Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 39, Issue 4
Displaying 1-15 of 15 articles from this issue
  • Hiroshi NAGASHIMA, Hirohito YAMAKAWA, Nobuo NOMURA, Ryo HARASAWA, Tets ...
    1993 Volume 39 Issue 4 Pages 259-273
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Transgenic mice carrying activated v-Ha-ras under the control of mouse mammary tumor virus (MMTV) long terminal repeat were produced. The introduced gene was transmitted in the Mendelian fashion from a founder transgenic male to a total of 49 first-, 45 second- and 16 third-generation progeny, with a stochastic tumor occurrence ranging from 39 to 56%. The most common primary lesions were hyperplasia, adenoma and/or adenocarcinoma of Harderian lacrimal, salivary and mammary glands. Majority of primary tumors in the females occurred during their nulliparous state, though the MMTV promotor is known to be responsive to pregnancy/lactation-related hormones. The second-generation progeny segregated into 2 subgroups characterized by high (77%) and low (9%) risk of tumorigenesity, suggesting an influence of genotype on the appearance of the proliferative disorders caused by the activated v-Ha-ras oncogene.
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  • Yutaka FUKUI, Masashi FUJII, Yumiko TASHIRO
    1993 Volume 39 Issue 4 Pages 269-273
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    During the non-breeding season, a total of 176 Suffolk ewes at 3 sheep farms were treated with either a self-made, progesterone-impregnated intravaginal sponge (P sponge) or controlled internal drug release (CIDR®) for 9 days and an intramuscular injection of pregnant mare's serum gonadotropin (PMSG) 1 day before the cessation of progesterone treatment. At 44-52 h after treatment, 174 ewes were inseminated with 0.2, 0.1 or 0.05 ml of frozen-thawed ram semen per uterine horn with the aid of a laparoscope. There was no significant difference in lambing rate between P sponge (54.2%) and CIDR®(61.5%). The insemination dose did not also affect the lambing rates in both P sponge-treated (51.7, 66.7 and 44.4% for 0.2, 0.1 and 0.05 ml, respectively) and CIDR®-treated (58.1, 59.4 and 67.9%, respectively) ewes. Prolificacy was not significantly different between progesterone treatments nor among insemination doses. Lambing rates and prolificacy at the 3 sheep farms were not also significantly different. These results indicate that even a low insemination dose (0.05 ml) can result in similar rates of lambing compared with 0.1 or 0.2 ml of insemination dose per uterus in Suffolk ewes treated with either P sponge or CIDR® during the non-breeding season.
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  • Hiroharu MATSUSHITA, Kenji TUCHIHASHI, Shuichi ASANO, Toshiho NISHITA, ...
    1993 Volume 39 Issue 4 Pages 275-279
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    An bovine, amniotic-fluid protein, (bAP), was purified from amniotic fluid by gel filtration, anion exchange chromatography and isoelectric focusing. The isolation was monitored by a double immunodiffusion with an antiserum raised against amniotic fluid antigens and absorbed with maternal and fetal sera. The molecular weight of an amniotic fluid protein isolated was estimated as 60, 000 by SDS-PAGE, and the isoelectric point by isoelectric focusing was 3.4-3.8.
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  • Xihe LI, Setsuo IWASAKI, Tatsuo NAKAHARA
    1993 Volume 39 Issue 4 Pages 281-286
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    A part of trophectoderm cells was dissected microsurgically from bovine blastocysts fertilized in vitro for sex determination by chromosome analysis and its effects on the proliferation of inner cell mass (ICM) of fresh and frozen-thawed embryos were investigated by a differential fluorochrome staining technique. In total, 188 blastocysts were dissected and 85.5% (65/76) of embryos cultured in vitro for 24 h was repaired. Mitotic index (14.9%) and sexing rate (57.1%) of unrepaired embryos after dissection were lower than those (19.9% and 66.0%) of repaired embryos but not significant. Further culture resulted in significant decreases in the repair rates (48 h: 37.5%, 72 h: 32.5%, 96h: 28.6%) but not in embryos co-cultured with cumulus cells monolayer (96 h: 81.8%). The proliferation of ICM cells was inhibited up to 72 h after in-vitro culture and it was promoted thereafter. Survival rates of frozen-thawed embryos, which was repaired after the dissection decreased significantly from 12 h after the initiation of in-vitro co-culture. Total and live ICM cell numbers increased slightly with in-vitro culture but not significant. These results show that the proliferative ability of ICM cells becomes temporarily arrested by the dissection of trophectoderm cells but is resumed by the subsequent in-vitro culture. In addition, it was suggested that the applicable time of transplantation of dissected and frozen-thawed embryo is within 2 h after thawing.
