Hossain Md Sharoare, Afrose Sadia, Sawada Tomio, Hamano Koh-Ichi, Tsujii Hirotada
Laboratory of Animal Biotechnology, Faculty of Agriculture Shinshu University Minamiminowa-mura 399-4598 Nagano Japan.
The Sawada Women's Clinic Nagoya Reproduction Center Chikusaku Nagoya Aichi Japan.
Reprod Med Biol. 2009 Oct 27;9(1):23-31. doi: 10.1007/s12522-009-0036-7. eCollection 2010 Mar.
For understanding the roles of fatty acids on the induction of acrosome reaction which occurs under association of cholesterol efflux and PKA or PKC pathways in boar spermatozoa, metabolic fate of alone and combined radiolabeled C-oleic acid and H-linoleic acid incorporated in the sperm was compared, and behavior of cholesterol and effects of PKA and PKC inhibitors upon fatty acid-induced acrosome reaction were examined.
Semen was collected from a Duroc boar, and the metabolic activities of fatty acids in the spermatozoa were measured using radioactive compounds and thin layer chromatography. Cholesterol efflux was measured with a cholesterol determination assay kit. Participation of fatty acids on the AR through PKA and PKC pathways was evaluated using a specific inhibitor of these enzymes.
Incorporation rate of C-oleic acid into the sperm lipids was significantly higher than that of H-linoleic acid ( < 0.05). The oxidation of C-oleic acid was higher in combined radiolabeling rather than in one. The highest amounts of H-linoleic acid and C-oleic acid were recovered mainly in the triglycerides and phospholipids fraction, and C-oleic acid distribution was higher than the H-linoleic acid in both labeled ( < 0.05) sperm lipids. In the H-linoleic and C-oleic acid combined radiolabeling, the incorporation rate of the radioactive fatty acids in all the lipid fractions increased 15 times more than the alone radiolabeling. Boar sperm utilize oleic acid to generate energy for hyperactivation ( < 0.05). Supplementation of arachidonic acid significantly increased ( < 0.05) cholesterol efflux in sperm. When spermatozoa were incubated with PKA or PKC inhibitors, there was a significant reduction of arachidonic acid-induced acrosome reaction (AR) ( < 0.05), and inhibition by PKA inhibitor is stronger than that by PKC inhibitor.
Incorporation of unsaturated fatty acids, especially oleic acid, into triglycerides and phospholipids provides prerequisite energy for AR. Cholesterol efflux by arachidonic acid triggers AR. Arachidonic acid activated PKA and PKC pathway participate in induction of the AR.
为了解脂肪酸在公猪精子中胆固醇外流与蛋白激酶A（PKA）或蛋白激酶C（PKC）途径联合作用下诱导顶体反应中的作用，比较了单独和联合掺入精子中的放射性标记C - 油酸和H - 亚油酸的代谢命运，并检测了胆固醇的行为以及PKA和PKC抑制剂对脂肪酸诱导的顶体反应的影响。
从一头杜洛克公猪采集精液，使用放射性化合物和薄层色谱法测量精子中脂肪酸的代谢活性。用胆固醇测定试剂盒测量胆固醇外流。使用这些酶的特异性抑制剂评估脂肪酸通过PKA和PKC途径参与顶体反应的情况。
C - 油酸掺入精子脂质的速率显著高于H - 亚油酸（<0.05）。联合放射性标记时C - 油酸的氧化高于单一标记。H - 亚油酸和C - 油酸的最高含量主要在甘油三酯和磷脂部分中回收，并且在两种标记的精子脂质中C - 油酸的分布均高于H - 亚油酸（<0.05）。在H - 亚油酸和C - 油酸联合放射性标记中，所有脂质部分中放射性脂肪酸的掺入率比单独放射性标记增加了15倍以上。公猪精子利用油酸产生能量以实现超活化（<0.05）。添加花生四烯酸显著增加了精子中的胆固醇外流（<0.05）。当精子与PKA或PKC抑制剂孵育时，花生四烯酸诱导的顶体反应（AR）显著降低（<0.05），并且PKA抑制剂的抑制作用强于PKC抑制剂。
不饱和脂肪酸，尤其是油酸，掺入甘油三酯和磷脂中为顶体反应提供了前提能量。花生四烯酸引起的胆固醇外流触发顶体反应。花生四烯酸激活的PKA和PKC途径参与顶体反应的诱导。
