Masaharu Kamada

Significance of D-mannose as a sperm receptor site on the zona pellucida in human fertilization

The role of monosaccharides in human fertilization was studied by testing their effects on penetration of spermatozoa into mature human oocytes (zona penetration test). When oocytes were pretreated with concanavalin A, wheat germ agglutinin, or Ricinus communis agglutinin-I at a concentration of 100 micrograms/ml, no spermatozoa were found to bind to or penetrate through the zona pellucida. Penetration of spermatozoa was restored when the zona pellucida pretreated with wheat germ agglutinin and Ricinus communis agglutinin-I were rinsed with N-acetyl-D-glucosamine (wheat germ agglutinin inhibitor) and D-galactose (Ricinus communis agglutinin inhibitor), respectively. Conversely, the blocking effect of concanavalin A on sperm penetration was not reversed by treatment with D-mannose (concanavalin A inhibitor). Furthermore, pretreatment of spermatozoa with D-mannose (50 mmol/L) completely inhibited sperm penetration through the zona pellucida. However, sperm penetration was clearly demonstrated when the zona pellucida was pretreated with D-mannose. These data suggest that D-mannose residues are essential in, or sterically closely related to, the sperm receptor site on the human zona pellucida.

A repertoire of cytokines in human seminal plasma.

The pathophysiological significance of seminal cytokines in sperm function is still controversial. We determined the repertoire of cytokines in seminal plasma obtained from men with or without abnormalities in semen and assessed the pathophysiological significance of seminal cytokines. After conventional analysis of semen samples obtained from 86 men, levels of seminal cytokines (interleukin [IL]-1alpha, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor-alpha [TNF-alpha], interferon-gamma, granulocyte colony-stimulating factor [G-CSF], macrophage CFS [M-CSF]) and granulocyte elastase were measured by an enzyme-linked immunosorbent assay. Leukocytospermia was defined as seminal plasma, which has > or =1000 ng/ml granulocyte elastase. Leukocytospermia was found in nine of 62 of the subjects in the normozoospermic group but in none of the 24 subjects showing abnormal sperm parameters (azoospermia, n=5; oligozoospermia, n=4; asthenozoospermia, n=15). The IL-8 level in the leukocytospermic group was significantly higher than those in the normal and oligozoospermic groups. IL-1alpha and TNF-alpha levels in the leukocytospermic group were significantly higher than those in the normal and asthenozoospermic groups. Although the G-CSF level in the leukocytospermic group was significantly higher than that in the normal group, high levels of M-CSF were detected in all groups. The IL-8 level was strongly correlated with IL-1alpha (r=0.935, P<0.0001) and G-CSF (r=0.916, P<0.0001) levels. Cytokines detected in seminal plasma are associated with the pathogenesis of leukocytospermia but not with the pathogenesis of asthenozoospermia and oligozoospermia.

Etiological implication of autoantibodies to zona pellucida in human female infertility.

PROBLEM: Autoantibodies to zona pellucida (ZP) have been implicated as a cause of infertility in woman by three lines of clinical and laboratory evidence.

Postmenopausal changes in production of type 1 and type 2 cytokines and the effects of hormone replacement therapy.

ObjectiveAn appropriate defense against infective agents or malignant cells is attributed to the exquisitely balanced T helper 1 type (cellular) and T helper 2 type (humoral) immune reactions. We investigated the effect of hormone replacement therapy (HRT) on postmenopausal changes in the production of interferon (IFN)-&ggr; and interleukin (IL)-10, a type 1 and a type 2 cytokine, respectively. DesignBoth cytokines were measured by ELISA in the supernatant of lipopolysaccharide-stimulated whole blood cells from 72 untreated and 44 HRT-treated women. Thirteen women were examined before and during HRT. ResultsThe production of IFN-&ggr; in women in their 40s and in postmenopausal women was significantly higher compared with that of younger women. However, IFN-&ggr; fell to the lowest level in the late postmenopausal stage, whereas the production of IL-10 increased gradually with age and in parallel with the postmenopausal period. Thus, in women in the mid-and late postmenopausal period, excessive production of type 2 cytokine (IL-10) compared with type 1 cytokine (IFN-&ggr;) occurred. The IFN-&ggr; levels of women on HRT were significantly lower than those of untreated women in the early and mid-postmenopausal stages, and IL-10 levels of women on HRT were significantly lower than those of untreated women in the mid-and late postmenopausal stages. HRT induced a significant decrease in the production of IL-10 and tended to lower the level of IFN-&ggr;. ConclusionsProduction of IL-10 is augmented in postmenopausal women. HRT probably prevents postmenopausal women from an aberration of the immune system by improving the balance of type 1 and type 2 immune reactions.

