Abnormal proliferation of Leydig cells in Sox30 knockout mice

Abstract

Objective To investigate the effect of Sox30 gene knockout on the proliferation of mouse Leydig cells. Methods The pathological morphology of the testis of 10-week-old Sox30+/+, Sox30-/+ and Sox30-/- mice was observed with HE staining. Immunohistochemical assay was adopted to detect the expression level of 3β-HSD in the Leydig cells. The cell models of Sox30 gene overexpression and knockdown were constructed by lentivirus infection and siRNA interference in TM3 cell line, respectively. On the basis of these cell models, CCK-8 assay, flow cytometry, RT-qPCR and Western blotting were used to detect the cell survival rate, cell cycle distribution, and expression of cell cycle-related genes and proteins. The JASPAR database was used to predict the binding sites of Sox30 protein in promoter region of cycle related genes. Results Compared with Sox30+/+ and Sox30-/+ mice, HE staining indicated that the number of Leydig cells were significantly increased, and immunohistochemical assay showed the expression of 3β-HSD were increased the Leydig cells in the testis tissue of Sox30-/- mice. Compared with the blank vector group, Sox30 overexpression led to the decrease of cell count (P < 0.05), cell cycle arrest in G1 phase(P < 0.05), and significantly decreased expression of CyclinD1 and Cdk2 at mRNA and protein levels (P < 0.05). On the contrary, Sox30 knockdown resulted in opposite phenomena (P < 0.05). JASPAR database predicted that Sox30 may bind to the promoter of CyclinD1 and Cdk2. Conclusion Knockout of Sox30 induces abnormal proliferation of Leydig cells in mice. CyclinD1 and Cdk2 may be important downstream targets of Sox30 gene in the regulation of cell cycle in Leydig cells.

Keywords

• sox30
• mouse leydig cells
• tm3 cells
• cell cycle