Md Sharoare Hossain1, Sadia Afrose1, Tomio Sawada2, Koh-Ichi Hamano1, Hirotada Tsujii1. 1. Laboratory of Animal Biotechnology, Faculty of Agriculture Shinshu University Minamiminowa-mura 399-4598 Nagano Japan. 2. The Sawada Women's Clinic Nagoya Reproduction Center Chikusaku Nagoya Aichi Japan.
Abstract
PURPOSE: 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 14C-oleic acid and 3H-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. METHODS: 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. RESULTS: Incorporation rate of 14C-oleic acid into the sperm lipids was significantly higher than that of 3H-linoleic acid (P < 0.05). The oxidation of 14C-oleic acid was higher in combined radiolabeling rather than in one. The highest amounts of 3H-linoleic acid and 14C-oleic acid were recovered mainly in the triglycerides and phospholipids fraction, and 14C-oleic acid distribution was higher than the 3H-linoleic acid in both labeled (P < 0.05) sperm lipids. In the 3H-linoleic and 14C-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 (P < 0.05). Supplementation of arachidonic acid significantly increased (P < 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) (P < 0.05), and inhibition by PKA inhibitor is stronger than that by PKC inhibitor. CONCLUSIONS: 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.
PURPOSE: 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 14C-oleic acid and 3H-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. METHODS: 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. RESULTS: Incorporation rate of 14C-oleic acid into the sperm lipids was significantly higher than that of 3H-linoleic acid (P < 0.05). The oxidation of 14C-oleic acid was higher in combined radiolabeling rather than in one. The highest amounts of 3H-linoleic acid and 14C-oleic acid were recovered mainly in the triglycerides and phospholipids fraction, and 14C-oleic acid distribution was higher than the 3H-linoleic acid in both labeled (P < 0.05) sperm lipids. In the 3H-linoleic and 14C-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 (P < 0.05). Supplementation of arachidonic acid significantly increased (P < 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) (P < 0.05), and inhibition by PKA inhibitor is stronger than that by PKC inhibitor. CONCLUSIONS: 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.
Entities:
Keywords:
Acrosome reaction; Boar sperm; Cholesterol efflux; Incorporation and oxidation of fatty acid; PKA and PKC
Authors: F M Flesch; J F Brouwers; P F Nievelstein; A J Verkleij; L M van Golde; B Colenbrander; B M Gadella Journal: J Cell Sci Date: 2001-10 Impact factor: 5.285