Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 40, Issue 2
Displaying 1-14 of 14 articles from this issue
  • Norihiro TADA, Masahiro SATO, Kazuhiro KASAI, Shyoso OGAWA
    1994 Volume 40 Issue 2 Pages 65-70
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    We substantially improved the cryosurvival rate of unfertilized mouse oocytes by em-ploying a pre-vitrification 2-step equilibration with sucrose (0.25 M and 0.5 M for 5 min each) at room temperature. As a result, 96.4% of the oocytes vitrified in a DPS-1 solution (2.75 M dimethylsulphoxide, 2.75 M propylene glycol, and 1.0 M sucrose) were recovered normally after warming, and 85.2% of the recovered oocytes were successfully fertilized by fresh spermatozoa. The in vitro developmental rate of these fertilized eggs into the blastocysts was 81.4% which was comparable to that (83.0%) of its solution control. Using this technique, we next examined the developmental rate of vitrified mouse oocytes that were fertilized by frozen mouse epididymal spermatozoa. Though the in vitro fertilization rate was relatively low (55.8%), the in vitro develop-ment of these fertilized eggs into the 2-cell stage was 93.5%, comparable to the solution control (95.6%). The in vivo developmental rate after transfer into recipient females was 54.2%, which was also comparable to the rate (57.8%) of eggs derived from fertilization between fresh oocytes and spermatozoa. We successfully demonstrated that fetal development is possible from cryopreserved oocytes and spermatozoa in mice.
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  • Norihiro TADA, Masahiro SATO, Kazuhiro KASAI, Shyoso OGAWA
    1994 Volume 40 Issue 2 Pages 71-78
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Transgenic mouse embryos at the 2-cell stage were vitrified by the method described by Nakagata with slight modifications [J Mamm Ova Res 1989; 6: 23-26 (in Japanese)]. In vivo cryosurvival was examined by transferring the vitrified-warmed transgenic embryos into recipi-ents. A total of 107 embryos out of 118 (90.7%) were recovered normally after warming; 45.8% of the normal embryos developed to full term. When the integration pattern of the transgene into the host genome and its expression level were compared between offspring derived from vitrified and unvitrified (control) transgenic embryos, there was no significant alteration with respect to each parameter. From these results, it was indicated that the vitrification procedure we employed does not affect the expression level of the transgene or its integration pattern in chromosomal DNA.
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  • Hiromichi TAMADA, Mika OHNO, Tsutomu SAWADA, Junichi MORI
    1994 Volume 40 Issue 2 Pages 79-84
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Immunohistochemical staining of insulin-like growth factor-I (IGF-I) in the corpora lutea (CL) during estrous cycle and pregnancy was compared among mice, rats, hamsters, guinea pigs and cattle. During the estrous cycle, while clear staining was not detected in the hamster CL, the CL of the mouse, rat and guinea pig showed modest staining except for the mouse CL 1 day after ovulation which showed very weak staining. In contrast, immunoreactive IGF-I in the bovine CL showed distinct changes. While very weak staining was noted during the follicular and early luteal phases, the intensity increased markedly thereafter, peaking at the late luteal phase. During preg-nancy, the mouse and rat CL showed positive staining, and the intensity increased at late preg-nancy, especially in the rat CL intense focal staining was noted. In the hamster CL, clear positive staining was detected only at late pregnancy. In contrast, in the guinea-pig and bovine CL, al-though intense staining was detected during mid-pregnancy, the staining decreased at late preg-nancy. The results revealed species differences in the accumulation of immunoreactive IGF-I in CL.
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  • Shiro KURUSU, Tomoko OHYAMA, Hitoshi WATANABE, Kazuyuki MIYAMOTO, Kohj ...
    1994 Volume 40 Issue 2 Pages 85-89
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    To investigate the role of locally produced prostaglandin F (PGF) in luteal function, luteal PGF and plasma levels of progestins, progesterone, and 20α-hydroxypregn-4-en-3-one (20α-OHP) were measured during pseudopregnancy in PMSG-primed immature rats. Plasma pro-gesterone increased from the basal level at 0700 h on day 1 of diestrus (D1) and reached the peak level at 1800 h on D2. The plateau level was maintained until D6 and decreased to the basal level on D7. 20α-OHP in plasma increased temporally from D1 to D3 and showed a gradual increase until D8. PGF content in the corpus luteum was low (5.2-7.7 pg/CL) until D3 and increased significantly (P<0.05) on D4 and D5. After that, luteal PGF declined to a basal level as low as that before D3. These results show that a rise in luteal PGF preceded the decline of progesterone levels and suggest that the increase in endogenous PGF may initiate the functional regression of the corpus luteum in immature pseudopregnant rats.
