenic acute regulatory protein (STAR) and lowered FSH-stimulated estradiol and progesterone production. Mechanistic experiments demonstrated that T-2 toxin decreased FSH-stimulated cyclic adenosine monophosphate (cAMP) production. Addition of total PDE inhibitor 3-isobutyl-1-methylxanthine prevented T-2 toxin's action on LHCGR, STAR, and CYP19A1 mRNA expression in FSH-stimulated human cumulus granulosa cells. Furthermore, T-2 toxin partially decreased 8-bromoadenosine 3'5'-cyclic monophosphate (8-Br-cAMP)-stimulated LHCGR and STAR, but did not affect 8-Br-cAMP-stimulated CYP19A1 mRNA expression in human cumulus granulosa cells. Overall, our data indicate that environmentally relevant dose of T-2 toxin decreases steroidogenesis and ovulatory potency in human cumulus granulosa cells probably through activation of PDE, thus posing a significant risk for female fertility.


Assuntos
Aromatase/genética , Células do Cúmulo/efeitos dos fármacos , AMP Cíclico/metabolismo , Hormônios Esteroides Gonadais/biossíntese , Fosfoproteínas/genética , Receptores do LH/genética , Toxina T-2/farmacologia , Adulto , Aromatase/metabolismo , Células Cultivadas , Gonadotropina Coriônica/metabolismo , Células do Cúmulo/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Fosfoproteínas/metabolismo , Progesterona/metabolismo , RNA Mensageiro/metabolismo , Receptores do LH/metabolismo , Adulto Jovem
11.
Syst Biol Reprod Med ; 65(5): 400-408, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30958034

RESUMO

Altered folliculogenesis and reproductive anomalies in polycystic ovary syndrome (PCOS) suggest that variations of genes involved in folliculogenesis might influence etiopathogenesis of this syndrome. The objective of this study was to assess the association of LHß (rs1056917) and lutropin receptor (LHR) (rs61996318) polymorphism with polycystic ovarian syndrome and to interrelate the levels of luteinizing hormone (LH) with severity of clinical manifestations of PCOS. Three hundred women of reproductive age were enrolled in this retrospective case-control study. Rotterdam Criteria was used to diagnose PCOS patients. Nucleotide mutations of LH and LHR gene was analyzed using polymerase chain reaction-restriction fragment length polymorphism. High LH levels were found in 88% of PCOS patients. LHß TC and CC genotypes were significantly associated with PCOS risk (OR [odds ratio] 13.95, CI [confidence interval] 6.30-30.86, p < 0.0001 and OR 3.31, CI 1.30-8.41, p = 0.01). The frequency of the C allele was 0.31 in PCOS and 0.02 in controls (OR 18.80, CI 8.54-41.37, p < 0.0001). LHR CA and AA genotype conferred a significant risk in development of PCOS (OR 5.07, CI 2.50-10.31, p < 0.0001). The frequency of the A allele was 0.51 in PCOS and 0.03 in controls (OR 26.62, CI 13.99-50.65, p < 0.0001). The results show an association between polymorphism of LHß, LHR and PCOS, indicating that variants of these genes may affect the metabolic pathways involved in this syndrome. Majority of the affected women were found to have elevated LH levels. This study sheds new light in the diagnosis, treatment and management of PCOS syndrome. Abbreviations: AUC: area under curve; BMI: body mass index; C: cholesterol; CI: confidence interval; DBP: diastolic blood pressure; DHEAS: dehydroepiandrosterone sulfate; FG: Ferriman-Gallway; FSH: follicle stimulating hormone; GHQ: general health questionnaire; HA: hyperandrogenism; HDL-C: high-density lipoprotein cholesterol; HOMA-IR: homeostatic model assessment for insulin resistance; HWR: hip waist ratio; LDL-C: low-density lipoprotein cholesterol; LH: luteinizing hormone; LH: luteinizing hormone; LHR: lutropin receptor; O: oligomenorrhea; OR: odds ratio; PCO: polycystic ovaries; PCO: polycystic ovary; PCOS: polycystic ovary syndrome; PCR: polymerase chain reaction; ROC: receiver operating curve; SBP: systolic blood pressure; SE: standard error of coefficient; SNP: single nucleotide polymorphism; TG: triglycerides; TSH: thyroid stimulating hormone; VD: vitamin D.


