Rihachi Iizuka

Separation of human X- and Y-bearing sperm using Percoll density gradient centrifugation

Human X- and Y-bearing sperm, which were detected by the quinacrine mustard fluorescent staining method, were separated in density gradient centrifugation using Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden). The apparent percentages of Y-bearing sperm in normo- and oligospermia were 46.7% +/- 3.4% (n = 13) and 46.6% +/- 1.2% (n = 10), respectively. The separation was observed to some extent in Ficoll Paque (Pharmacia Fine Chemicals); then it was further achieved using Percoll. After centrifugation, sperm were distributed widely on various interfaces of the discontinuous Percoll density gradient (1.06 to 1.11 gm/ml). The content of Y-bearing sperm in the fraction lighter than 1.06 gm/ml was 73.1% +/- 3.3% and was reduced gradually along the density increment. Finally, it was found to be 27.4% +/- 3.4% in the sediment (more than 1.11 gm/ml).

Human X- and Y-bearing sperm differ in cell surface sialic acid content

Analysis of cell surface charge of human sperm by means of free-flow electrophoresis revealed that the electrophoretic mobility of X-bearing sperm was faster than that of Y-bearing sperm, indicating that X-bearing sperm have a higher net negative charge on the cell surface than do Y-bearing sperm. Sialidase treatment of X- and Y-bearing sperm caused an obvious and progressive reduction in the electrophoretic mobilities of the two types of cells, until they finally merged into a single peak. This observation suggests that the difference in net negative charge between X- and Y-bearing sperm is due mainly to sialic acid content on the cell surface.

Menstrual cycle-associated alteration of sulfogalactosylceramide in human uterine endometrium: Possible induction of glycolipid sulfation by sex steroid hormones

The human uterine endometrium is a tissue in which cell proliferation and differentiation are strictly controlled by sex steroid hormones, and these hormone-controlled cellular events occurring in association with the menstrual cycle of the uterine endometrium should be accompanied by characteristic molecular and metabolic changes. To characterize the menstrual cycle at the molecular level, we analyzed the glycolipids of human uterine endometrium in the proliferative and secretory phases of the menstrual cycle. Neutral glycosphingolipids from uterine endometrium comprised globo-series glycosphingolipids, such as GlcCer, LacCer, Gb3Cer, and Gb4Cer, and the relative concentrations remained constant in the two phases. However, in the case of acidic glycosphingolipids, although the concentrations of sialoglycosphingolipids remained at constant levels in the two phases, sulfatide, I3-SulfoGalCer, dramatically increased from the proliferative to the secretory phase, amounting to 7-17 nmol/g dry weight in the proliferative phase and 115-245 nmol/g dry weight in the secretory phase. Since sulfatide was the only glycolipid that changed in association with the menstrual cycle, it is likely that the sulfotransferase responsible for the synthesis of sulfatide might be induced by sex steroid hormones, estrogen and progesterone, and that sulfatide might play an essential biological role in the secretory phase of the menstrual cycle in the uterine endometrium.

Continuous-step density gradient centrifugation for the selective concentration of progressively motile sperm for insemination with husband's semen

To increase the fertilizability of sperm for use in artificial insemination with husbands semen, it is effective to concentrate progressively motile sperm from whole ejaculated semen. The simple procedure, a continuous-step density gradient technique, was developed to selectively concentrate progressively motile sperm. The ejaculated semen was placed on 6.0 ml of 80% Percoll solution, and the density gradient was formed by mixing the semen and Percoll with an L-shaped rod for two or three strokes. After centrifugation at 400 x g for 30 min, progressively motile sperm were concentrated in the sediment, whereas the immotile sperm and other types of cells remained in the upper part of the density gradient. In most specimens sperm motility was improved by more than 80% regardless of the original motility. Thus, the fertility index (sperm concentration/ml x percentage of motility x 10(-8)). was increased to 7.7 +/- 3.7 times (n = 15).

Survey of 1120 Japanese women with a history of recurrent spontaneous abortions

Our recent 7-year clinical survey showed that among the 1120 women with repeated spontaneous abortions registered in this clinic, 2898 out of a total of 3216 pregnancies (90.1%) had terminated in spontaneous abortion. Among these wastages, 84.2% occurred before 12 weeks of gestation, and 11.1 percent occurred between 12 and 15 weeks. Through routine examination of reproductive wastage, 82 (9.9%) of the 825 Japanese couples examined were shown to have either a chromosomal abnormality or normal variants in the wife and/or husband, thus demonstrating no racial difference in the incidence of chromosomal abnormalities in infertile patients in comparison with studies performed in other countries. One hundred and forty-seven congenital uterine anomalies (14.7%) were found in 1000 hysterosalpingographies, and 12 of 148 examined females were positive for anti-cardiolipin antibody. 393 other females with no major abnormalities likely to induce spontaneous abortions were indicated for immunotherapy. Ample time spent on genetic counseling prevented further reproductive wastage, and ideal metroplasty resulted in a successful post-operative pregnancy course in more than 85% of cases. Immunosuppressant and anticoagulant therapy decreased the serum titer of anti-cardiolipin antibody, enabling pregnancies to be maintained to term. Immunotherapy utilizing the husbands lymphocytes also brought more than 80% of pregnancies to successful completion, with 200 deliveries achieved with this therapy. In contrast, 64.1% of pregnancies again terminated spontaneously in patients who were indicated for immunotherapy but did not receive treatment. The findings of the present study suggest that the causes of reproductive wastage, especially the etiology of early recurrent spontaneous abortion, are complex.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody (MSN-1) against a newly established uterine endometrial cancer cell line (SNG-II) and its application to immunohistochemistry and flow cytometry

