|Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥1,400,000 (Direct Cost: ¥1,400,000)
To clarify the relationship between the functions of gonadotropins and the maturation of sperm in testis gland of male bullfrog, Rana catesbeiana, we had isolated lutropin (LH) and follitropin (FSH), studied their biological activities such as ovulation activity and enhance the increase of the weight of testis gland. We also determined the amino acid sequence, and the sugar chain structures bound to the bullfrog LH and FSH.
We attempted to isolate the testis-specific protein or transition protein (TP), which believed to appear and disappear during the maturation of sperm cell, and protamine. Testis glands kept in cold acetone or flesh, were delipidated by homogenizing with acetone. Basic proteins were extracted by two methods, one was directly extracted with 0.25M HCL,and other was after washing with Na-citrate buffer containing Na-bisulfite and p-amidinophenyl methanesullfonyl fluoride as the protease inhibitor. Two preparations were compared by 15% polyacrylamide SDS or acid urea gel electrophoresis system. As the references, liver, kidney, spleen glands were also treated with the same ways. One band observed only in the preparation from testis with histones seemed to be TP or protamine. This protein and histone H1s were completely precipitated with 73% acetone by volume, whereas other histones were with 90% acetone. Materials precipitated with 73% acetone were further fractionated by reversed phase HPLC using C4, C8, C10 and phenyl columns. Proteins were separated into 8 peaks. TP or protamine was eluted in 6 peaks having the same mobilities in SDS gel electrophoresis. It is not known how many spices of TP or protamine are present.