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Biomedical subjects

S S Koide

Publications and source records attributed to S S Koide.

At least 109 records · Page 6Linked to original sources

Unique variants of hCG in sera of choriocarcinoma patients.

Human chorionic gonadotropin (hCG) of pregnancy can be separated into several variants by an isoelectric focusing (IEF) method. The hCG produced by choriocarcinoma consists of components found in pregnancy sera (pI 3.9-6.0) and, in addition, unique variants with more acidic isoelectric points ranging from 3.2 to 3.7. The sera of all five patients with advanced choriocarcinoma contained these unique acidic variants of hCG and the immunoreactive levels were significantly higher in these patients than in normal pregnant women or patients afflicted with hydatidiform mole or invasive mole. When the acidic variants of hCG were treated with neuraminidase they migrated to the alkaline region (pI 9-11) of the IEF column. In conclusion, sera of choriocarcinoma patients contain unique variants of hCG. The finding that these highly acidic components can be converted to molecules with an alkaline pI by neuraminidase digestion suggests that this high negative charge may be due to an increased sialic acid content. Patients possessing the acidic variants of hCG in their sera had poor survival rate. Determination of the variants may be useful as a prognosis marker and in evaluating the efficacy of therapy.

Chorionic Gonadotropin↗

Cyclic nucleotide content and protein phosphorylation during maturation of Spisula oocytes.

Cyclic nucleotide levels in the oocytes of the surf clam Spisula solidissima were measured during germinal vesicle breakdown (GVBD) induced by fertilization. The level of cAMP and cGMP in untreated oocytes was 8.23 +/- 0.95 and 4.89 +/- 0.39 pmol/10(6) oocytes. The ratio of cAMP to cGMP ranged from 1.5 to 2.0. The cAMP level in Spisula oocytes fluctuated after fertilization and before GVBD. The cGMP level showed minimal fluctuation, with a tendency to decrease initially followed by a subsequent rise to the basal level in a nonsynchronous manner. These changes were not statistically significant. There was a general increase in protein phosphorylation during the period after fertilization and before GVBD. The greatest increase occurred with proteins of estimated molecular weights of 52, 18, and 12 kD, analyzed by gel electrophoresis and autoradiography.

Animals↗

Prevention of spontaneous degeneration of mouse oocytes in culture by ovarian glycosaminoglycans.

Mouse follicular oocytes cultured for more than 72 h in modified Krebs-Ringer solution in vitro undergo spontaneous segmentation to form "blastomeres" with or without nuclear fragments or chromatin. Glycosaminoglycans (GAGS) isolated from bovine follicular fluid (bFF) and added to the suspending medium at concentrations of 0.25 mg/ml or greater prevented the occurrence of spontaneous segmentation in isolated cumulus-free mouse oocytes in a dose-dependent manner. The active material was isolated from bFF by chromatography on Dowex 1-X2. The fraction eluted with 0.5 M NaCl possessed the greatest biological activity. Preparations of purified bFF-GAGs and hyaluronic acid and chondroitin sulfate from a commercial source prevented the occurrence of segmentation. Hyaluronic acid was more potent than chondroitin sulfate. The present results show that GAGs prevent segmentation of oocytes in vitro.

Animals↗

Identification of antigen in rat spermatogenic cells interacting with an anti-human sperm monoclonal antibody.

A monoclonal antibody (MAb) raised against human sperm protein, designated YWK-II, was used to determine the distribution of antigens in rat spermatozoa and rat testicular germ cells. By an indirect immunofluorescent method, the antibody localized over the rat spermatozoal head, except for the postacrosomal region. In paraffin sections of adult and immature rat testis, germ cells, at every developmental stage, and Sertoli cells stained, while interstitial cells and peritubular myoid cells remained unstained. When cocultures of Sertoli and germ cells were tested, only the germ cells stained intensely. Sertoli cells and peritubular myoid cells in cultures did not stain. In the epididymal sections, strong staining occurred with spermatozoa in the lumen and epididymal epithelial cells, with moderate staining in the myoid layers of epididymis. To determine the sperm antigen interacting with the YWK-II antibody, rat spermatozoa proteins were prepared and analyzed by an immunoblot technique. The monoclonal antibody interacted with a single protein, with an estimated molecular weight of 115,000, present in the cauda epididymal spermatozoa. Among the proteins of the caput epididymal spermatozoa, however, the antibody interacted with a major and a minor band with molecular weights of 115,000 and 88,000, respectively. On the other hand, with proteins prepared from the membrane fraction of adult and immature rat testis, the antibody reacted with two bands with estimated molecular weights of 88,000 and 115,000. In the lysate prepared from germ cells dissociated from Sertoli-germ cell cocultures, the antibody recognized only the 88,000 protein. The present results show that the YWK-II MAb interacts with two proteins with different molecular weights. The amount of the interacting proteins in spermatozoa varied with their location within the epididymis.

