Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 54, Issue 4
August
Displaying 1-13 of 13 articles from this issue
Full Paper
  • Hitoshi USHIJIMA, Kiyoshi AKIYAMA, Toshio TAJIMA
    Article type: -Full Paper-
    2008 Volume 54 Issue 4 Pages 239-243
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: April 28, 2008
    JOURNAL FREE ACCESS
    The total cell numbers (TCNs) of bovine embryos collected from superovulated donors (VIVO embryos) were counted 0-9 d after ovulation to quantify the developmental process. Using numerical analysis of embryo development, we also compared the developmental process of VIVO embryos, in vitro-fertilized (IVF) embryos and nuclear transfer (NT) embryos obtained from enucleated oocytes and blastomere nuclei. The TCNs of embryos were measured using the air-dry method. Cleavage divisions (CD) of the embryos were obtained using logarithmic transformation of the TCN. The TCN of the VIVO embryos increased significantly (P<0.001) with time. The relationship between the CD of the VIVO embryos at 0-9 d after ovulation and age in days was described by a linear equation with a high correlation (y=1.03x+0.16, r=0.99), showing that CD occurs about once each day for all blastomeres. However, compared to the VIVO embryos, the TCN of the IVF embryos did not increase from 3-4 d nor after 7 d; the TCN of the NT embryos did not increase after 7 d (P>0.05). The results suggest a delay in development at these developmental stages. The slopes of regression lines of the IVF and NT embryos were significantly (P<0.001) smaller, indicating that quantification of the developmental process of VIVO embryos according to TCN and CD would be useful as criteria for numerical evaluation of the developmental process of bovine in vitro produced embryos.
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  • Tomoko NAKANISHI, Naoko ISHIBASHI, Haruka KUBOTA, Kimiko INOUE, Narumi ...
    Article type: -Full Paper-
    2008 Volume 54 Issue 4 Pages 244-249
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: May 19, 2008
    JOURNAL FREE ACCESS
    Sperm-specific phospholipase C, PLCζ, is a candidate for the Ca2+ oscillation-inducing factor that is introduced into the ooplasm upon sperm-egg fusion. In addition to the 647-residue full-length PLCζ, s-PLCζ lacking the N-terminal 110 amino acids is known to be present in the mouse testis. In this study, we attempted to obtain full-term offspring from s-PLCζ-activated eggs by round spermatid injection. Metaphase II-arrested eggs injected with a high RNA concentration of s-PLCζ RNA normally developed to blastocysts. When the round spermatid nucleus was injected into telophase II-stage eggs previously activated by s-PLCζ RNA, three live offspring were successfully obtained by transfer of the developed 4-cell embryos to pseudopregnant mice. These three offspring all grew to be normal adults and reproduced healthy second-generation mice.
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  • Chi-Gu KIM, Hwanyul YONG, Gene LEE, Jaejin CHO
    Article type: -Full Paper-
    2008 Volume 54 Issue 4 Pages 250-253
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: May 19, 2008
    JOURNAL FREE ACCESS
    In this study, we investigated the effect of polyvinylpyrrolidone (PVP) concentration on in vitro and in vivo development of 2 cell stage, vitrified ICR mouse embryos using a cryoprotectant consisting of ethylene glycol (EG) and sucrose. M2 was selected as the basic medium for vitrification and thawing. After equilibration with 4% (v/v) EG at 37 C for 15 min, the embryos were vitrified with 35% EG, 5, 6 or 7.5% (w/v) PVP and 0.4 M sucrose at 37 C for 30 sec. One week later, the cryotubes of cryopreserved embryos in liquid nitrogen were directly immersed into a 37 C water bath for 1 min and transferred serially into 300 μl of 0.5 or 0.3 M sucrose at room temperature for 5 min and M2 medium at 37 C for 10 min. The surviving embryos were cultured in KSOM (potassium simplex optimized medium) for 96-120 h in an atmosphere of 5% CO2 in humidified air. Survival was evaluated by morphological appearance, including membrane integrity and presence of apoptotic blastomeres after thawing. For in vivo evaluation, blastocysts were transferred to the uteri of pseudopregnant mice. The survival rates of the 5 and 7.5% PVP concentration groups showed a significantly higher difference compared with that of the 6% PVP group (85.5 and 86.5 vs. 71.2%), respectively. Each pup in the of 5 and 6% groups was cannibalized immediately after parturition. A litter of live pups was obtained from only the 7.5% PVP groups. Our study indicated that supplementation of EG and sucrose cryoprotectant solution with 7.5% PVP is optimal for successful vitrification of 2-cell stage ICR mouse embryos.