To determine a phenotypic difference between normal endometrium and endometrial adenocarcinoma, a new monoclonal antibody (MSN-1) was produced by immunizing a new endometrial cancer cell line (SNG-II), which was established in 1981 from a 43-year-old Japanese woman with stage II uterine endometrial cancer. MSN-1 recognized the Lewis-b carbohydrate moiety on the cell surface glycolipid and seldom reacted immunohistochemically with normal endometrium but with about 90% of endometrial cancer cases. By application of MSN-1 to flow cytometry, the possibility of differentiating endometrial normal cells from cancer cells was demonstrated.

Differences in lectin binding patterns of normal human endometrium between proliferative and secretory phases

SummaryLectin binding patterns in normal human endometrium were examined by light and electron microscopy using seven different lectins (ConA, WGA, RCA, PNA, UEA-1, DBA, and SBA). For light microscopic observations, criteria based on the incidence and intensity of cells positive for the lectin staining were adopted to evaluate the different staining patterns of the proliferative and secretory endometria obtained by the avidin-biotin-peroxidase complex (ABC) technique. At the light microscopic level, ConA, WGA, and RCA stained endometrial glandular cells in both phases. The number of PNA-positive cells with the binding sites entirely limited to the apical surface tended to be reduced slightly in the secretory phase. UEA-1 weakly stained the apical surface of glandular cells in the proliferative phase but not in the secretory phase. Among the lectins used in this study, DBA and SBA displayed remarkable changes between the phases. That is, in the proliferative phase they produced only a faint or slight positive stain at the apical surface, but the incidence and intensity of DBA- and SBA-positive glandular cells increased in the secretory phase. By electron microscopy, the reaction product of ConA was observed in the plasma membrane, endplasmic reticulum, nuclear envelope, and the Golgi apparatus, and the binding sites of RCA and DBA were observed in the plasma and, Golgi membranes. Between both phases, the reactivity of ConA and RCA showed almost no change. However, the secretory endometrial cells containing the DBA-positive Golgi apparatus were markedly increased in number compared with the proliferative ones bearing the lectin-positive organelles. These results indicate that DBA and SBA are useful for detection of hormone-mediated changes of normal human endometrium, and suggest that such changes are closely related to the Golgi apparatus.

Washing and Concentration of Human Semen by Percoll Density Gradients and Its Application to Aih

Human sperm for use in artificial insemination with husbands semen (AIH) were prepared by two methods: the monolayer Percoll method and the discontinuous Percoll density gradient of four steps. The former focused mainly on the oligozoospermic semen concentration, with the sperm concentration and motility being improved 4.29 +/- 3.7 times and 1.92 +/- 1.1 times, respectively (n = 41). The latter was used principally for the selective isolation of forward motile sperm, with the sperm motility being increased from 42.3 +/- 29.5% to 76.3 +/- 11.0% after centrifugation. The clinical application of these procedures resulted in 35 (the monolayer Percoll method) and 10 (the discontinuous Percoll density gradient) successful deliveries with greater enhancement of sperm qualities. The number of females was predominate over the males in the delivered cases: 31:34 (100:45.1) females to males.

A possible mechanism of immunotherapy for patients with recurrent spontaneous abortions

ABSTRACT: The mechanism of the beneficial effect of immunotherapy for human reproductive wastage remains to be elucidated. Induction of blocking antibodies such as anti‐HLA class II antibodies and anti‐idiotypic antibodies was investigated as the mechanism of specific immunosuppression in pregnancy. We reported the changes in the mixed lymphocyte reaction (MLR), T‐cell subsets, and generation of anti‐idiotypic antibodies after immunotherapy compared to before immunotherapy. MLR was significantly (P < 0.001) inhibited after the immunization. The mean inhibition rate was 50.2%, suggesting that MLR blocking antibodies were induced by immunotherapy. Binding of autoantibodies to alloactivated maternal lymphoblasts against the paternal lymphocytes was detected in postimmunization cases in two‐color flow‐cytometric experiments. This suggests that anti‐idiotypic antibodies were induced by the immunotherapy. The percentage of cytotoxic T‐cells was significantly decreased (P < 0.05) and the percentage of suppressor T‐cells was significantly increased (P < 0.01) after the immunotherapy, suggesting that a cell‐mediated immune response was induced by the immunotherapy.

IMMOBILIZED DATURA STRAMONIUM AGGLUTININ COLUMN CHROMATOGRAPHY, A NOVEL METHOD TO DISCRIMINATE THE URINARY hCGs OF PATIENTS WITH INVASIVE MOLE AND CHORIOCARCINOMA FROM THOSE OF NORMAL PREGNANT WOMEN AND PATIENTS WITH HYDATIDIFORM MOLE

Datura stramonium agglutinin (DSA) binds the asparagine‐linked sugar chains commonly found in the human chorionic gonadotropin (hCG) purified from urine of patients with invasive mole or choriocarcinoma, but not in that of normal pregnant women or patients with hydatidiform mole. With the use of an immobilized DSA column, a novel method to discriminate urinary hCGs from various trophoblastic diseases was developed.