Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 45, Issue 3
June
Displaying 1-7 of 7 articles from this issue
Original Articles
  • Tie Zhu An, Kana Ikegami, Keisuke Edashige, Takashi Sakurai, Magosabur ...
    1999 Volume 45 Issue 3 Pages 191-196
    Published: June 20, 1999
    Released on J-STAGE: March 27, 2000
    JOURNAL FREE ACCESS
    To develop a model for utilizing germ cells collected from dead animals, male mice of ICR and BDF1 were euthanized and refrigerated at 4-6 C for various periods, then the epididymal spermatozoa collected from the carcasses were frozen in liquid nitrogen, and the viability of the spermatozoa after thawing was examined by in-vitro fertilization, embryo culture and embryo transfer. When males had not been refrigerated, 41-52% of freshly collected oocytes were fertilized. The fertilization rate after 1 day of refrigeration was 29-49%, and the rate dropped as the refrigeration period increased, reaching 1-3% after 3 days. Partial zona dissection improved the fertilization rate when males were refrigerated for 2 days (5-9% vs 12-24%). High proportions of the resulting embryos developed in vitro, regardless of the strain of male mice, the refrigeration period and the partial zona dissection of oocytes. Embryos derived from 2-day refrigerated male sperm developed to young after transfer to recipients. The present system, using carcass refrigeration and sperm freezing, should be applicable to rescuing valuable genetic variants in laboratory animals or livestock animals as well as wild species in the future.
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  • Shixiong Xi, Hiroyuki Suzuki, Koji Toyokawa
    1999 Volume 45 Issue 3 Pages 197-204
    Published: June 20, 1999
    Released on J-STAGE: March 27, 2000
    JOURNAL FREE ACCESS
    The embryonic development and changes of the uterine tissue during pregnancy were examined morphologically to reveal causes of the age-related decline in litter size in the golden hamster. Uteri were obtained from young (9-12-week old, n=48) and aged hamsters (43-53-week old, n=47) at estrus and on Days 2 to 16 of pregnancy. Viability of conceptuses were examined in half of implants from Day 6 onwards by a dissecting microscope. The remaining implants were evaluated histologically. The number of resorbing implants was significantly larger in the aged hamsters than in the young females on Day 6 (4.4 vs. 1.2, p<0.05), although no difference was found in the overall mean number of ova ovulated (14.8 vs. 14.2, respectively). Delays in the embryonic development and uterine response were observed in all aged hamsters throughout gestation. Resorbing implants noted in the aged hamsters were accompanied with stunted decidual cells having pale cytoplasm (incomplete decidualization) and heavy infiltration of endometrial granulocytes. It is suggested that the embryonic wastage may occur much crucially during the peri-implantaion period rather than late gestational period, due to the retarded development of embryos and less-developed decidualization, and that endometrial granulocytes may function in resorption of greatly retarded embryos in the aged hamsters.
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  • Eiichi Hondo, Tohru Kobayashi, Naotaka Ishiguro, Masamichi Kurohmaru, ...
    1999 Volume 45 Issue 3 Pages 205-212
    Published: June 20, 1999
    Released on J-STAGE: March 27, 2000
    JOURNAL FREE ACCESS
    The effect of prolactin on transcription during spermatogenesis was examined by subtractive DNA hybridization and differential screening. A protamine 2 cDNA fragment was isolated from one of the genes up-regulated in testes of rat 6 h after prolactin injection. In situ hybridization to detect protamine 2 mRNA was also performed using digoxigenin-labeled cRNA probes. Weak signals were detected from preleptotene to early pachytene spermatocytes, elongated spermatids and spermatozoa; strong ones were recognized from mid- to late-pachytene, diplotene, and secondary spermatocytes, and early round spermatids. Localization of protamine 2 mRNA coincided with that of prolactin receptor mRNA described in our previous report. We then compared the time-dependent expression of protamine 2 mRNA with that of luteinizing hormone receptor mRNA after prolactin administration, using Northern blot analysis. Protamine 2 mRNA was up-regulated 1 h after the prolactin treatment, and stable levels were maintained for another 13 h. In contrast, luteinizing hormone receptor mRNA levels increased much later, at 13 h after prolactin injection. Therefore, protamine 2 gene responds rapidly to prolactin administration and up-regulation of expression appears to be independent of the luteinizing hormone pathway.
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  • Shuichi Yoshimura, Eiichi Hondo, Hiroshi Murabayashi, Nobuo Kitamura, ...
    1999 Volume 45 Issue 3 Pages 213-222
    Published: June 20, 1999
    Released on J-STAGE: March 27, 2000
    JOURNAL FREE ACCESS
    The changes in localization of inhibin α-subunit in bovine ovarian follicles during follicular development were assessed by immunohistochemical staining with monoclonal anti-inhibin α-subunit antibody. The changes in inhibin, estradiol-17β (E2), progesterone (P) and testosterone (T) levels in the follicular fluid during follicular development were also investigated. The follicles obtained from the bovine ovaries in mid-luteal stage were histologically classified into 7 stages. Stages I, II and III were growing healthy follicles, and stages IV, V, VI and VII were atretic follicles. In both growing and atretic follicles, inhibin α-subunit were histochemically demonstrated in the granulosa cells. Strong or moderate immunostaining for inhibin α-subunit were found only in the granulosa cells of stages II and III follicles (the strongest staining was found in stage III), and trace staining was seen in a small number of theca interna cells of stages II and III follicles. The granulosa cells of early atretic follicles (stage IV) also have the ability to produce and secrete inhibin, but finally they lost the ability. No positive staining was demonstrated in any cells of progressed atretic follicles (stage V, VI and VII). Inhibin level in the follicular fluid was higher in the growing follicles (highest level at stage II) than in the atretic follicles. Follicular fluid E2 level increased from stages I to III, but decreased from stage IV onward. The P levels remained low from stages I to VI, but increased only in stage VII. The T levels in stages III and VII were lower than in the other stages. Follicular fluid inhibin level were related to E2 level but not P or T levels. In conclusion, the inhibin α-subunit is produced by follicular granulosa cells in growing follicles of the bovine ovaries, and inhibin may stimulate E2 secretion in growing but not in atretic follicles.