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  • Koichiro KANO, Takashi MIYANO, Seishiro KATO
    1994 Volume 40 Issue 2 Pages 91-97
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    The present study was carried out to examine ovarian response to a combination of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), oocyte maturation in vivo or in vitro, and the in vitro fertilization rate of in vivo and in vitro matured oocytes of prepubertal Jinhua pigs at 70-75 days of age. Eighteen of the 19 pigs treated with gonadotropins had follicles larger than or equal to 7 mm in diameter, and 4 pigs ovulated 36-42 h after hCG injection. The rate of nuclear maturation in the oocytes from large ( ?? 7 mm) follicles (81%) was similar to that in oocytes from small (2-6 mm) follicles of control animals and cultured in vitro (83%), and the rate of sperm penetration in oocytes matured in vivo (88%) did not differ significantly from that oocytes matured in vitro (97%). However, the rates of male pronucleus formation and monospermy were significantly higher in oocytes matured in vivo (89% and 31%) than in oocytes matured in vitro (56% and 9%). These results show that follicular oocytes in prepubertal Jinhua pigs at 70-75 days of age can mature equally in vivo and in vitro, but the fertilizability of oocytes matured in vivo is superior to that of oocytes matured in vitro.
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  • Yuji TAKAHASHI, Hiroshi IMAHIE, Kae KAMIO, Sadahiro AZUMA, Eimei SATO, ...
    1994 Volume 40 Issue 2 Pages 99-105
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    The functional role of the cumulus oophorus during fertilization was examined. It was found that the sperm concentration necessary for successful fertilization was markedly different between cumulus-intact and cumulus-free oocytes; only 5 sperm/μl were required for the maxi-mum level of fertilization of cumulus-intact oocytes, while 150 sperm/μl were needed for cumu-lus-free oocytes. Fertilization rate did not increase by co-culturing cumulus-free oocytes with detached cumulus cells in the fertilization medium. On the other hand, supplementing the fertili-zation medium with soluble factor(s) vortexed out of cumulus-oocyte complexes significantly improved the rate of fertilization (56% in the treated group vs 36% in controls; P<0.05) when spermatozoa were preincubated in BSA-supplemented medium. No significant effect, however, was observed when spermatozoa were preincubated in protein-free medium. These results strongly suggest that soluble factor(s) sustained in cumulus-oocyte complexes promote fertilization, and the integrity of the three dimensional structure of the cumulus oophorus is important for efficient sperm-egg interaction.
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  • Hiroshi SUZUKI, Mamoru TOGASHI, Yoshiyuki MORIGUCHI, Jiro ADACHI
    1994 Volume 40 Issue 2 Pages 107-116
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    A decline in fertility rate is observed in aged mice of an inbred IVCS strain. This study was conducted if the rate of in vitro fertilization is also decreased with age. If male and female mice of the same age were used for natural matings, the highest (7.0) reproduction efficiency (no. of neonates / no. of mated mice) was obtained at 4 months of age. No live neonates were born from dams over 9 months of age. The frequency of post-implantation embryonic death was as high as 43-63% at 6-8 months of age.
    From females at 9 or 10 months of age, 6.3 or 2.0 fertilized eggs were recovered after natural matings. Of these eggs, 92-100% developed into blastocysts in culture. Superovulation was induced in females at age of 2-11 months, and 51-95% of the recovered oocytes were fertilized in vitro. The majority of these embryos passed through the 2-cell stage within 24 h and 70-99% of them developed to the blastocyst stage 96 h after insemination. Of these embryos obtained from 5, 10 or 11 months old mice, 52, 25 or 21% of the transferred embryos were developed to neonates. These results indicate that most of eggs are still viable, even if they are derived from "infertile" aged mice. Thus, infertility occurring in aged mice of this strain over 9-months of age is primarily due to the dysfunction of the reproductive tract rather than the low quality of eggs. In addition, neonates can be obtained from aged mice by transferring in vitro fertilized embryos to younger recipients.