Animals↗

The antibody from an infertile woman that induces sperm agglutination interacts with rat testicular cells and sperm.

The serum obtained from an infertile woman induced a specific head-to-head agglutination of human and rat sperm. The immunoglobulin G (IgG) fraction of the serum was obtained and found to interact with the proteins of rat sperm in testis and epididymis. Using an indirect immunofluorescent method with rat sperm from vas deferens, we determined that the antibody recognized the protein on the convex and concave regions of the acrosome and over the entire tail. However, with testicular spermatozoa, the antibody recognized only the distal end of the tails. In paraffin sections of adult rat testis, sperm tails located at the luminal region of the seminiferous tubules stained intensely. Weak but significant staining also occurred on late spermatids. In the epididymal sections, staining was restricted to spermatozoa in the lumen. On the other hand, sections of testes from 25-day-old rats containing spermatogonia and early spermatocytes had a completely negative reaction. Testicular somatic cells, including Sertoli cells, peritubular myoid cells and interstitial cells, did not stain. To identify the testicular protein interacting with the antibody, adult rat testis proteins were prepared and analyzed by a sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) immunoblot technique. The antibody interacted with a protein with an estimated molecular weight of 82,000 in the testicular homogenate and particulate fraction, whereas the reaction was considerably weaker with the testicular cytosol fraction.

Animals↗

Properties of a monoclonal antibody interacting with human sperm.

A monoclonal antibody (MAB) interacting with human sperm was purified by ammonium sulfate fractionation and gel filtration on Sephadex G-200. The MAB designated as YWK-II belongs to the IgM class and is composed of mu heavy chains and kappa light chains. The MAB localized to the equatorial region of the sperm head, midpiece, and tail. The sperm antigen interacting with the YWK-II MAB was purified by immunoaffinity chromatography on Sepharose 4B conjugated with YWK-II antibodies. Two interacting proteins, estimated Mr of 60 and 72 kD, were identified.

Animals↗

Follicular fluid constituents influencing spontaneous maturation of cultured mouse oocytes.

Different concentrations of cyclic adenosine 3',5'-pyrophosphate (cAPP), adenosine, hypoxanthine, 17 beta-estradiol and testosterone were tested to determine their effect on spontaneous germinal vesicle breakdown (GVBD) in mouse oocytes. Individually, these compounds at concentrations of 200 microM or less, had no or minimal inhibitory effect on the spontaneous GVBD of mouse oocytes. Dibutyryl 3',5' cyclic adenosine monophosphate (dbcAMP) at a concentration of 50 microM or higher blocked GVBD but had no effect at concentrations of 20 microM or less. With dbcAMP at a concentration of 20 microM, cAPP blocked GVBD at a concentration of 10 microM. To affect inhibition of GVBD with adenosine, 17 beta-estradiol and testosterone and with hypoxanthine, concentrations of 100-200 microM and 500 microM, respectively, were required. In conclusion cAPP is the most potent compound among those tested in blocking spontaneous GVBD of mouse oocytes when combined with dbcAMP.

Adenosine↗

Suppression of natural killer cell activity by human seminal plasma in vitro: identification of 19-OH-PGE as the suppressor factor.

Human seminal plasma (SP) was found to contain a potent suppressor of human natural killer (NK) cell cytotoxic activity against the K562 erythroleukemia target in vitro. Pooled and filter-sterilized SP reduced the NK cell activity of normal blood donors in a concentration-dependent manner; strong reduction of target cell lysis was observed at a final dilution of 1/400. Possible toxic effects of SP on NK cells were ruled out, because donor leukocytes incubated in a final SP dilution of 1/100 remained greater than 99 percent viable as determined by trypan blue exclusion. SP did not affect the lysability of the tumor targets, but suppressed the cytotoxic activity of the effector leukocytes, an effect that was reversed after washing the leukocytes free of the SP. The suppressive action of SP was retained after heating to 95 degrees C for 10 min, but was removed after adsorption with activated charcoal (Norit-A). Lipids extracted from SP were tested for suppression of NK cell cytotoxic activity, and the active principle was identified with the acidic lipid fraction. Components of the acidic lipid fraction of a single freshly obtained SP sample were separated by high pressure liquid chromatography, and suppression of NK cell cytotoxic activity was found to be associated with the predominant prostaglandins (PG) in this fraction, 19-OH-PGE1 and 19-OH-PGE2. Suppression of cellular immune functions by SP has been described; however, the identity of the suppressor factor (or factors) is unknown. The 19-OH-PGE are present in high concentration in primate semen, and may minimize immunologic sensitization to sperm in females after insemination. In addition, these compounds may suppress NK cell antitumor and antiviral activities in the vagina, as well as in their tissues of origin.