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  • Shoji NAGATANI, Kiyoshi OKUDA, Masataka YUHARA
    1993 Volume 39 Issue 4 Pages 287-291
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Changes in the luteinizing hormone-releasing hormone-degrading activities (LHRH-DA) in the anterior pituitary and the hypothalamus after ovariectomy (OVX) were examined in mice. Mice were decapitated at 1000 h on diestrus day 1 (D1) or on day 7, 14 and 21 after OVX. Another group of mice was also killed at 1000, 1200, 1500 and 1700 h on D1 or on day 7 after OVX to determine the change in LHRH-DA during the day. Trunk blood, the anterior pituitary and hypothalamic tissue were collected. LHRH-DA were determined by incubating the exogenous LHRH with tissue extracts at 37C for 30 min. The changes of LHRH-DA in the anterior pituitary were inversely correlated with those of LH secretion: LHRH-DA decreased after OVX when plasma LH levels increased, but did not change when plasma LH levels were kept at a stable level throughout the day on D1 or day 7 after OVX. The present results suggest that the LHRH-DA in the anterior pituitary is involved in its hypersensitivity to the LHRH stimulation after OVX.
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  • Noboru MURAKAMI, Osamu AKAGUMA, Takayuki MURAKAMI, Kouei SUGITA, Miki ...
    1993 Volume 39 Issue 4 Pages 293-299
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Intact preantral follicles were isolated from immature rat ovaries by enzymatic dissociation and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. The globular shape of the follicles was maintained during culture for 96 h on agar-coated plates. When the follicles were placed near the center of the plates, follicles assembled, being in close contact with each other 24 h after culture and after 48 h large spheroidal tissues had formed. Histological examination revealed that these tissues possessed many intact follicles with preserved oocytes and many differentiated cells filled the gaps between follicles. The layer resembled squamous epithelium covered this spheroidal tissue. Culture in serum-free DMEM resulted in follicle assemblage but not formation of ovary-like multitissue spheroids (ovarioids). On the other hand, fetal calf serum induced ovarioid formation in a concentration-dependent manner as did the fraction containing the 30-100-kDa protein from fetal calf serum. Protein and RNA synthesis inhibitors inhibited ovarioid formation. The formation of ovarioids from isolated ovarian follicles may prove to be a useful model to study the ovarian cell-cell or tissue-tissue attachment and the cell differentiation in vitro.
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  • Tomohiro KONO, Miyoko OGAWA, Tatsuo NAKAHARA
    1993 Volume 39 Issue 4 Pages 301-307
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Thymocytes from fetal, newborn, and adult mice were transferred into enucleated oocytes by standard micromanipulation. The developmental ability of oocytes receiving a thymocyte was examined in vitro. Most of the reconstituted eggs were successfully activated and formed a pronucleus-like structure. The percentage of reconstituted eggs developing to the 2-cell stage was high, 75-84%, but development of embryos to more advanced stages was limited. Only 8 and 1% of the reconstituted eggs receiving a thymocyte from fetal and newborn mice developed to the blastocyst stage, respectively. None developed to the 8-cell stage when a thymocyte from adult mice was transferred. Analysis of newly synthesized proteins of the reconstituted embryos showed that 68-70 kDa heat shock proteins, initial markers of embryonic genome activation, were synthesized at the late 2-cell stage.
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  • Kazuei ITO, Yasuhisa YASUDA
    1993 Volume 39 Issue 4 Pages 309-317
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    In a previous study, we suggested that bovine EPF had a molecular weight of 21.5 KD because a 21.5 KD polypeptide was not found in the nonpregnant serum, and the isoelectric point was near 5.0 by 2D SDS-PAGE using non-equilibrium pH gradient electrophoresis (NEPHGE). We extended the study to characterize the biochemical nature of purified bovine EPF. As a result, the isoelectric point of bovine EPF turned out to be 6.3 by 2D SDS-PAGE using isoelectric focusing (IEF). Also, the purified EPF was not reduced by the addition of 2-mercaptoethanol. Accordingly, it was inferred that bovine EPF is a monomeric peptide. Amino acid analysis of EPF was attempted, but a definitive sequence could not be confirmed. In the present study, the crude anti-EPF lgG fraction was purified by adsorption with CNBr-activated Sepharose 4B coupled with nonpregnant bovine whole serum. The purified anti-EPF IgG decreased the rosette inhibition titer of pregnant serum from 6 to 3. The Sepharose 4B affinity column coupled with anti EPF-IgG effectively isolated the EPF from pregnant bovine serum.