Assuntos
Hormônio Luteinizante/genética , Síndrome do Ovário Policístico/genética , Polimorfismo de Nucleotídeo Único , Receptores do LH/genética , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Índia , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto Jovem
12.
J Steroid Biochem Mol Biol ; 190: 183-192, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30954507

RESUMO

Luteinizing hormone and human chorionic gonadotropin (hCG) bind to the luteinizing hormone/chorionic gonadotropin receptor (LHCGR). LHCGR is required to maintain corpus luteum function but the mechanisms involved in the regulation of LHCGR in human luteal cells remain incompletely understood. This study aimed to characterize the expression of LHCGR mRNA in primary human luteinized granulosa cells (hLGCs) obtained from patients undergoing in vitro fertilization and to correlate LHCGR expression with the response of hLGCs to hCG by assessing the expression of genes known to be markers of hCG actions. The results show that LHCGR expression is low in freshly isolated cells but recovers rapidly in culture and that hCG maintains LHCGR expression, suggesting a positive feedback loop. The activity of a LHCGR-LUC reporter increased in cells treated with hCG but not with follicle-stimulating hormone. Treatment with hCG also stimulated the expression of genes involved in steroidogenesis in a time-dependent manner. LHCGR promoter expression was found to be regulated by SP1, which we show is highly expressed in hLGCs. Moreover, SP1 inhibition prevented the stimulation of steroidogenic genes and the increase in LHCGR-LUC reporter activity by hCG. Finally, we provide evidence that a complex formed by SP1 and GATA4 may play a role in the maintenance of LHCGR expression. This report reveals the mechanisms involved in the regulation of the LHCGR and provides experimental data demonstrating that the proximal region of the LHCGR promoter is sufficient to drive the expression of this gene in primary hLGCs.


Assuntos
Regulação da Expressão Gênica , Células Lúteas/metabolismo , Receptores do LH/genética , Fator de Transcrição Sp1/metabolismo , Células Cultivadas , Gonadotropina Coriônica/metabolismo , Feminino , Fertilização In Vitro , Humanos , Esteroides/metabolismo
13.
Gen Comp Endocrinol ; 280: 123-133, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31009604

RESUMO

Receptors for follicle-stimulating hormone (Fshr), luteinizing hormone (Lhcgr1 and Lhcgr2) and androgens (Ara and Arb) transduce the hormonal signals that coordinate spermatogenesis, but the factors that regulate the abundance of these transducers in fish testes remain little-understood. To mend this paucity of information, we first determined changes in transcript abundance for these receptors (fshr, lhcgr1, ara and arb) during spermatogenesis induced by human chorionic gonadotropin (hCG) injection in the eel, Anguilla australis. We related our findings to testicular production of the fish androgen, 11-ketotestosterone (11-KT), and to the levels of the transcripts encoding steroidogenic acute regulatory protein (star) and 11ß-hydroxylase (cyp11b), and subsequently evaluated the effects of hCG or 11-KT on mRNA levels of these target genes in vitro. Testicular 11-KT production was greatly increased by hCG treatment, both in vivo and in vitro, and associated with up-regulation of star and cyp11b transcripts. In situ hybridization indicated that testicular fshr mRNA levels were higher in the early stages of hCG-induced spermatogenesis, while lhcgr1 transcripts were most abundant later, once spermatids were observed. In vitro experiments further showed that hCG and its steroidal mediator 11-KT significantly increased fshr transcript abundance. These data provide new angles on the interactions between gonadotropin and androgen signaling during early spermatogenesis. Increases in levels of 11-KT following hCG injection elevated testicular fshr mRNA levels augmenting Fsh sensitivity in the testis. This evidence is suggestive of a positive feedback loop between gonadotropins and 11-KT that may be key to regulating early spermatogenesis in fish.