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  • Mayuko KUROME, Hisashi HISATOMI, Shirou MATSUMOTO, Ryo TOMII, Satoshi ...
    Article type: -Full Paper-
    2008 Volume 54 Issue 4 Pages 254-258
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: May 19, 2008
    JOURNAL FREE ACCESS
    The aim of the present study was to examine the production efficiency of cloned pigs by serial somatic cell nuclear transfer (SCNT) and to ascertain any changes in the telomere lengths of multiple generations of pigs. Using fetal fibroblasts as the starting nuclear donor cells, porcine salivary gland progenitor cells were collected from the resultant first-generation cloned pigs to successively produce second- and third-generation clones, with no significant differences in production efficiency, which ranged from 1.4% (2/140) to 3.3% (13/391) among the 3 generations. The average telomere lengths (terminal restriction fragment values) for the first, second and third generation clones were 16.3, 18.1 and 20.5 kb, respectively, and were comparable to those in age-matched controls. These findings suggest that third-generation cloned pigs can be produced by serial somatic cell cloning without compromising production efficiency and that the telomere lengths of cloned pigs from the first to third generations are normal.
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  • Bing YAO, Mitsumori KAWAMINAMI
    Article type: -Full Paper-
    2008 Volume 54 Issue 4 Pages 259-264
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: May 27, 2008
    JOURNAL FREE ACCESS
    The distribution and regulation of annexin A5 expression, a gonadotropin releasing hormone (GnRH) receptor regulated protein in gonadotropes and luteal cells, in the testes of rats were examined. Immunocytochemical staining revealed high levels of annexin A5 in the Leydig and endothelial cells and lower levels in the primary spermatocytes and sperm. Hemicastration significantly increased the annexin A5 content of the remaining testis within 24 h. Annexin A5 immunoreactivity was increased mainly in interstitial tissues including the peritubular cells, while some spermatocytes also showed higher intensity of annexin A5 in the remaining testis. Administration of hCG (50 IU) enhanced the testicular content of annexin A5 after 24 h. This treatment expanded the area of interstitial tissue in the testis and increased annexin A5 immunoreactivity, but the area of the of endothelial cells was unchanged. Similarly, human chorionic gonadotropin (hCG) enhanced annexin A5 expression in a primary culture of testis cells that consisted of mainly interstitial cells. Because GnRH stimulates the expression of annexin A5 in the gonadotropes and luteal cells, we examined the effect of GnRH on annexin A5 expression in the testes. We found that des-Gly10 [Pro9]-GnRH ethylamide (100 nM), a GnRH agonist, increased annexin A5 expression in cultured testis cells and that Cetrorelix (100 nM), a GnRH antagonist, inhibited the effect of hCG on annexin A5 expression. These results suggest that pituitary luteinizing hormone promotes annexin A5 synthesis in Leydig cells and that this effect could be mediated by local GnRH in the testis.
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  • Bo JIN, Chihiro YAMASAKI, Naoko YAMADA, Shinsuke SEKI, Delgado M. VALD ...
    Article type: -Full Paper-
    2008 Volume 54 Issue 4 Pages 265-269
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: May 30, 2008
    JOURNAL FREE ACCESS
    To improve the cryopreservation protocol for mouse sperm, we attempted to estimate the type and extent of cryoinjury at various steps of the process. First, we demonstrated that mouse sperm are sensitive to chilling at -15 C and that the sensitivity is dependent on the length of exposure. To estimate cryoinjuries, sperm suspensions were ice-seeded at -5 or -15 C, frozen with liquid nitrogen (LN2) gas and then frozen in LN2. In one experiment, sperm seeded at -5 C were cooled slowly to -15 C before deep freezing. At various steps of the cryopreservation process, the sperm were warmed and their viability was assessed based on motility and the integrities of the plasma membrane and acrosome. The motility of frozen-thawed sperm was higher on seeding at -5 C (28%) than at -15 C (9%). The motility did not decrease when the sample was transferred from LN2 gas to LN2. To estimate cryoinjury of sperm, we presumed the viability of frozen sperm to be decreased by chilling, hypertonic stress and formation of intracellular ice. When the sperm suspension was cooled and seeded at -5 C, the motility decreased by 25% due to hypertonic stress. When the sperm were cooled in LN2 gas, the motility decreased by 17% with the formation of intracellular ice. When the sperm were cooled to -15 C, the motility decreased by 51% from chilling. After seeding, the motility decreased by 18% due to formation of intracellular ice and by 7% due to hypertonic stress. Considering the results, it would be preferable to seed samples at a higher temperature to prevent intracellular ice from forming and to cool seeded samples rapidly enough to minimize chilling injury and hypertonic stress, but not too rapidly to allow intracellular ice to form.