Transient Increase in the Levels of T-Helper 1 Cytokines in Postmenopausal Women and the Effects of Hormone Replacement Therapy

The aim of this study was to determine, at least in part, T-cell function in postmenopausal women and the effects of hormone replacement therapy (HRT). Levels of T-helper 1 (Th1) cytokines (IL-2 and IFN-γ) and T-helper 2 (Th2) cytokines (IL-4 and IL-10) produced by phytohemagglutinin-stimulated whole blood cells from 72 untreated and 44 HRT-treated women were measured by ELISA. Thirteen of the 44 HRT-treated women were examined before and during HRT. The production of IL-2 increased gradually with advance of the postmenopausal period. The levels of IL-2 in women in the early (≤10 years) and mid (>10 and <30 years) postmenopausal stages were significantly higher than those in women in their second, third and fourth decades. The level in women in the late (≧30 years) postmenopausal stage, however, was significantly lower than those in women in the early and mid postmenopausal stages. The level of IFN-γ was highest in women in the mid postmenopausal stage. On the other hand, the levels of Th2 cytokines did not change with age or after menopause until the mid postmenopausal period but were significantly lower in women in the late postmenopausal stage. IFN-γ levels in women on HRT were significantly lower than those in untreated postmenopausal women at all postmenopausal stages. HRT induced a significant decrease in the production of IL-2 and IL-4. In conclusion, production of Th1 cytokines is augmented in women after menopause. HRT prevents this increase, thereby improving the aberration of Th1/Th2 balance that is implicated in an inadequate immune response and pathological conditions.

Identification of IgG and Fc-Binding Proteins in Human Seminal Plasma and Sperm

Human seminal plasma contains two novel soluble proteins capable of binding IgG and Fc, but not Fab. The IgG- and Fc-binding proteins were identified by immunoblotting using IgG of various species, Fc and Fab fragments. Their estimated molecular sizes are 16-kD and 20-kD. The monoclonal antibody (MAb), Leu 11b, raised against Fc gamma RIII interacts with the 16-kD protein, whereas other mAbs (32.2, IV.3, and 3G8) raised against FcR did not. The 16-kD protein is capable of binding IgG of several species (human, mouse, rabbits, and goat), whereas the 20-kD protein interacted only with human IgG-Fc fragment. The 16-kD IgG and the 20-kD Fc-binding proteins were found in the PBS extract of human sperm. Human seminal plasma/sperm contain an IgG- and a Fc-binding protein with estimated Mr of 16 and 20 kD, respectively.

High implantation rate and consequently high pregnancy rate by in vitro fertilization-embryo transfer treatment in infertile women with antisperm antibody

OBJECTIVE To examine the effect of antisperm immunity on postfertilization steps, such as implantation of embryos and fetal growth in IVF-ET treatment of women with sperm-immobilizing antibodies. DESIGN Retrospective analysis of clinical laboratory data. SETTING The IVF-ET program of the Department of Obstetrics and Gynecology. The University of Tokushima, School of Medicine. PATIENTS Eighteen women with sperm-immobilizing antibodies and 122 infertile patients with nonimmune etiology as controls. Infertile couples due to a male factor and with unknown etiology were excluded. INTERVENTIONS All patients received the same IVF-ET program with GnRH agonist. MAIN OUTCOME MEASURES Rates of fertilization and cleavage, implantation rate per embryo transferred and pregnancy rate (PR) in both test and comparison groups. RESULTS The rate of fertilization in the antisperm group (61.3%) was significantly lower than that in the comparison group (76.8%). But the implantation rate per embryo transferred (23.5%) and consequently the modified PR per oocyte recovery procedure (34.4%) in immunologically infertile women were significantly higher than those in the comparison group (7.9% and 17.8%, respectively). CONCLUSIONS Although sperm-immobilizing antibodies prevent sperm-egg interaction, they do not seem to have any adverse effects on achievement of pregnancy. Moreover, the existence of antisperm immunity in woman with antisperm antibodies is suggested to be favorable for successful pregnancy by the IVF-ET procedure.