Assuntos
Anguilla/genética , Regulação da Expressão Gênica , Receptores Androgênicos/genética , Receptores da Gonadotropina/genética , Testículo/metabolismo , Androgênios/metabolismo , Anguilla/sangue , Animais , Gonadotropina Coriônica/administração & dosagem , Gonadotropina Coriônica/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/metabolismo , Receptores do FSH/genética , Receptores do FSH/metabolismo , Receptores da Gonadotropina/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Espermatogênese/efeitos dos fármacos , Espermatogênese/genética , Esteroide 11-beta-Hidroxilase/genética , Esteroide 11-beta-Hidroxilase/metabolismo , Testículo/efeitos dos fármacos , Testosterona/análogos & derivados , Testosterona/sangue
14.
Nutrients ; 11(4)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018574

RESUMO

Age-related male sexual dysfunction covers a wide variety of issues, together with spermatogenic and testicular impairment. In the present work, the effects of cordycepin (COR), an active constituent of a nutrient powerhouse Cordyceps militaris Linn, on senile testicular dysfunction in rats was investigated. The sperm kinematics, antioxidant enzymes, spermatogenic factors, sex hormone receptors, histone deacetylating sirtuin 1 (SIRT1), and autophagy-related mammalian target of rapamycin complex 1 (mTORC1) expression in aged rat testes were evaluated. Sprague Dawley rats were divided into young control (2-month-old; YC), aged control (12-month-old; AC), and aged plus COR-treated groups (5 (COR-5), 10 (COR-10), and 20 (COR-20) mg/kg). The AC group showed reduced sperm kinematics and altered testicular histomorphology compared with the YC group (p < 0.05). However, compared with the AC group, the COR-treated group exhibited improved sperm motility, progressiveness, and average path/straight line velocity (p < 0.05-0.01). Alterations in spermatogenesis-related protein and mRNA expression were significantly ameliorated (p < 0.05) in the COR-20 group compared with the AC group. The altered histone deacetylating SIRT1 and autophagy-related mTORC1 molecular expression in aged rats were restored in the COR-20 group (p < 0.05). In conclusion, the results suggest that COR holds immense nutritional potential and therapeutic value in ameliorating age-related male sexual dysfunctions.


Assuntos
Envelhecimento , Cordyceps/química , Desoxiadenosinas/farmacologia , Testículo/efeitos dos fármacos , Animais , Desoxiadenosinas/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores do FSH/genética , Receptores do FSH/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Espermatogênese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Anim Sci J ; 90(4): 473-480, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30793438

RESUMO

This study was aimed to address melatonin receptor expression, mRNA level of hypothalamus and hypophysis hormone receptors (GnRHR, FSHR, and LHR), steroidogenesis, cell cycle, apoptosis, and their regulatory factors after addition of melatonin for 24 hr in cultured buffalo granulosa cells (GCs). The results revealed that direct addition of different concentrations of melatonin (100 pM, 1 nM, and 100 nM) resulted in significant upregulation (p < 0.05) of mRNA level of melatonin receptor 1a (MT1) without affecting melatonin receptor 1b (MT2). Melatonin treatment significantly downregulated (p < 0.05) mRNA level of FSH and GnRH receptors, whereas 100 nM dose of melatonin significantly increased mRNA level of LH receptor. Treatment with 100 nM of melatonin significantly decreased the basal progesterone production with significant decrease (p < 0.05) in mRNA levels of StAR and p450ssc, and lower mRNA level of genes (Insig1, Lipe, and Scrab1) that affect cholesterol availability. Melatonin supplementation suppressed apoptosis (100 nM, p < 0.05) and enhanced G2/M phase (1 nM, 100 nM, p < 0.05) of cell cycle progression which was further corroborated by decrease in protein expression of caspase-3, p21, and p27 and increase in bcl2. Our results demonstrate that melatonin regulates gonadotrophin receptors and ovarian steroidogenesis through MT1. Furthermore, the notion of its incorporation in apoptosis and proliferation of buffalo GCs extends its role in buffalo ovaries.


Assuntos
Apoptose/efeitos dos fármacos , Estradiol/metabolismo , Hormônio Foliculoestimulante/metabolismo , Células da Granulosa/metabolismo , Células da Granulosa/patologia , Melatonina/farmacologia , Progesterona/metabolismo , Animais , Búfalos , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Hormônio Foliculoestimulante/genética , Expressão Gênica/efeitos dos fármacos , Melatonina/fisiologia , RNA Mensageiro/metabolismo , Receptor MT1 de Melatonina/genética , Receptor MT1 de Melatonina/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Receptores LHRH/metabolismo , Regulação para Cima/efeitos dos fármacos
16.
Pathol Res Pract ; 215(4): 748-754, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30712886

