摘要
为探究铬元素在猪精子体外获能培养过程中,对猪精子活力、蛋白磷酸化信号通路的影响,研究采取八头杜洛克猪新鲜精液,分别在体外获能培养液中添加不同浓度的6价铬离子(0、0.1、0.5、1、2、10、100μmol/mL)以及双丁酰环腺苷酸(dbcAMP)、异丁基黄嘌呤(IBMX)、PKA抑制剂(H-89)等cAMP-PKAs信号通路调节因子进行调控培养2h。利用精子活力计算机检测(CASA)、蛋白质免疫印迹(WB)技术,分析测定猪精子体外铬暴露获能培养过程中精子活力、蛋白磷酸化水平,并用相应试剂盒检测细胞中甘油醛-3-磷酸脱氢酶(GAPDH)活性、腺苷-3′,5′-环化一磷酸(cAMP)及ATP水平。结果表明:铬离子对猪精子活力、蛋白磷酸化水平有抑制作用,且随铬离子浓度增加抑制作用增强。当铬浓度达到2μmol/mL时精子活力MOT(58.4%)显著低于获能培养组(73.6%)(P<0.05);10μmol/mL铬处理明显抑制蛋白磷酸化水平,且细胞内GAPDH活性、cAMP及ATP水平显著降低(P<0.05)。而10μmol/mL铬处理中分别添加葡萄糖(5.0μmol/mL)及dbcAMP(1.0 mmol/L)和IBMX(0.1 mmol/L),能缓解铬对蛋白酪氨酸磷酸化(PTP)、蛋白丝氨酸/苏氨酸磷酸化(P-PKAs)及GAPDH活性的抑制作用。结果提示铬可能是通过干扰糖代谢及cAMP/PKA信号通路影响猪精子活力及蛋白磷酸化水平。本研究首次探究了体外培养过程中铬离子对家畜精子活力的影响,并初步探究了其作用机理,为进一步研究铬离子引起繁殖毒性的分子机制及家畜繁育提供一定的理论基础。
To explore the effects of chromium on sperm motility and protein phosphorylation signal pathway in boar sperm during capacitation in vitro,fresh semen from 8 Duroc boars were incubated in capacitation medium with Cr6＋（0,0.1,0.5,1,2,10,100μmol/mL）,3＇-5＇-cyclic adenosine（cAMP）analog dibutytyl-cAMP（dbcAMP）,phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine（IBMX）,and N-[2-（p-Bromocinnamylamino）ethyl]-5-isoquinolinesufonamide·2HCl hydrate（H-89）for 2hours.Computerassisted sperm analysis（CASA）and Western Blot（WB）were used to determine the sperm motility,protein phosphorylation,the activity of glyceraldehyde 3-phosphate dehydrogenase（GAPDH）,the level of cAMP and ATP of boar sperm were measured with commercial kits.The results showed that sperm motility and protein phosphorylation decreased as the Cr6＋concentration increased.Incubated with 2μmol/mL Cr6＋,sperm MOT（54.8%）in the control group was significantly lower than the control（73%）（P0.05）.Moreover,10μmol/mL Cr6＋dramatically inhibited protein phosphorylation,GAPDH activity,cAMP level and adenosine 5＇-triphosphate（ATP）level in boar sperm（P〈0.05）.However,the capacitation medium with 10 μmol/mL Cr6＋could alleviate the inhibition of protein tyrosine phosphorylation（PTP）,phosphorylation of protein kinase A（PKA）substrates（P-PKA）and GAPDH activity after adding dbcAMP,IBMX and glucose（5.0μmol/mL）separately.The results indicated that chromium may inhibit sperm motility and protein phosphorylation through impairing glycolysis and then blocking cAMP-PKAs pathway in boar spermatozoa.The present study,to our knowledge,is the first time to explore the effects of chromium on sperm motility and the underlying mechanisms in domestic animal in vitro,providing molecular mechanism for further study on reproductive toxicity and then facilitating the study of domestic animal breeding.
出处
《上海交通大学学报（农业科学版）》
2015年第3期1-8,共8页
Journal of Shanghai Jiaotong University(Agricultural Science)
基金
农业部公益性行业专项(200903056)
上海市科技兴农攻关项目[沪农科攻字(2009)第5-1号]
关键词
铬
猪
精子获能
信号通路
糖酵解
chromium
boar
sperm capacitation
signal pathway
glycolysis
