Effect of inhibiting Gprc6a gene on testosterone production in fluoridated Leydig cells

Background Fluoride induces aberrant osteoblast activation and promotes osteocalcin expression in multiple ways, while excess fluoride decreases testosterone production in Leydig cells, and the osteocalcin-specific receptor Gprc6a expressed on Leydig cells can regulate testosterone biosynthesis by binding to osteocalcin.

Objective To investigate the effect of Gprc6a gene on testosterone production in Leydig cells with NaF intervention.

Methods Normal mouse testicular interstitial TM3 cell line and Gprc6a-/- TM3 cell line were used, they were divided into TM3 group, Gprc6a^-/- TM3 group, TM3(NaF) group, Gprc6a^-/- TM3(NaF) group, TM3(NaF) + OCN group, and Gprc6a^-/- TM3(NaF) + OCN group, respectively. After 48 h of intervention, the cell morphology was observed by inverted fluorescence microscope, the expression of Creb, Star, Cyp11a1 and 3β-HSD gene transcription was detected by fluorescence quantitative PCR, and the expression of testosterone secretion was detected by enzyme-linked immunosorbent assay.

Results MTT assay for cell proliferative activity suggested that Gprc6a^-/-TM3 cell activity was lower in each experimental group than those in the corresponding groups of TM3 (P=0.01), and cells in the TM3(NaF) + OCN and Gprc6a^-/-TM3(NaF) + OCN groups were more viable than those in the TM3(NaF) group; Gprc6a inhibition only affected the number of proliferating TM3 cells and did not affect cell morphology under fluorescence microscopy. After single NaF stimulation, Gprc6a^-/- TM3 was more prone to desmoplasticization, and some of the cells retracted significantly; Fluorescence quantitative PCR detection of genes showed that compared with the TM3(NaF) group, Creb, Star and Cyp11a1 gene expression was downregulated in the Gprc6a^-/-TM3(NaF) group (P=0.032); compared with the TM3(NaF) + OCN group, the gene expression of Creb, Star, Cyp11a1 and 3β-HSD in the cells of the Gprc6a^-/-TM3(NaF) + OCN group was decreased (P=0.029); while they were up-regulated in the Gprc6a^-/-TM3(NaF) + OCN group compared to the Gprc6a^-/-TM3 (NaF) group (P=0.012); and gene expression of Gprc6a, which was originally suppressed, was up-regulated after OCN intervention (P=0.010). The results of enzyme-linked immunosorbent assay showed that in normal culture, the amount of testosterone secreted in the Gprc6a^-/-TM3 group was less than that in the TM3 group (P=0.004); after NaF stimulation, the amount of testosterone secreted by the cells in each group was reduced (P=0.001), and testosterone secretion was rebounded in the TM3 group by NaF + OCN stimulation (P=0.003); testosterone secretion was reduced in the Gprc6a^-/-TM3 cells conmpared with that of the same stimulation in the TM3 group (P=0.007).

Conclusion Inhibiting Gprc6a gene accelerates excess fluorine-induced reduction in testosterone production in Leydig cells. Gprc6a is involved in excess fluoride-osteocalcin regulation of testosterone production in Leydig cells.