Alprostadil↗

Müllerian inhibiting substance as oocyte meiosis inhibitor.

Müllerian inhibiting substance (MIS) inhibited the resumption of meiosis in both denuded and cumulus cell-enclosed rat oocytes in vitro. Spontaneous germinal vesicle breakdown was prevented in both types of oocytes treated by a purified MIS preparation at protein concentrations of 15 micrograms to 150 pg/ml. The inhibiting effect of MIS on the resumption of meiosis was dose dependent, reversible and cyclic AMP independent. Neither follicular-stimulating hormone, luteinizing hormone, progesterone, estradiol, nor testosterone acted significantly to influence MIS-mediated inhibition of rat oocyte maturation. In contrast, MIS had no influence on meiosis in the mouse, where other protein has been reported to inhibit the cumulus cell-enclosed oocyte in a cyclic AMP-dependent fashion. Thus MIS may be yet another inhibitor of oocyte meiosis, acting in the rat by a mechanism different from those inhibitors known, in the mouse ovary, to exert their effect in a cyclic AMP-dependent manner.

1-Methyl-3-isobutylxanthine↗

Requirement of serum components for the preservation of primordial germ cells in testis cords during early stages of testicular differentiation in vitro in the mouse.

Mouse gonadal primordia were isolated from embryos on the 11th day of gestation and cultured in vitro. They developed into either testes or ovaries after 7 days of culture in Eagle's minimum essential medium (MEM) supplemented with horse serum, whereas they did not differentiate in MEM alone. We studied how serum components are required for testicular development in vitro. When gonadal primordia were cultured in MEM alone for the first 1-3 days and subsequently in MEM supplemented with serum, testis cords developed while germ cells disappeared or only a few remained in the testis cords. In contrast, when serum was present in the medium during the first day of culture and omitted thereafter, germ cells were retained within testis cords. These results suggested that some serum component(s) is specifically required by germ cells independent of testis cord organization. Of more than 10 serum components tested, low and very low density lipoprotein fractions increased the number of germ cells in testicular explants.

Animals↗

Dibutyryl adenosine cyclic monophosphate regulates differentiation of type A spermatogonia with vitamin A in adult mouse cryptorchid testis in vitro.

Surgically prepared cryptorchid mouse testes containing only type A spermatogonia were cultured with (Bu)2cAMP in combination with vitamin A (retinol). Treatment with (Bu)2cAMP and retinol for 12-24 h and with basal medium for an additional 8 days stimulated mitotic activity in type A spermatogonia and induced differentiation of germ cells. However, (Bu)2cAMP alone did not induce differentiation of type A spermatogonia. Moreover, when cryptorchid testes were treated with (Bu)2cAMP for longer than 3 days in the presence or absence of retinol, differentiation of type A spermatogonia did not take place; disintegration of the seminiferous tubules occurred instead. When the cryptorchid testes were cultured for 24 h in a medium containing a fixed concentration of retinol and varying concentrations of (Bu)2cAMP from 0.001-0.4 mM, there was a dose-dependent increase in the number of differentiated and mitotic germ cells and type A spermatogonia. Likewise, at a fixed dose of (Bu)2cAMP and increasing concentrations of retinol, a dose-dependent increase in the number of differentiated and mitotic germ cells occurred. However, the number of type A spermatogonia was decreased. The addition of puromycin, cycloheximide, and actinomycin D to the medium completely blocked retinol-(Bu)2cAMP-induced differentiation of the germ cells. The present results suggest that cAMP and retinol trigger biochemical events promoting the synthesis of specific macromolecules involved in the proliferation and differentiation of type A spermatogonia.

Animals↗

Isolation and characterization of a rabbit sperm tail protein.