Tn antigen, a marker of potential for metastasis of uterine cervix cancer cells.

Background. The expressions of Thomsen–Friedenreich antigen (T‐Ag) and Tn antigen (Tn‐Ag), precursors of MN blood group antigens, were examined in the tissues of squamous cell carcinoma of the uterine cervix from 111 patients to determine their clinicopathologic significance with regard to the biologic behaviors of cancer cells and the clinical course of the patients.

Production and Regulation of Cytokine-Induced Neutrophil Chemoattractant in Rat Ovulation

Abstract A cytokine-induced neutrophil chemoattractant (CINC/gro), which belongs to the interleukin (IL)-8 family, acts as a functional chemoattractant for neutrophils in rats. In the present study, we examined whether CINC/gro contributes to the ovulation process in the rat ovulation system. In rat ovaries, CINC/gro was immunohistochemically recognized in the theca layer of the antral follicle but not in the granulosa cells. To clarify the role of CINC/gro in the ovulation process, CINC/gro protein and mRNA were examined during pregnant mare serum gonadotropin (PMSG)-hCG treatment. CINC/gro protein did not increase as a result of PMSG injection. However, it increased rapidly after hCG injection and peaked at 6 h after hCG. CINC/gro mRNA was also strongly expressed after hCG injection. The increase of CINC/gro protein followed increases in IL-1β and tumor necrosis factor α (TNFα). In the whole ovarian dispersate culture, FSH, hCG, IL-1β, and TNFα stimulated the production of CINC/gro protein in a dose-dependent manner. In particular, the stimulatory effects of IL-1β and TNFα were stronger than those of gonadotropins. These results suggest that CINC/gro plays an important role in the rat ovulation process by attracting neutrophils. CINC/gro increased just prior to ovulation, and it may be regulated directly by cytokines such as IL-1β and TNFα and indirectly by gonadotropins.

Lysophosphatidic acid stimulates nuclear and cytoplasmic maturation of golden hamster immature oocytes in vitro via cumulus cells.

Lysophosphatidic acid (LPA), a member of the phospholipid autacoid family, is induced in incubated human follicular fluid by lysophospholipase D. It is well known that LPA functions as a growth factor and the hypothesis that LPA in human follicular fluid takes a part in meiosis of oocytes is quite plausible. We studied the effects of LPA on the maturation of golden hamster immature oocytes in vitro. Hamster oocytes with a germinal vesicle were cultured in Tyrodes albumin lactate pyruvate (TALP) medium with 10(-5) M LPA, 10 ng/ml epidermal growth factor (EGF), 30 ng/ml insulin-like growth factor-1, 1 ng/ml tumor growth factor-alpha or 1 ng/ml basic fibroblast growth factor. The nuclear maturation rates in the LPA and EGF groups were significantly higher than in the control group and the other growth factors did not show any stimulatory effect (LPA group; 74.3% [75/101], EGF group; 82.4% [89/108] vs. control group; 60.2% [59/98], p < 0.05, p < 0.01, respectively). When the cells of cumulus were removed, EGF and LPA did not increase the nuclear maturation rates. Cotreatment EGF and LPA did not significantly enhance the stimulatory effect observed with LPA alone on maturation in vitro. The penetration rate determined by the zona-free hamster oocyte test was significantly higher in the LPA group than in the control group (26.7% vs. 13.2%, p < 0.05) and was comparable with that of oocytes matured in vivo. In conclusion, LPA stimulates the nuclear and cytoplasmic maturation of hamster immature oocytes via cumulus cells.