2型糖尿病雄性小鼠生殖细胞形态学改变及凋亡机制研究

Influence of type 2 diabetes mellitus on the morphology and apoptosis of germ cells in male mice

摘要:

目的 通过构建2型糖尿病（type 2 diabetes mellitus，T2DM）雄性小鼠模型探究2型糖尿病对睾丸组织形态学、睾酮分泌、精子生成等方面的影响。方法 将24只8周龄C57BL/6J雄性小鼠随机分为2组：对照组10只和T2DM组14只。对照组小鼠基础饲料喂养，T2DM组给予高脂喂养。8周后对照组小鼠腹腔注射柠檬酸盐缓冲液，T2DM组小鼠腹腔注射60 mg/kg链脲佐菌素（streptozocin，STZ），持续5 d。饲养小鼠4周后断颈处死，分离出小鼠的血清、睾丸、附睾。通过精子计数、睾酮水平检测评估雄性小鼠生殖细胞生精功能；HE染色分析睾丸组织形态学变化；TUNEL染色检测睾丸生殖细胞的凋亡情况。ELISA检测雄性小鼠血清中超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）、血红素加氧酶-1（heme oxygenase-1，HO-1）水平；Western blot检测雄性小鼠睾丸组织中B细胞淋巴瘤/白血病-2（B cell lymphoma/leukemia-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2-associated X protein，BaX）、半胱氨酸天冬氨酸蛋白酶-3（cysteinyl aspartate specific proteinase-3，Caspase-3）蛋白表达水平。结果 与对照组相比，T2DM组小鼠体质量（26.00±2.85） g、精子数目（4.15±1.01）×10⁶个/L、睾酮水平（0.99±0.41） ng/mL均降低（P=0.000，P=0.009，P=0.041）；TUNEL结果显示：T2DM组小鼠睾丸生精小管各级生精细胞都有不同程度的凋亡，凋亡细胞数量（21.60±5.11，P=0.033）增加；ELISA结果显示：T2DM组小鼠血清氧化应激SOD（4.42±0.47） pg/mL和HO-1（2 968.00±536.10） pg/mL表达水平增加（P=0.000，P=0.008）。Western blot检测发现BaX（1.270±0.059）、Caspase-3（1.94±0.47）蛋白表达水平增加（P=0.008，P=0.041），且BaX与Bcl-2比值增大。结论 2型糖尿病雄性小鼠血清睾酮含量显著降低，生精细胞凋亡增加，成熟精子数量减少，影响生殖能力。

Abstract:

Objective To investigate the influence of type 2 diabetes mellitus（T2DM） on testicular histomorphology，testosterone secretion，and spermatogenesis by establishing a male mouse model of T2DM.Methods A total of 24 male C57BL/6J mice，aged 8 weeks，were randomly divided into control group with 10 mice and T2DM group with 14 mice. The mice in the control group were fed a basal diet，and those in the T2DM group were fed a high-fat diet. After 8 weeks，the mice in the control group were given intraperitoneal injection of citrate buffer，and those in the T2DM group were given intraperitoneal injection of 60 mg/kg streptozotocin（STZ），for 5 consecutive days. The mice were sacrificed after 4 weeks of feeding，and the serum，testis，and epididymis of the male mice were isolated. Sperm count and testosterone level were measured to assess the spermatogenic function of germ cells in male mice；HE staining was used to observe the morphological changes of the testis；TUNEL staining was used to observe the apoptosis of testicular germ cells. ELISA was used to measure the serum levels of superoxide dismutase（SOD），malondialdehyde（MDA），and heme oxygenase-1（HO-1），and Western blot was used to measure the protein expression levels of B cell lymphoma/leukemia-2（Bcl-2），Bcl-2-associated X protein（BaX），and cysteinyl aspartate specific proteinase-3（caspase-3） in testicular tissue of male mice.Results Compared with the control group，the T2DM group had significant reductions in body weight [（26.00±2.85） g vs. （29.71±4.28） g，P=0.000]，sperm number [（4.15±1.01） ×10⁶/L vs. （5.56±0.70）×10⁶/L，P=0.009]，and testosterone level [（0.99±0.41） ng/mL vs. （19.85±10.23） ng/mL，P=0.041]. TUNEL staining showed that the T2DM group had varying degrees of apoptosis in spermatogenic cells at all levels of testicular seminiferous tubules，with a significant increase in the number of apoptotic cells（21.60±5.11，P=0.033）. ELISA showed that the T2DM group had significant increases in the expression levels of the serum oxidative stress markers SOD（4.42±0.47） pg/mL and HO-1（2 968.00±536.10） pg/mL（P=0.000 and 0.008）. Western blot showed significant increases in the protein expression levels of BaX（1.270±0.059） and caspase-3（1.94±0.47）（P=0.008 and 0.041） and BaX/Bcl-2 ratio.Conclusion Male mice with T2DM have a significant reduction in serum testosterone level，a significant increase in the apoptosis of spermatogenic cells，and a significant reduction in the number of mature spermatogenic cells，which affect reproductive capacity.