RESUMO

In addition to its critical role during pregnancy, human chorionic gonadotropin (hCG) has been shown to be expressed by various tumor types. Recent studies have similarly documented the presence of the luteinizing hormone (LH)/hCG receptor (LHCGR) in a variety of nongonadal organs; however, its clinicopathological significance in ovarian cancer remains unclear. The present study used a combination of immunohistochemical, real-time PCR, and western blot analyses to examine hCG and LHCGR expression in normal and cancerous tissues collected from patients with epithelial ovarian cancer (EOC). hCG and LHCGR expression levels were resultantly shown to be significantly increased and decreased in cancerous versus normal (or benign) ovarian tissues, respectively (P < 0.05), and both expression pattern changes were associated with more advanced tumor stages and a higher rate of metastasis. Furthermore, patients with tumors with high or low levels of hCG and LHCGR, respectively, experienced a worse overall survival (OS) rate than those with low hCG or high LHCGR expression levels (P < 0.05). In fact, hCG and LHCGR expression levels were independent prognostic factors of patient OS (P < 0.05) for EOC. Collectively, these findings indicate that hCG and LHCGR expression pattern changes are associated with EOC occurrence and progression. Thus, hCG and LHCGR represent promising potential targets to improve the diagnosis, treatment, and prognosis of patients with EOC.


Assuntos
Carcinoma Epitelial do Ovário/patologia , Gonadotropina Coriônica/metabolismo , Neoplasias Ovarianas/patologia , Ovário/metabolismo , Receptores do LH/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/metabolismo , Gonadotropina Coriônica/genética , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Ovário/patologia , Receptores do LH/genética , Adulto Jovem
17.
Prog Mol Biol Transl Sci ; 161: 69-89, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30711030

RESUMO

Accumulating evidence showed that the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is an essential regulator of sexual development and reproduction from zebrafish to human. Activating and inactivating mutations of LHCGR gene have been identified from patients of different phenotypes. Familial male-limited precocious puberty, Leydig cell hypoplasia, and empty follicle syndrome are caused by LHCGR mutations. More than 50 mutations have been reported from subjects of different ethnic backgrounds. Functional analyses of the mutant LHCGR revealed multiple defects, including cell surface expression, ligand binding, and signaling. The difference of the two native ligands and signaling pathway activated by LHCGR are illustrated. Potential therapeutic implications from the analyses of the naturally occurring LHCGR mutations, such as pharmacological chaperones, are highlighted.


Assuntos
Doença/genética , Hormônio Luteinizante/genética , Mutação/genética , Receptores do LH/genética , Sequência de Aminoácidos , Sequência de Bases , Humanos , Receptores do LH/química , Transdução de Sinais
18.
Andrology ; 7(1): 110-123, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30515996

RESUMO

BACKGROUND: Metformin has long been used for glycemic control in diabetic state. Recently, other benefits of metformin beyond blood glucose regulation have emerged. OBJECTIVES: To investigate the effect of metformin on the expression of testicular steroidogenesis-related genes, spermatogenesis, and fertility of male diabetic rats. MATERIALS AND METHODS: Eighteen adult male Sprague Dawley rats were divided into three groups, namely normal control (NC), diabetic control (DC), and metformin-treated (300 mg/kg body weight/day) diabetic rats (D+Met). Diabetes was induced using a single intraperitoneal injection of streptozotocin (60 mg/kg b.w.), followed by oral treatment with metformin for four weeks. RESULTS: Diabetes decreased serum and intratesticular testosterone levels and increased serum but not intratesticular levels of luteinizing hormone. Sperm count, motility, viability, and normal morphology were decreased, while sperm nuclear DNA fragmentation was increased in DC group, relative to NC group. Testicular mRNA levels of androgen receptor, luteinizing hormone receptor, cytochrome P450 enzyme (CYP11A1), steroidogenic acute regulatory (StAR) protein, 3ß-hydroxysteroid dehydrogenase (HSD), and 17ß-HSD, as well as the level of StAR protein and activities of CYP11A1, 3ß-HSD, and 17ß-HSD, were decreased in DC group. Similarly, decreased activities of epididymal antioxidant enzymes and increased lipid peroxidation were observed in DC group. Consequently, decreased litter size, fetal weight, mating and fertility indices, and increased pre- and post-implantation losses were recorded in DC group. Following intervention with metformin, we observed increases in serum and intratesticular testosterone levels, Leydig cell count, improved sperm parameters, and decreased sperm nuclear DNA fragmentation. Furthermore, mRNA levels and activities of steroidogenesis-related enzymes were increased, with improved fertility outcome. DISCUSSION AND CONCLUSION: Diabetes mellitus is associated with dysregulation of steroidogenesis, abnormal spermatogenesis, and fertility decline. Controlling hyperglycemia is therefore crucial in preserving male reproductive function. Metformin not only regulates blood glucose level, but also preserves male fertility in diabetic state.