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  • Teruhiko WAKAYAMA, Yuko MARUYAMA, Kenkichi IMAMURA, Katsuhiro FUKUTA
    1993 Volume 39 Issue 4 Pages 319-323
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Penetration of the zona pellucida of heterologous eggs by spermatozoa of Japanese field vole, Microtus montebelli, was examined in vitro. Spermatozoa from the vole epididymis were more active and survived longer in mKRB medium than did mouse spermatozoa. When vole spermatozoa were used for insemination, they passed through the zona pellucida of mouse and hamster eggs at a high rate. Although many spermatozoa invaded the perivitelline space of such eggs, penetration of the vitelline membrane was not confirmed. The present result suggests that vole spermatozoa have a high ability to penetrate zona pellucida of heterologous eggs.
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  • Toshio TSUBOTA, Hiroshi KANAGAWA
    1993 Volume 39 Issue 4 Pages 325-331
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Ovaries having corpora lutea and uteri of 6 female Hokkaido brown bears (Ursus arctos yesoensis), which were legally killed by hunters from October to December in Hokkaido, Japan, were examined macroscopically and histologically. Four of the 6 female bears had unimplanted embryos in the uterine lumen during the period of delayed implantation between October 19 and November 20. Ovaries of bears which had unimplanted embryos contained the corpora lutea with extensive vacuolation or numerous granules in the cytoplasm of luteal cells. These bears had well-developed uterine glands which had tall epithelial cells with vacuoles in the cytoplasm of glandular cells and coagula in the glandular lumina. It is concluded that Hokkaido brown bears exhibit obligate delayed implantation and that this period of delayed implantation continues at least until late November.
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  • Katsushi SUZUKI, Yoji HAKAMATA, Hiroetsu SUZUKI, Kazuyoshi TAYA, Shuji ...
    1993 Volume 39 Issue 4 Pages 333-346
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    In order to determine the initial alterations in histopathological and hormonal milieu in the male hypogonadism mutant rats (gene symbol: hgn, a single autosomal recessive trait), postnatal morphological development of the affected testis, and testicular testosterone (T) contents, plasma concentrations of T, follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin (PRL) were examined in the hgn/hgn and phenotypically normal male littermates (+/?) on days 0 to 21 after birth. The processes of postnatal differentiation of seminiferous tubules from primitive sex cords; inhibition of germ cells entering mitosis, proliferation and distribution of the Sertoli cells, colonization of the gonocyte and elongation of the tubules, were defective in the hgn/hgn testis. These histopathological alterations were present at birth. These defects might result in the loss of normal spermatogonia, leading failure of spermatogenesis in the adult. Severe progressive degenerations were observed in the tubular diameter and cells within the tubules on day 7 and afterwards, and the affected status seen in the adult was achieved by day 18. There have been marked elevations in the plasma FSH and LH levels from early postnatal age in the hgn/hgn male rat, while testicular and plasma T contents and PRL values were almost comparable between hgn/hgn and +/?.
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  • Taku NAGAI, Katsuhiro MIURA, Kazuhiro KIKUCHI, Naomichi OKAMURA
    1993 Volume 39 Issue 4 Pages 347-352
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Experiments were conducted to assess the effects of caffeine on the ability of frozen-thawed boar epididymal spermatozoa to penetrate oocytes that had been matured in culture. The spermatozoa were preincubated in modified TCM199 medium and subsequently incubated for 2 h in either a fertilization medium with 2 mM caffeine or in a fertilization medium without caffeine. Then a sample of the spermatozoa in the caffeine-containing medium was introduced into another caffeine-containing medium that contained matured oocytes, while another sample was introduced into a medium with matured oocytes without caffeine. This procedure was repeated for the spermatozoa that had been incubated in the caffeine-free medium. The two sets of manipulations resulted in higher rates of penetration of oocytes inseminated in the caffeine-containing medium than of oocytes in the caffeine-free medium. Furthermore, when oocytes with attaching spermatozoa were washed and transferred to caffeine-free medium after 1, 2 and 3 h of incubation in the presence of caffeine, penetration rates were 14%, 76% and 94%, respectively. Addition of caffeine to the fertilization medium caused as much as a 1.4-fold elevation in levels of cyclic AMP in sperm, the effect being maximal within about 0.5 h of incubation. These results indicate that during 2 to 3 h of incubation with oocytes in the caffeine-containing medium, the spermatozoa become highly fertile, and that a high concentration of cyclic AMP in sperm might not have any direct effects on the ability of sperm to penetrate oocytes.
