双酚A对大鼠睾丸支持细胞糖代谢的影响

Bisphenol A alters glucose metabolism in rat Sertoli cells in vitro

目的:观察不同浓度双酚A(BPA)对原代培养大鼠睾丸支持细胞(SC)糖代谢及乳酸脱氢酶(LDH)表达的影响,探讨BPA致男性不育的机制。方法:采用两步酶消化法分离雄性Wistar大鼠睾丸SC,建立SC原代培养模型,免疫组化鉴定Fas L。传代后SC随机分为对照组和实验组(100 nmol/L、10μmol/L和1 mmol/L BPA)。培养48 h后,CCK-8法测定细胞增殖活性,核磁共振波谱法测定细胞内代谢物浓度,RT-PCR及Western印迹检测LDH的表达。结果:体外分离培养SC的纯度为(96.05±1.28)%(n=10),CCK-8实验结果显示:与对照组相比,暴露于100 nmol/L BPA组[(98±8)%]、10μmol/L BPA组[(96±3)%]和1 mmol/L BPA组[(95±3)%]的细胞增殖率无明显变化(P>0.05);核磁共振波谱显示:与对照组相比,10μmol/L和1 mmol/L BPA作用下SC内葡萄糖及乳酸浓度明显降低(P<0.05);RT-PCR及Western印迹结果显示:LDH mRNA的表达随BPA浓度升高呈现降低趋势(100 nmol/L、10μmol/L及1 mmol/L组P均<0.05),而LDH蛋白的表达只在1 mmol/L组明显降低(P<0.05)。结论:较高浓度BPA降低LDH,影响SC糖代谢过程。推测BPA通过影响SC糖代谢,减少向生殖细胞乳酸的供给,影响精子发生过程。

Objective： To observe the influence of different concentrations of bisphenol A （BPA） on glucose metabolism and lactate dehydrogenase （LDH） expression in rat Sertoli cells in vitro and investigate the mechanisms of BPA inducing male infertility. Methods ： Using two-step enzyme digestion, we isolated Sertoli cells from male Wistar rats and constructed a primary Sertoli cell system, followed by immunohistochemical FasL staining. We randomly divided the Sertoli cells into a control group to be cultured in the serum-free minimal essential medium （MEM） plus dimethyl sulfoxide （DMSO） and three experimental groups to be treated with 100 nmol/L, 10 μmol/L, and 1 mmol/L BPA, respectively, in the MEM plus DMSO. After 48 hours of treatment, we measured the proliferation of the cells by CCK-8 assay, determined the concentrations of metabolites by NMR spectroscopy, and detected the expression of LDH in the Sertoli cells by RT-PCR and Western blot. Results ： The purity of the isolated Sertoli cells was （96.05±1.28） % （n = 10）. Compared with the control group, the 100 nmol/L, 10 μmol/L, and 1 mmol/L BPA groups showed no remarkable changes in the proliferation of Sertoli cells （[98±8 ] %, [96±3]%, and [ 95 ± 3 ] %, P 〉0.05 ）, but the 10 μmol/L and 1 mmol/L of BPA groups exhibited significantly decreased concentrations of intracellular glucose （ [ 3.89±0. 07 ] vs [ 3.36 ± 0.24 ] and [ 3.04 ± 0.21 ] pmol/cell, P 〈 0.05 ） and lactate （ [ 0.43 ± 0.06 ] vs [ 0.29 ± 0.05 ] and [0.20±0. 03 ] pmol/cell, P 〈 0.05 ）. The expression of LDI-I mRNA was decreased with the increased concentration of BPA, while that of LDH protein reduced only in the 1 mmol/L BPA group （P 〈0.05）. Conclusion： High-concentration BPA decreases the expression of LDH and alters glucose metabolism in Sertoli cells, and therefore may reduce the provision of lactate for germ cells and impair spermatogenesis.