Assuntos
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Hipoglicemiantes/farmacologia , Infertilidade Masculina/genética , Metformina/farmacologia , Espermatogênese/efeitos dos fármacos , Animais , Sobrevivência Celular/fisiologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Fragmentação do DNA , Diabetes Mellitus Experimental/induzido quimicamente , Hidroxiesteroide Desidrogenases/genética , Hiperglicemia/prevenção & controle , Hormônio Luteinizante/sangue , Masculino , Estresse Oxidativo/efeitos dos fármacos , Fosfoproteínas/genética , Ratos , Ratos Sprague-Dawley , Receptores Androgênicos/genética , Receptores do LH/genética , Contagem de Espermatozoides , Motilidade Espermática/fisiologia , Espermatogênese/fisiologia , Estreptozocina/toxicidade , Testosterona/sangue
19.
Reprod Toxicol ; 83: 54-62, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30508572

RESUMO

Perfluorooctanoic acid (PFOA) is a persistent organic pollutant, which may possess endocrine disrupting properties. Herein, we investigated the possible mechanism(s) of toxicity and steroidogenesis in mouse Leydig cells. MLTC-1 (mouse Leydig tumour cells) cells were exposed to 0, 50, 100 or 200 µM PFOA for 48 h to ascertain their effects on the nuclear (membrane) receptor responses, steroidogenesis pathway and related regulated gene expression and steroid hormone secretion profiles. Our results reveal that nuclear receptors PXR, SR-B1 and LHR are sensitive to PFOA exposure. PFOA can accumulate in mitochondria and alter cholesterol precursor (fatty acid) mitochondrial transport process-related gene expression and thus inhibit steroid hormone precursor (cholesterol) production. In particular, PFOA exhibits biphasic effects on testosterone and progesterone production at differing levels of exposure. These findings indicate the potential endocrine-related effects of PFOA on steroid hormone secretion in Leydig cells and point to a novel disruption model.


Assuntos
Caprilatos/toxicidade , Disruptores Endócrinos/toxicidade , Fluorcarbonetos/toxicidade , Tumor de Células de Leydig/metabolismo , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ácidos Graxos/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Receptor de Pregnano X/genética , Receptores do LH/genética , Receptores Depuradores Classe B/genética
20.
Reprod Fertil Dev ; 31(4): 735-742, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30509341

RESUMO

Luteinising hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) are pituitary-derived hormones and mediate their functions through LH receptor (LHR), FSH receptor (FSHR) and PRL receptor (PRLR) respectively. This study aimed to investigate the seasonal expression patterns of LHR, FSHR and PRLR in the epididymis of the male wild ground squirrel during the breeding and non-breeding seasons. Histologically, principal cells, basal cells, cilia and mature spermatozoa were found in the lumen of caput, corpus and cauda epididymidis in the breeding season, whereas in the non-breeding season, cilia and basal cells were rarely found and the epididymidal duct was devoid of spermatozoa. Immunohistochemical results showed that LHR, FSHR and PRLR were mainly present in the filamentous cytoplasm layer of epithelial cells of the caput, corpus and cauda epididymidis and FSHR and PRLR displayed stronger staining in the breeding season than in the non-breeding season. Furthermore, the mRNA and protein levels of FSHR and PRLR in all regions of epididymis as well as the levels of LHR in the caput and cauda epididymidis were higher during the breeding season. The protein levels of FSHR, LHR and PRLR were positively correlated with epididymal weight. Together, these results suggest that LHR, FSHR and PRLR may regulate epididymal functional changes in the male wild ground squirrel during its seasonal breeding cycle.


Assuntos
Epididimo/metabolismo , Receptores do FSH/metabolismo , Receptores do LH/metabolismo , Receptores da Prolactina/metabolismo , Estações do Ano , Animais , Masculino , Receptores do FSH/genética , Receptores do LH/genética , Receptores da Prolactina/genética , Reprodução/fisiologia , Sciuridae , Testículo/metabolismo
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