Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 38, Issue 4
Displaying 1-11 of 11 articles from this issue
  • Kohji SHIMODA, Eimei SATO, Hajime MIYAMOTO, Yutaka TOYODA
    1992 Volume 38 Issue 4 Pages 243-247
    Published: 1992
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    A modified lectin angiography method was applied to identify ovarian vasculature in 32-34 days old mice treated with gonadotropins. Blood was washed out by perfusion until the inferior vena cava became translucent. Horseradish-peroxidase (HRP)-conjugated Concanavalin A (Con A) solution (10-15 ml) was perfused into the systemic circulation via the left ventricle at the rate of 2-3ml/min. The animals were detained for a 30 min reaction interval. The lumina of the blood vessels were flushed with 5-10 ml of phosphate buffered saline (pH 7.4). The ovaries were excised and fixed by immersion in 4% paraformaldehyde and sectioned serially at thickness of 100, 200 or 400μm using a Microslicer. The binding of HRP-conjugated-Con A to endothelial cells was visualized by using 3, 3'- diaminobenzidine-4 HCl (DAB-4 HCl) reaction. By examining various sections the three-dimensional architecture of the vascular networks of the preovulatory Graafian follicles and corpora lutea can be established. Capillary wreaths of preovulatory Graafian follicles were identified in sections of ovaries removed 11 h after hCG injection. Then, capillary wreaths of the surrounding Graafian follicles became elevated due to the hypertrophic growth of the theca interna and extension of capillary branches into the follicles. Well-developed capillary networks of corpora lutea were found in ovaries removed 24 h after hCG injection. For these observations the 200μm-section was the most useful. Taken together, the present modified lectin angiography is a useful method for visualizing the microvasculature of mouse ovaries.
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  • Ryuichi MIURA, Ken NODA, Kunio SHIOTA, Michio TAKAHASHI
    1992 Volume 38 Issue 4 Pages 249-254
    Published: 1992
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    20α-Hydroxysteroid dehydrogenase (20α-HSD) catabolizes progesterone into 20α-dihydroprogesterone (20α-OHP), which is considered to be a biologically inactive metabolite. In this study, using a Xenopus oocyte translation system, changes in 20α-HSD mRNA content were assessed in the ovary of immature female rats bearing a single generation of corpora lutea, which were induced to form by treatment with pregnant mare serum gonadotropin (PMSG) at the age of 26 days and human chorionic gonadotropin (hCG) at the age of 28 days. The day of the hCG treatment was designed as day 0. Although both enzymatic activities and amount of immunoreactive 20α-HSD continued to increase up to day 9, the ovarian mRNA content remained relatively constant. The content of 20α-HSD mRNA in the ovary abruptly began to increase from day 9, while both enzymatic activity and the amount of immunoreactive 20α-HSD stayed almost constant. Thus, the biphasic change in 20α-HSD mRNA content did not parallel the change in either the enzymatic activities or the degree of immunoreactiv-ity. These results indicate that the mechanism for regulation of 20α-HSD biosynthesis in the ovary during a period of pseudopregnancy involves not only transcriptional but also post-transcriptional processes, probably including stabilization of the mRNA.
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  • Hiroshi IMAI, Clayton E WALTON, Jim T MARSHALL, Colin D NANCARROW
    1992 Volume 38 Issue 4 Pages 255-262
    Published: 1992
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Spermatozoa were incubated in PBS with [32P] labeled DNA fragments at 37 C. After 10 min incubations, DNA fragments were tightly bound to spermatozoa. Pre-treatment of sperm with 3% glutaraldehyde prevented this binding ability. Bound DNA could not be removed from sperm, even after treatment of the sperm with 0.1% Triton X-100 or DNase. After the induction of capacitation and the acrosome reaction of the spermatozoa, which are prerequisites for normal fertilization, more than 40% of the DNA fragments remained bound. The site of binding of DNA on spermatozoa was determined by membrane fractionation on discontinuous sucrose gradient and electronmicroscopic observation and found to be on the plasma membrane of the post-acrosomal region. Artificial insemination was carried out with spermatozoa incubated with DNA fragments, and 20 fetuses at 35 days of pregnancy and 11 lambs were collected. The possibility that DNA integration into the chromosome had occurred was determined by Southern hybridization. Under these experimental conditions, DNA did not integrate into the fetal genome in any sample tested..