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  • Kwang-Wook Park, Hiroaki Funahashi, Koji Niwa
    1999 Volume 45 Issue 3 Pages 223-231
    Published: June 20, 1999
    Released on J-STAGE: March 27, 2000
    JOURNAL FREE ACCESS
    The effects of maturation inhibitors and follicular cells on germinal vesicle (GV) development and meiotic resumption in bovine oocytes were examined. Evaluation of GV development of immature oocytes revealed that oocytes from larger diameter follicles were at more advanced stages. Although culture of cumulus-oocyte complexes (COCs) in the presence of dibutyryl-cAMP, 8-bromo-cAMP or N6-monobutyryl-cAMP did not inhibit meiotic maturation, culture in the presence of cycloheximide (CX) did. Cycloheximide also completely inhibited development of the GV as assessed by changes in morphology. Co-culture of COCs with theca cells (TH) or theca and granulosa cells (TG) also inhibited meiotic resumption but allowed GV development to the most advanced stage, GV-V. Thus co-culture with TH was able to synchronize oocytes at the stage just prior to GV breakdown. When removed from co-culture, the majority of oocytes were able to resume meiosis if co-cultured in a sufficient large volume of medium. The ability of TH to synchronize GV development could be used in a two-step protocol for oocyte maturation, the first step promoting GV development and the second inducing resumption of meiosis. Such an approach might improve the developmental potential of embryos obtained following in vitro fertilization of in vitro matured oocytes.
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  • Masayuki Ohtani, Shu-ichi Kobayashi, Akio Miyamoto
    1999 Volume 45 Issue 3 Pages 233-238
    Published: June 20, 1999
    Released on J-STAGE: March 27, 2000
    JOURNAL FREE ACCESS
    There is general agreement that prostaglandin F (PGF) is a physiological luteolysin in the cow. Contrary to this fact, PGF has been shown to stimulate the progesterone (P) release from luteal cells in vitro, through the activation of intracellular protein kinase C (PKC) and free calcium ion (Ca2+). To examine the possibility that an essential factor or function(s) may be lost through the process of preparing cells from the corpus luteum (CL), we evaluated the direct effect of PGF, TPA and ionophore A23187 on local P release within the CL in the cow, by utilizing in vivo microdialysis system (MDS). Normal cyclic Holstein cows (n=4) were superovulated with FSH and the PGF analogue, and on Day 8 after estrus, they were surgically implanted with the MDS (cut-off Mr=1000 kDa) into multiple CL (1-3 line/CL). The system was continuously perfused with Ringer's solution from immediately after the surgery. On Days 10-11, several CL were assigned to the control, PGF (10-5 M), TPA (10-5 M), A23187 (10-5 M), PGF+A23187 and TPA+A23187. The stimulants were infused into the CL using the MDS for 6 h, and each line of the MDS was further perfused with Ringer's solution for 26 h. A 6-h infusion with PGF stimulated P release during infusion, but after infusion the P release was again stable around the baseline. A 6-h infusion with TPA or A23187 strongly stimulated the P release. Similarly, PGF+A23187 or TPA+A23187 stimulated the P release. During the last 8 h of the experiment, A23187 alone, PGF+A23187 or TPA+A23187 inhibited the P release, whereas TPA as well as PGF maintained the same level as in the control. The results indicate that the direct exposure of the microenvironment in the bovine CL to PGF in vivo does not result in the suppression of P release until 26 h after stimulation, and that the activation of cytosolic free calcium is more effective in inhibiting the P release than the stimulation of PKC during the following period. Thus, the results support the concept that PGF needs some local luteolytic intermediator(s) that may enhance the intracellular calcium concentration.
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Technical Note
  • Masashi Nagano, Yoshiyuki Takahashi, Seiji Katagiri
    1999 Volume 45 Issue 3 Pages 239-242
    Published: June 20, 1999
    Released on J-STAGE: March 27, 2000
    JOURNAL FREE ACCESS
    The efficacy of a water purification system including a high-intensity 185 nm ultra violet lamp with ultrafilter (UV-UF) for eliminating endotoxins was examined. The activity of endotoxins was below the detectable level in UV-UF water. In contrast, ultra-purified water from a system without a UV lamp (UP) showed a high level of endotoxins, despite water resistivity of more than 18 MΩ·cm. Bovine IVM/IVF zygotes were cultured for 174 h in protein-free media prepared with UV-UF and UP water. The developmental rate to blastocysts at 150 h was significantly higher in the medium prepared with UV-UF water. The results demonstrate that the UV-UF system is beneficial for eliminating endotoxins from ultra-purified water, and for preparing protein-free media for culture of bovine embryos.
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