Toshiyuki Katsumata

Characterization of the zona pellucida glycoproteins from bovine ovarian and fertilized eggs

Bovine zona pellucida (ZP) glycoproteins from ovarian egg emerged as three bands with molecular mass of 78 kDa, 64 kDa and 21 kDa in SDS-PAGE under reducing conditions. Endo-beta-galactosidase (E beta G) digestion of the glycoproteins yielded five products with molecular mass of 76 kDa (E beta G-76), 68 kDa (E beta G-68), 63 kDa (E beta G-63), 47 kDa (E beta G-47) and 21 kDa (E beta G-21) under the same conditions. The N-terminal amino acid sequences of E beta G-76 and E beta G-21 were identical. This fact together with the results of diagonal SDS-PAGE indicated that E beta G-21 (N-terminal region) is linked to E beta G-63 (C-terminal region) through disulfide bond to form E beta G-76. Immunoblot analysis using anti-pig ZP protein antibodies revealed that bovine E beta G-76, E beta G-68 and E beta G-47 correspond to pig PZP2, PZP3 alpha and PZP3 beta glycoproteins, respectively. The E beta G-76 and E beta G-68 components were shown to be specifically cleaved during fertilization.

Abnormal biantennary sugar chains are expressed in human chorionic gonadotropin produced in the choriocarcinoma cell line, JEG-3

Over the past two decades, sugar chain structures of human chorionic gonadotropin (hCG) produced in healthy people, three types of trophoblastic disease, and some types of cell lines have been analyzed. The abnormal biantennary structure of hCG is a good marker for the diagnosis of malignant choriocarcinoma. In spite of much research, hCG with an abnormal biantennary structure is only detected in the urine of choriocarcinoma or pregnant diabetic patients. We hypothesized that the formation mechanism of the abnormal biantennary sugar chain structure is mainly caused by high GnT-IV activity. To confirm this, we measured the N-acetylglucosaminyltransferase (GnT)-IV activity and hCG productivity in three choriocarcinoma cell lines, and selected JEG-3 cells. hCG samples were purified from medium conditioned by JEG-3 cells, and their sugar chain structures were analyzed. We detected an abnormal biantennary structure, and the proportions were different from those previously reported in the urine samples of choriocarcinoma patients. These findings proved our hypothesis and suggest the usefulness of JEG-3 cells for further analyses of abnormal biantennary structure formation. Published in 2004.

Activity of exoglycosidases in ejaculated spermatozoa of boar and bull.

The activity of exoglycosidases in extracts from freshly ejaculated boar and bull spermatozoa with 0.2% Brij-35/2% acetic acid was measured. The results show that beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase are the major glycosidases; much higher levels of activity were found in boar spermatozoa than in bull spermatozoa. When compared on a per spermatozoon basis, the ratios of the activities of beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase in boar spermatozoon relative to those in bull spermatozoon were approximately 13000:1, 1700:1 and 400:1, respectively. Liberation of these glycosidases from bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) was low, in contrast to liberation of alpha-mannosidase from boar spermatozoa previously found by the same means. The possibility that the exoglycosidases present in large amounts in boar spermatozoa play a role in the process of binding to the zona pellucida glycoprotein of the egg is discussed.