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  • Masao YAMAMOTO, Tatsuyuki SUZUKI, Masato OOE, Mitsuhiro TAKAGI
    1993 Volume 39 Issue 4 Pages 353-356
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Abstract. This study was designed to establish the efficacy of 30% polyvinylpyrrolidone solution (PVP; molecular weight 40, 000) as a solvent for follicle stimulating hormone (FSH) as a single intramuscular (i.m.) injection for superovulating beef cattle and to compare its efficacy in inducing superovulation with descending doses of FSH dissolved in saline. In experiment 1, 16 Japanese Black cows were randomly alloted to 2 treatment groups. Each of the 8 cows in the PVP group or saline group were given a single i.m. injection with 30 mg of FSH dissolved in either 10 ml of PVP or 10 ml of saline, respectively. In Experiment 2, the efficacy of a single FSH injection was compared between 15 Japanese Black cows and 12 heifers. In experiment 1, a larger number of ova and embryos was observed with FSH dissolved in PVP than in saline (9.8±2.4 versus 0). In experiment 2, the average number of embryos and transferable embryos in the control group (12.0±8.4 and 6.0±4.7) and experimental cows (10.4±7.6 and 5.2±2.2) were significantly higher (P<0.01) than in heifers (2.8±3.1 and 1.1±1.9).9).
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  • Naohito KUSAKARI, Mutsuo OHARA
    1993 Volume 39 Issue 4 Pages 357-361
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Abstract. Suffolk rams were fed melatonin pellets in 2 experiments to control their reproductive seasonality. In Exp. 1, 6 rams were randomly divided into 2 groups: Group 1M rams (n=3) were fed 4 mg of melatonin daily for 100 days from February 17 (Day 0), and Group 1C rams were untreated. Scrotal circumference in Group 1M was significantly larger than that in Group 1C on Day 45 and 75 (P<0.05). Libido tests, performed on Day 104, indicated that mating behaviour in the Group 1M rams was more active compared to Group 1C rams. Group 1M rams had more sperm per ejaculation than control rams. In Exp. 2, 4 rams were divided into 2 groups: Group 2M rams (n=2) were fed 4 mg of melatonin daily for 45 days from May 16 (Day 0), and Group 2C rams were untreated. The timing of seasonal development in scrotal circumference was advanced in Group 2M rams; the circumference reached maximum on Day 45. However, an abrupt decrease in scrotal size was seen in Group 2M 45 days after cessation of melatonin feeding. These results suggest that melatonin feeding either from February or May can increase the reproductive activity of Suffolk rams during periods of seasonal regression.
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  • Kouichi JISHAGE, Hiroshi SUZUKI
    1993 Volume 39 Issue 4 Pages 363-367
    Published: 1993
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Capacitated mouse spermatozoa were stored for 24 to 72 h under the following conditions: 4C in air, 24C in air, 24C in 5% CO2 in air, or 37C in 5% CO2 in air. None or few of the oocytes inseminated with the capacitated spermatozoa stored for 24 h at 37C in 5% CO2 in air or 4C in air were fertilized. Fertilization rates of oocytes inseminated with capacitated spermatozoa stored for 24 h at 24C in 5% CO22 in air and 24C in air were 46% and 37%, respectively. When capacitated spermatozoa were stored at high concentrations, over 2×104 cells/μl, 87% of the oocytes were fertilized. Seventy-one percent of the fertilized eggs developed to term after transfer into the oviduct of recipients. These results indicate that capacitated mouse spermatozoa stored for 24 h at 24C can fertilize ova that subsequently develop normally through pre-and post-implantation stages.
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