• Animal Bioscience
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• v.35 no.3
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• pp.399-409
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• 2022

Objective: Follicle-stimulating hormone (FSH) is the central hormone involved in mammalian reproduction, maturation at puberty, and gamete production that mediates its function by control of follicle growth and function. The present study investigated the mutations involved in the regulation of FSH receptor (FSHR) activation. Methods: We analyzed seven naturally-occurring mutations that were previously reported in human FSHR (hFSHR), in the context of equine FSHR (eFSHR); these include one constitutively activation variant, one allelic variant, and five inactivating variants. These mutations were introduced into wild-type eFSHR (eFSHR-wt) sequence to generate mutants that were designated as eFSHR-D566G, -A306T, -A189V, -N191I, -R572C, -A574V, and -R633H. Mutants were transfected into PathHunter EA-parental CHO-K1 cells expressing β-arrestin. The biological function of mutants was analyzed by quantitating cAMP accumulation in cells incubated with increasing concentrations of FSH. Results: Cells expressing eFSHR-D566G exhibited an 8.6-fold increase in basal cAMP response, as compared to that in eFSHR-wt. The allelic variation mutant eFSHR-A306T was not found to affect the basal cAMP response or half maximal effective concentration (EC₅₀) levels. On the other hand, eFSHR-D566G and eFSHR-A306T displayed a 1.5- and 1.4-fold increase in the maximal response, respectively. Signal transduction was found to be completely impaired in case of the inactivating mutants eFSHR-A189V, -R572C, and -A574V. When compared with eFSHR-wt, eFSHR-N191I displayed a 5.4-fold decrease in the EC₅₀ levels (3,910 ng/mL) and a 2.3-fold decrease in the maximal response. In contrast, cells expressing eFSHR-R633H displayed in a similar manner to that of the cells expressing the eFSHR-wt on signal transduction and maximal response. Conclusion: The activating mutant eFSHR-D566G greatly enhanced the signal transduction in response to FSH, in the absence of agonist treatment. We suggest that the state of activation of the eFSHR can modulate its basal cAMP accumulation.

• Development and Reproduction
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• v.21 no.2
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• pp.111-120
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• 2017

Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG ($eCG{\beta}/{\alpha}$) and mutant eCG ($eCG{\beta}/{\alpha}{\Delta}56$) with an N-linked oligosaccharide at $Asn^{56}$ of the ${\alpha}-subunit$. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of $rec-eCG{\beta}/{\alpha}$. The dose-dependent response was highest when 10 ng of $rec-eCG{\beta}/{\alpha}$ was used. The deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

• Clinical and Experimental Reproductive Medicine
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• v.47 no.3
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• pp.194-206
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• 2020

Objective: The aim of this study was to investigate microRNAs (miRNAs) related to follicle-stimulating hormone (FSH) responsiveness using miRNA microarrays and to identify their target genes to determine the molecular regulatory pathways involved in FSH signaling in KGN cells. Methods: To change the cellular responsiveness to FSH, KGN cells were treated with FSH receptor (FSHR)-specific small interfering RNA (siRNA) followed by FSH. miRNA expression profiles were determined through miRNA microarray analysis. Potential target genes of selected miRNAs were predicted using bioinformatics tools, and their regulatory function was confirmed in KGN cells. Results: We found that six miRNAs (miR-1261, miR-130a-3p, miR-329-3p, miR-185-5p, miR-144-5p and miR-4463) were differentially expressed after FSHR siRNA treatment in KGN cells. Through a bioinformatics analysis, we showed that these miRNAs were predicted to regulate a large number of genes, which we narrowed down to cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and estrogen receptor alpha (ESR1) as the main targets for miR-4463. Functional analysis revealed that miR-4463 is a regulatory factor for aromatase expression and function in KGN cells. Conclusion: In this study, we identified differentially expressed miRNAs related to FSH responsiveness. In particular, upregulation of miR-4463 expression by FSHR deficiency in human granulosa cells impaired 17β-estradiol synthesis by targeting CYP19A1 and ESR1. Therefore, our data might provide novel candidates for molecular biomarkers for use in research into poor responders.