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  • Nobuaki NISHIMURA, Noritoshi KAWATE, Tsutomu SAWADA, Junichi MORI
    1992 Volume 38 Issue 4 Pages 263-266
    Published: 1992
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Chemical castration by a single intratesticular injection of lactic acid, which is used for cattle, was studied in rats and dogs. The injection caused necrosis of the testicular tissue in both species. Lesions in the testes were observed as early as 24 h after the injection. Plasma testosterone levels declined rapidly after treatment and remained low thereafter. The results showed that a long-lasting and probably irreversible suppression of spermatogenesis can be brought about easily and immediately by an injection of lactic acid into the testes, and that the procedure could be used for the castration of dogs.
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  • Hiroshi IMAHIE, Shinji NITO, Takashi KOBAYASHI, Fumio ARIYUKI
    1992 Volume 38 Issue 4 Pages 267-270
    Published: 1992
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    To overcome the mouse 2-cell block, embryos resulting from in vitro fertilization (IVF) were co-cultured with established cell lines, namely, CHO-K1 (derived from Chinese hamster ovary), CHUIU (derived from Chinese hamster lung), and HEPM (derived from human embryonic palatal mesenchyme). Differences of embryonal development were observed in the different established cell line co-cultures. CHO-K1 were the best feeder cells for overcoming the block, while the CHL/IU exhibited this ability to a much lesser degree. HEPM was not able to overcome the block. Observations of the structure of these established cell lines, revealed variations in the morphology and density of the feeder cells. A large number of villi were observed on the surfaces of CHO-K1 and CHL/IU cells, and the cell density of CHO-K1 was much higher than that of CHL/IU. On the other hand, the surfaces of HEPM cells exhibited no villi or other structures. These findings suggest that the morphology and density of the feeder cells play a role in overcoming the 2-cell block.
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  • Helga SAUERWEIN, Heinrich H.D. MEYER, Dieter SCHAMS
    1992 Volume 38 Issue 4 Pages 271-278
    Published: 1992
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    This study describes 24 h profiles of growth hormone (GH) and of cortisol in male and female prepubertal calves before, during and after treatment with estradiol-17β (E2, silastic implants, 45 mg). Plasma concentrations of estradiol, testosterone and of insulin-like growth factor-1 (IGF-1) were also recorded. No significant (P<0.05) sex differences in GH, IGF-1 and in cortisol secretion were observed in untreated animals before E2-treatment. GH means, baselines and peak amplitudes increased during the treatment and remained elevated 1 week after removal of E2 in female calves, but were unchanged in male calves. In contrast, IGF-1 plasma concentrations were increased in both sexes during and after E2-treatment. In males maximal IGF-1 concentrations were higher than in females. Cortisol profiles showed a high individual variability and no consistant changes related to sex or E2-stimulation. Based on the observation that the male calves already had a significant testosterone secretion, we conclude that the observed sex differences of GH-secretion in response to E2 are related to endogenous testosterone. The E2-stimulated increase of IGF-1 plasma concentrations in males appears as related to an altered GH responsiveness rather than to changes in GH secretory patterns.
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  • Tetsuma MURASE, Kiyoshi OKUDA, Koji NIWA
    1992 Volume 38 Issue 4 Pages 279-284
    Published: 1992
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Cervical mucus was collected from Holstein cows in estrus. Frozen-thawed spermatozoa were separated from seminal plasma by swim-up method for 1h (ME spermatozoa) or penetration through fresh mucus for 50-70 min (MU spermatozoa). The separated spermatozoa were incubated for 5 and 11 h and assessed for sperm motility, viability and proportion of the acrosome reaction. Motility and viability were significantly increased in the separated spermatozoa than in those before separation. However, MU spermatozoa lost their motility and viability more rapidly than ME spermatozoa during incubation by for 11h. Proportion of the acrosome reaction was always higher in MU than in ME spermatozoa during the incubation. Exposure of ME and MU spermatozoa to lysophosphatidylcholine did not alter the proportion of the acrosome reaction during the incubation. These results indicate that cervical mucus selects only motile spermatozoa and induces sperm capacitation and the acrosome reaction during migration in it.
