Effects Of Retinol On In Vitro Characterization Of Sertoli Cells From Prepubertal Porcine Testis

Retinol and its derivatives are essential for the testis development and spermatogenesis. While sertoli cells are target cells for retinol, which play a critical role spermatogenic cells development and maturation. To analyze the characterization of Sertoli cells cultured in the presence or absence of retinol, sertoli cells were isolated from testis of 3-5 week-old porcine by digest method. And then we evaluated the expression of proliferation- and appoptosos-related gene, and cell growth facor by CCK-8, RT-PCR, and ELISA. The results are showed as follows: 1. Isolation of sertoli cellsThe cells obtained by digestion method were evaluated by the number of cells, cell viability and the relative expression of GATA-4 mRNA, the results show:first with 0.25% collagenase IV and 0.1% hyaluronidase,37℃ for 60min, and then with 0.25% trypsin and 0.1% DNase I,37℃ for 20 min, was in favor of sertoli cell enrichment. Adherent and suspension cells were collected at different times, and analyzed by GATA-4 mRNA relative expression, sertoli cells rarely adhere in 2 hours, and it may adhere mainly at the time of 3h-5h. After purified by differential plating and hypotonic treated, cells were identified by H&E staining, immunohistochemical staining, and RT-PCR. The result showed the purified cells with large body and fusiform or irregular shape. These cells were positive for GATA4, but negative for CAKP. And RT-PCR showed cell expressed sertoli cells markers GATA4, SOX9, INHB, not express leydig cell marker CYP17,3β-HSD and pertubular cell marker a-SMA. All these features were in line with sertoli cells. ELISA confirmed the fact of the isolated sertoli cells expressing GDNF, LIF, CSF-1, FGF2, EGF. However no SCF protein was detected.2 Influence of retinol to sertoli cell proliferation and apoptosisRetinol was added to the medium at a concentration of 0.005μmol/L,0.025μmol/L, 0.125μmol/L,0.625μmol/L,1.25μmol/L,2.5μmol/L,5μmol/L, 10μmol/L, respectively. Sertoli cells viability was assessed by the CCK-8 assay after a short-term culture for 24h and a long-term culture for 3d. After 24h, cellular viability is compromised by retinol 5μmol/L and 10μmol/L retinol, but there is no significant influence in cellular viability when the concentration is below 5μmol/L, showed these two concentration carried out cytotoxic effect. And after a 3-days’ culture, when the retinol concentrations at 0.005μmol/L and 0.025μmol/L no significant effect was seen on Sertoli cell viability, while cell viability was significantly inhibited when reached to 0.125μmol/L and 0.625μmol/L (P <0.05), and the concentration reached to 1.25μmol/L performance a very significantly inhibited (P<0.01) in cellular viability. Analyzed by the short-term and long-term culture, the effect of retinol on sertoli cells viability was showed in a dose-and time-dependent manner.We conducted further experiments using retinol at 1,25μmol/L, the impact is detected by RT-PCR on the expression of the proliferation-related gene (PCNA, C-MYC and BCL-2) and apoptosis-related genes(BAX and CASP3) in sertoli cells, the results were as follows:the level of proliferation-related genes were significantly lower in retinol treated sertoli cells. However, the apoptosis-related genes level were significantly higher than in control. Thereby, retinol inhibited Sertoli cells proliferation by inhibiting the expression of proliferation-related genes and stimulating the expression of apoptosis-related genes. 3 The effects of retinol on the secretion of cell growth factor from Sertoli cellsAdd 1.25μmol/L retinol to medium, the expression and secretion of cell growth factor were tested by RT-PCR and ELISA, respectively. By RT-PCR analysis,1.25μmol/L retinol significantly inhibited expression of GDNF, CSF-1 and EGF mRNA, while no difference was seen in LIF and FGF2 mRNA expression. And by ELISA, retinol significantly inhibited Sertoli cells secretion GDNF, LIF, CSF-1, FGF2 and EGF protein.Thereby, the way retinol inhibiting Sertoli cells viability and proliferation is accomplished by reducing the expression of proliferation-related genes and stimulating the expression of apoptosis-related genes, as well as by decreasing the synthesis and secretion of cell groth factor.