Rabbit sperm tails were obtained by a nitrogen cavitation procedure and separated by discontinuous sucrose gradient centrifugation. Proteins were extracted from sperm tails with 3[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) and separative disk electrophoresis and DEAE-trisacryl column chromatography. A protein with an estimated mol wt of 20.1 +/- 1.1 kD was isolated and found to be homogeneous by SDS-PAGE and designated as rSMP-B. The isoelectric point of rSMP-B was in the range of pH 4.4-4.7. The amino acid composition was determined, and glycine was identified as the N-terminal residue. Antisera were raised against purified rSMP-B. Using a peroxidase-antiperoxidase method, the rSMP-B antigen was located on the surface of the midpiece and tail of the sperm. Testis sections showed intense staining of late spermatids located within the seminiferous tubules. Adult male rabbits were inoculated with rSMP-B protein and Freund's adjuvant. The testis and epididymis of the immunized animals showed absence of sperm. The immunolocalization findings and the immunization data suggest that rSMP-B is formed in late spermatid and that it is an essential structural component of sperm.

Amino Acids↗

Testicular cell differentiation in fetal mouse ovaries following transplantation into adult male mice.

Testicular development is a complicated process involving differentiation and arrangement of several cell types. To analyze the process of testicular organization we examined the sequence of the appearance of testicular structures induced in fetal ovaries following transplantation. Fetal mouse ovaries on the twelfth day of gestation were transplanted beneath the kidney capsules of adult male mice. They continued to develop morphologically as ovaries until the eleventh day after transplantation, when seminiferous cord formation and testosterone production began in addition to follicle development (ovotestes). Between the eleventh and fourteenth day after transplantation, ovarian grafts frequently contained transitional structures consisting of Sertoli cells, pregranulosa cells, a third type of cells which show intermediate characteristics between Sertoli and pregranulosa cells, and oocytes enclosed by common basal lamina. Leydig cells or peritubular myoid cells were not found in the transitional area, whereas these cells were present around seminiferous cords composed only of Sertoli cells. Oocytes were absent or degenerated in the well-developed seminiferous cords. The present findings suggest that, in ovarian grafts, pregranulosa cells can differentiate into Sertoli cells, which are responsible for the organization of the seminiferous cords, degeneration of oocytes, and differentiation of other testicular somatic cell types.

Animals↗

In vitro induction of Xenopus oocyte maturation by 4-pregnene-17 alpha, 20 beta-diol-3-one.

Maturation in Xenopus oocyte is initiated by progesterone and other steroids. The possibility that a metabolite of progesterone is the active agent was explored. In the present study the 20 beta-dihydro metabolite of progesterone, 4-pregnene-17 alpha, 20 beta-diol-3-one was found to be as potent as progesterone at concentrations of 0.5 to 3.0 microM in inducing germinal vesicle breakdown (GVBD) in Xenopus oocytes. The order of relative potencies of the steroids tested were progesterone approximately equal to 17 alpha-hydroxy-20 beta-dihydroprogesterone greater than 17 alpha-hydroxy-20 alpha-dihydroprogesterone greater than 17 alpha-hydroxyprogesterone greater than 11-deoxycorticosterone. 17 alpha-hydroxy-20 beta-dihydroprogesterone induces the production of maturation-promoting factor leading to GVBD. The 20 beta-dihydro metabolite may be the active metabolite under in vivo condition.

17-alpha-Hydroxyprogesterone↗

Modulation of oocyte maturation by cyclic adenosine 3', 5'-pyrophosphate.

The germinal vesicle (GV) of follicle-enclosed oocytes in mammals remains arrested at the dictyate state of meiosis. Upon releasing the oocytes from the follicles, the meiotic process resumes, leading to dissolution of the GV (GVBD), suggesting that factors in the follicular fluid sustain the meiotic arrest of oocytes. In the present study the spontaneous resumption of meiosis was blocked by the addition of cyclic adenosine 3', 5'-pyrophosphate (cAPP) plus dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP), at final concentrations of 25 and 50 microM, respectively. These compounds were ineffective when added separately at these concentrations. None of the other related compounds tested with dbcAMP blocked GVBD. Bovine follicular fluid (BFF) was analyzed for inhibitors of GVBD. BFF was extracted with 70% ethanol and the ethanolic extract chromatographed on Dowex 1-X8 column. The fraction eluted with 0.1 N HCl markedly inhibited GVBD of isolated mouse oocytes in combination with dbcAMP. The active BFF substance and cAPP block spontaneous GVBD of mouse oocytes and may be related substances. The present study supports the thesis that meiotic arrest at the dictyate stage in oocytes is sustained by factors present in follicular fluid and may act in association with cAMP.

Animals↗