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  • Hitoshi USHIJIMA, Tetsuo ETO
    1994 Volume 40 Issue 2 Pages 117-124
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    This study investigates the remodeling of a bovine donor nucleus after fusion with a parthenogenetically activated oocyte and its developmental ability following embryo transplant in cows. Recipient ooplasts were prepared by enucleating in vitro-matured oocytes after culturing for 24-27 h. These enucleated oocytes were activated with an ethanol treatment and then electrically fused with a 16-cell stage donor blastomere at 0, 6, or 12 h after activation. Nuclei that were fused 0 h after activation (0-h nuclei) and fixed 2 h later had a significantly higher (P<0.05) frequency of abnormal karyomorphism (26%) than the 6-h nuclei (2%). Sixty-five percent of the 6-h nuclei showed nuclear remodeling, i.e., nucleus swelling, whereas corresponding 12-h nuclei showed only 8%. Development into the blastocyst stage was significantly higher (P<0.05) in the 6 h group (10%) than that occurring in both the 0 h and 12 h groups (2% and 2%). Eight blastocysts obtained from embryos fused 6 h after activation were nonsurgically transferred to 6 recipient cows (1-2 embryos each), with 4 recipients becoming pregnant and 3 producing a single healthy offspring. Results indicate that an activated bovine ooplast has the ability to reprogram a donor nucleus.
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  • G. CARBONNEAU, Marc-Andre SIRARD
    1994 Volume 40 Issue 2 Pages 125-132
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    We evaluated the importance of the attachment of the oocyte to the granulosa layer, using co-culture of follicular hemi-sections with groups of 10 COCs (cumulus-oocyte complexes) or 10 DOs (denuded oocytes) and oocytes cultured within their own half-follicle. Various prod-ucts acting on protein phosphorylation (IBMX, PMA, H-7) and cellular differentiation (estrous serum, FSH, LH or estradiol) involved in follicular and nuclear maturation were also used to evaluate their effect on the capacity of the follicular wall to inhibit meiosis resumption. When oocyte were cultured within their own half-follicles, none of the treatments used in this study could significantly reverse the inhibitory action of the follicular wall on the meiotic resumption. When the contact between the oocyte and the hemi-section was ruptured (co-culture of COCs and DOs), oocytes responded differently according to whether they were surrounded or not with their cumulus cells, and inhibition of meiosis was lowered particularly with LH and PMA with COCs. These results show that the communication junctions between the oocyte and the follicle are important for the inhibitory action of the follicle and this contact between both overcome the effect of any treatment applied.
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  • Hiroaki HARA, Shin-ichi SASAKI
    1994 Volume 40 Issue 2 Pages 133-139
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Changes in proliferation of rabbit placental cells during pregnancy were evaluated by bromodeoxyuridine (BrdU) incorporation into the DNA. Cells were isolated and purified from rabbit placental tissues at different stages of pregnancy (day 11, 17, 23 or 29). BrdU incorporation as detected by an ELISA, indicated that cells isolated on day 11 were mitotically active in growth factor free medium. However, BrdU incorporation decreased as pregnancy progressed; this paral-leled the change in placental weight gain. These results suggest that rabbit placental growth may be regulated not only by maternal factors but also by local factors.
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  • Joachim BRAUN, Yasunari MORIUCHI, Satomi NAKAGAWA, Shin-ichi HOCHI, Ak ...
    1994 Volume 40 Issue 2 Pages 141-147
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Follicular development, incidence of double ovulations, and steroid hormone levels were examined in Hokkaido Native Pony (HNP) and Thoroughbred (TB) mares during control and FSH-stimulated cycles. Daily FSH treatment (HNP 8 mg FSH/day and TB 16 mg FSH/day; i.m.) was initiated on Day 7 of the estrous cycle following PGF, administration on Day 6 (ovulation = Day 0). Follicular development and ovulation was monitored by transrectal ultrasonography. Peripheral blood concentrations of progesterone and estradiol-17β were evaluated by EIA. FSH treatment had no effect on the maximum follicular diameter attained during the estrous period and the diameter of the biggest follicle before ovulation. The incidence of a second follicle >10 mm before ovulation was 0/12 (HNP) and 7/9 (TB) during control cycles. During FSH-stimulated cycles, 2/7 (HNP) and 5/6 (TB)mares had a second follicle >10 mm before ovulation. No double ovulations could be observed in HNP mares during 16 control cycles and 12 FSH-stimulated cycles. In TB mares, double ovulations occurred in 2 out of 14 control cycles and 6 out of 11 FSH-stimulated cycles (P<0.05). Progesterone levels after ovulation were not influenced by FSH treat-ment. Mean concentrations of estradiol-17β during estrus were not different in control and FSH-stimulated cycles. However, maximum estradiol-17β levels were higher in control cycles (6.4 pg/ ml vs. 4.1 pg/ml; P<0.05). It is concluded that treatment with daily injections of FSH is effective in inducing double ovulations in Thoroughbred mares.
