Ichiro Takai

A new method for preparation of pure zonae pellucidae in large quantities from porcine ovaries

A new method for preparation of large quantities of zona substance in pure form from porcine ovaries has been described. In addition to the previously reported techniques of glass wool treatment and sieving by saran meshes, the following three significant improvements have been introduced: disruption of ovaries by an electric machine equipped with two multi-needed disks, which resulted in a considerably accelerated recovery of follicular oocytes with a high yield; low-speed centrifugation at 170 X g for 15 s was found to be an obligatory step to eliminate light particulate material from the crude oocyte suspension; use of 50% sucrose solution in discontinuous density gradient centrifugation permitted complete separation of homogeneous samples at two stages of the final preparation of zona-encased oocytes or of oocyte-free zonae. Microscopic examination revealed no contaminating components in the zona preparation. With this method 93 mg of lyophilized zona preparation were obtained from 24 553 porcine ovaries. Analysis of a solubilized zona substance by Sephacryl S-200 column chromatography showed the presence of two major glycoproteins which could not be separated completely from each other. By analysis of the two components with SDS-PAGE, only a single, but broad, band of glycoprotein was found, indicating the successful isolation of a major component(s) from porcine zonae.

Three types of polycystic ovarian syndrome in relation to androgenic function

OBJECTIVE To re-evaluate the concept of polycystic ovarian syndrome (PCOS) in view of androgenic function. DESIGN Patients were studied endocrinologically and ultrasonographically. SETTING This study was performed as a clinical investigation. PATIENTS, PARTICIPANTS Sixty-nine euprolactinemic anovulatory patients with luteinizing hormone (LH) hypersecretion and 18 normal volunteers were selected. INTERVENTIONS One hundred micrograms of LH-releasing hormone were administered. MAIN OUTCOME MEASURE(S) It was possible to divide PCOS patients into three types. RESULTS Patients with neither hirsutism nor elevation of serum androstenedione (delta 4) and/or testosterone (T) were designated as type I PCOS (n = 20). Patients without hirsutism but with elevated delta 4 and/or T were referred to as type II PCOS (n = 33). Patients with both hirsutism and elevation of delta 4 and/or T were defined as type III PCOS (n = 16). Concentrations of delta 4 appeared gradedly increasing in types I, II, and III in that order, whereas T concentrations were significantly higher in types II and III than in control. Ultrasonographically, cystic ovaries were found in 88%, 84%, and 100% of types I, II, and III patients, respectively. CONCLUSIONS It is postulated that each type may represent a subset of whole spectrum of PCOS from Stein-Leventhal syndrome to simple anovulation with LH hypersecretion.

Characterization of glycoproteins isolated from porcine zonae pellucidae

Two glycoproteins, tentatively designated as PZ-alpha and PZ-beta, have been isolated and purified to homogeneity from porcine zonae pellucidae by a simple purification procedure producing a high yield. The procedure included the dissolution of the zona material in 0.1 M sodium borate buffer pH 10.0, Sephadex G-100 column chromatography and preparative SDS-polyacrylamide gel electrophoresis. The purified glycoproteins gave a single band on polyacrylamide gel and had molecular weights of 60 000 (PZ-alpha) and 96 000 (PZ-beta). Glutamic acid was detected as the NH2-terminal residue in both glycoproteins, using the dansyl chloride method. Though their amino acid compositions were similar, their carbohydrate contents were slightly different (PZ-alpha: 24.9%; PZ-beta; 19.6%), but these components contained the same types of monosaccharides: fucose, mannose, galactose, NAcGlc and sialic acid. The antigenic properties of the two glycoproteins were indistinguishable by immunodiffusion tests. The PZ-beta could be converted in part to smaller molecular weight components, though not to PZ-alpha, by treatment with beta-mercaptoethanol. Thus clear differences between PZ-alpha and PZ-beta could not be detected by chemical or immunological analyses except for the difference in the behaviour on SDS-polyacrylamide gel electrophoresis.

A new method for detection of anti-zona activity in human sera using latex agglutination reaction

A slide latex test for detection of anti-zona pellucida activity in human sera was developed using a latex agglutination reaction. The latex reagent, made of polystyrene particles coated with solubilizing zona antigen(s), was found to give results comparable in sensitivity as well as specificity to those of the indirect immunofluorescence method as tested with anti-pig-zona antiserum and with human sera. Thus, the slide latex test was judged to be adequate for use instead of the indirect immunofluorescence technique.

A Method for Specific Detection of Autoantibodies to the Zona Pellucida in Infertile Women**Supported by Grant 77166 from the World Health Organization and Grant 137055 from the Ministry of Education, Japan.

Human antibodies to porcine erythrocytes were found to bind to porcine but not to human zonae pellucidae, whereas human isohemagglutinins bound to human but not to porcine zonae. On the basis of the binding behavior of agglutinins, it was found that the immunofluorescence of porcine zonae produced by selected sera from infertile women was due to an antibody of different specifity from that which agglutinated pig red blood cells. However, no serum component other than heteroagglutinin was contained in selected sera from control subjects which fluoresced porcine zonae. Since the component was present in the immunoglobulin G fraction, bound to human zonae, and was reactive with zona-specific antigen(s), it was judged an autoantibody to zonae. Thus a method for specific detection of autoantibodies to zonae has been developed by indirect membrane immunofluorescence using porcine ova as targets.