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

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 oxygen⁃ase-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)×10^(6)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(2968.00±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。

Objective:To investigate the influence of type 2 diabetes mellitus(T2DM)on testicular histomorphology,testosterone se⁃cretion,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 intraperito⁃neal 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 iso⁃lated.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 dis⁃mutase(SOD),malondialdehyde(MDA),and heme oxygenase-1(HO-1),and Western blot was used to measure the protein expres⁃sion levels of B cell lymphoma/leukemia-2(Bcl-2),Bcl-2-associ⁃ated 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)×10^(6)/L vs.(5.56±0.70)×10^(6)/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(2968.00±536.10)pg/mL(P=0.000 and 0.008).Western blot showed significant increases in the protein expres⁃sion 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.