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  • Akihiko KUDO, Min Kyun PARK, Seiichiro KAWASHIMA
    1994 Volume 40 Issue 2 Pages 149-158
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Expression of rat GnRH receptor mRNA in female rat pituitary was examined by in situ hybridization using 35S-labeled antisense RNA probe. Specific signals were detected in the pars distalis. For detailed analysis of the cellular expression of GnRH receptor mRNA, the pituitaries were double-stained by in situ hybridization of GnRH receptor mRNA and antisera against some pituitary hormones (LH, FSH, TSH and prolactin). The expression of GnRH receptor mRNA was detected in all the immunoreactive gonadotropes, but was not detectable in TSH or prolactin cells. Cellular expression of GnRH receptor mRNA in immunoreactive gonadotropes was compared during the estrous cycle and after ovariectomy. In the pituitary of normal cycling rats, the mean value of the cellular expression of the mRNA in estrus was significantly smaller than those of other phases of the cycle. In ovariectomized rats, the cellular expression was significantly in-creased 4 days after the operation as compared with normal cycling rats, and the elevated level of cellular expression was maintained until 21 days after ovariectomy. These results suggest that GnRH receptor expression in the pituitary is preferentially localized in gonadotropes, and the extent of cellular expression varies during the estrous cycle and after ovariectomy.
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  • Akiko YAMAJI, Yasuo KISO, Masatsugu SUZUKI, Mayumi YOKOYAMA, Fumihiko ...
    1994 Volume 40 Issue 2 Pages 159-165
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    Wild Sika deer (Cervus nippon yesoensis) are seasonal breeders and their reproductive behaviors change annually. Since reproductive activity is regulated by gonadotropins, gonadotro-pic cells (LH and FSH cells) in pituitaries in 19 male and 18 female wild Sika deer, which were collected from Ashoro town in Hokkaido, were investigated by immunohistochemical methods. Gonadotropic cells remarkably changed between August (the end of non-mating season) and October (mating season); their distribution was wider and their immunostaining reactivity was more intensive in October than in August in males and females. We found that gonadotropic cells existed also in the pars intermedia and that these cells showed seasonal changes similar to those in the pars distalis. The significances of such seasonal changes in gonadotropic cells in the wild Sika deer were discussed.
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  • Hiroshi TAKANO, Satoru SHIMIZU, Keisuke KOYAMA, Chiaki KOZAI, Yoko KAT ...
    1994 Volume 40 Issue 2 Pages 167-170
    Published: 1994
    Released on J-STAGE: May 15, 2008
    JOURNAL FREE ACCESS
    The present study was undertaken to examine whether the bovine nuclear transferred embryos produced by in vitro culture system could develop to young after transfer to recipients. Donor 8-to 16-cell stage embryos were obtained by in vitro maturation, in vitro fertilization and in vitro culture. Follicular oocytes maturated in vitro were used as recipient oocytes for nuclear transfer. Fusion of donor blastomere with recipient oocyte was induced by electric stimulation at 44 to 46 h of maturation. Of the 398 manipulated oocytes, 261 (66%) were fused with donor blastomere. Twenty-five (10%) embryos which developed into morula to blastocyst stage after co-culturing with cumulus cells in vitro. Fifteen embryos were transferred nonsurgically, 1 to 3 embryos per recipient, into the uterine horn of 9 heifers or cows on Day 7 to 9. The pregnancy rate was 44% (4/9) on Day 35 to 40. One recipient aborted on Day 60 to 70 and another on Day 50 to 60, respectively. Two normal offsprings were produced from the remaining 2 recipients. The production of calves for embryos transferred was thus low (13%). Nuclear transfer bovine em-bryos produced in vitro system developed to full-term.
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