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  • Tsuneatsu MORI, Mito TAKADA, Mao Wu GUO, Etsuko MORI, Mamoru ISEMURA
    1992 Volume 38 Issue 4 Pages 285-292
    Published: 1992
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    TTK-1 cell line from human first trimester decidual tissue carried the surface markers such as HNK-1 (Leu 7) and HLA-DR. This cell line was strongly suspected of being human natural suppressor cell line of bone marrow or lymphoid tissue origin. In addition, the cells were found to express laminin on its cell surface, as examined by indirect immunofluorescence (IIF) test, and also secrete it into culture supernates as examined by enzyme linked immunoassay (EIA) and immunostaining. The addition of the supernates from this cell line into the murine embryo culture induced the trophoblastic outgrowth of embryos in vitro in the same manner as murine EHS sarcoma derived laminin did. This phenomenon was considered to be due to laminin in the UK-1 supernates because the effect was completely blocked by the addition of anti-laminin antibodies into the embryo culture.
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  • Tsuneatsu MORI, Yukio KIKKAWA, Ko NOMURA, Mika KANAYA, Shoichi TANAKA, ...
    1992 Volume 38 Issue 4 Pages 293-301
    Published: 1992
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    We report here that the supernates of mixed lymphocyte reaction (MLR) enhanced the proliferation of trophoblast cells. The analysis of the factors in MLR supernates revealed that a series of CSF family cytokines such as IL-3/GM-CSF and M-CSF, which seemed to be responsible for stimulation of the proliferation of trophoblast cells, were significantly raised in MLR supernates. At the same time, the effects of recombinant human (rh) IL-3/GM-CSF and M-CSF on the proliferation of the trophoblast cells were investigated. The rhlL-3/GM-CSF gave slight proliferation-promoting effects while the rhM-CSF showed stronger stimulating effect which was comparable to the MLR supernates. Continuous addition of the MLR supernates into the primary culture of trophoblast cells resulted in the establishment of trphoblast cell line. The cells showed the polygonal epithelium like appearances and reacted positively with anti-cytokeratin antibodies but not with anti-vimentin antibodies by means of indirect immunofluorescence (IIF) test.
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  • P.K. SURESHKUMAR, K. JANAKIRAMAN
    1992 Volume 38 Issue 4 Pages 303-307
    Published: 1992
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    In experiment I series of blood collection was done from goats between the day of estrus (D1) to day 19 (D19) for analysis of serum concentrations of progesterone, prolactin and LH using radioimmunoassays. Experiment II consisted of pituitary LH estimation at different phases of estrous cycle. Progesterone level started rising from D5 and reached peak on D15. Prolactin remained elevated during estrus, and considerably lowered on D11 to D19. On D19, the stage prior to estrus, levels of prolactin, progesterone and LH were all low. The peak of serum LH was observed on D1 before the ovulation and reached basal level on D19 of the cycle. Pituitary LH level differed appreciably between different stages of estrous cycle. Highest level prevailed during the follicular stage of the estrous cycle.
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  • Eimei SATO, Yutaka TOYODA, Sheldon J. SEGAL, Samuel S. KOIDE
    1992 Volume 38 Issue 4 Pages 309-315
    Published: 1992
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Maturation of surf clam oocytes can be induced with sperm, KCl, serotonin or calcium ionophore. We now report that the oocytes treated with trypsin can also induce germinal vesicle breakdown (GVBD). Oocytes incubated in 0.1% trypsin solution for 0.5 to 1 h, subsequently washed and incubated in control medium for 1 to 2 h underwent maturation. Treatment with glycosidase, DNase and RNase did not induce GVBD. The optimal condition for the induction of maturation was to incubate oocytes in 0.1 % trypsin solution for 1 h and to transfer the oocytes to a medium composed of 0.4 M Tris-HCl, pH 7.4, 20 mM CaCl2. The occurrence of spontaneous GVBD was dependent upon the presence of Ca2+ in the suspending medium. The optimal concentration of Ca2+ was 20 mM. To show that a membrane component is involved in sustaining maturation arrest, oocytes were incubated in a medium containing 1 M urea, 5 mM EDTA, 10 mM Tris-HCl, pH 7.4, for 30 min. The extracted membrane protein at final concentrations of 0.4 to 0.8 mg/ml blocked GVBD of trypsin treated oocytes. To identify the active component, the extracted membrane protein preparations were fractionated by gel filtration through a Sephadex G-100 column. The active principle had an estimated Mr of 18 kD based on elution position of standard proteins on gel filtration. The partially purified product was active at a concentration of 0.1-0.2 mg/ml. The present findings suggest that maturation arrest is sustained by an oocyte membrane protein and the resumption of oocyte maturation results from the hydrolysis of this substance(s) by a protease in the presence of Ca2+.
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