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Research Note
  • Mitsuo ISHII, Syuichi KOBAYASHI, Tomas J. ACOSTA, Wataru MIKI, Takahir ...
    Article type: -Research Note-
    2008 Volume 54 Issue 4 Pages 270-274
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: May 22, 2008
    JOURNAL FREE ACCESS
    The aim of this study was to clarify the relationship between circulating oxytocin (OT) and PGF2α metabolite (PGFM) in mares at the third stage of labor and placental expulsion time in order to investigate a cause of retained placenta of which the incidence increase in a heavy draft mare. Blood was sampled every 5 min from foaling to expulsion of the placenta in 18 heavy draft mares to evaluate circulating OT and PGFM. The relationships between OT and PGFM concentration and recorded placental expulsion times were investigated. The results were as follows (1) The highest level of OT concentration was observed close to foaling in 15 mares. (2) The OT concentrations close to foaling were variable with a large difference from the lowest concentration, 22.1 pg/ml, to the highest concentration, 209.3 pg/ml. (3) The highest level of PGFM was observed close to foaling in 17 mares. (4) During the 60 min following foaling, the OT concentrations of the mares (n=11) that had a shorter placental expulsion time (i.e., <1 h), were significantly higher than those of the mares (n=7) that had a longer placental expulsion time (i.e., >1 h; P<0.05). Collectively, the OT concentration immediately after foaling is negatively related to the placental expulsion time. Deficiency of OT secretion at foaling have should be considered as one of the causes of retained placenta in heavy draft mares.
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  • Akihisa MAEDA, Fuko MATSUDA, Yasufumi GOTO, Yuan CHENG, Hiroshi GONDA, ...
    Article type: -Research Note-
    2008 Volume 54 Issue 4 Pages 275-280
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: May 23, 2008
    JOURNAL FREE ACCESS
    The apoptosis inhibitory ligand (Netrin-1) and its receptor (p53-regulated receptor for death and life: p53RDL1) play an important role in the regulation of selective apoptosis. When Netrin-1 binds to p53RDL1, p53-dependent apoptosis is inhibited. We identified porcine (Sus scrofa) cDNAs encoding Netrin-1 [pNetrin-1; 1,803 base pairs (bp) and 600 amino acids (aa)] and p53RDL1 (pp53RDL1; 2,838 bp and 945 aa). Porcine p53RDL1 (pp53RDL1) contains a death domain (DD), a tandem specific amino acid region, in its C-terminal, suggesting that it mediates death signaling by binding with other pro-apoptotic factors via the DD. Porcine Netrin-1 (pNetrin-1), pp53RDL1 and the DD in pp53RDL1 showed high levels of identity in aa sequence with human and murine Netrin-1 (98 and 97%, respectively), p53RDL1 (94 and 91%, respectively) and the DD in p53RDL1 (96 and 95%, respectively). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the levels of pNetrin-1 and pp53RDL1 mRNAs were moderate in granulosa cells compared with their expression in other tissues and that their levels during follicular atresia were stable. The Netrin-1 and p53RDL1 system may regulate the induction of apoptosis in porcine granulosa cells.
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  • Natsuki HAMA, Hideyasu KANEMITSU, Kensuke SAKAMOTO, Yujiro OYAMA, Toma ...
    Article type: -Research Note-
    2008 Volume 54 Issue 4 Pages 281-285
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: May 23, 2008
    JOURNAL FREE ACCESS
    To detect estrus for reproductive management, and to determine the relationship between urinary estrogen and estrous behavior, in a female giant panda, we developed and evaluated a rapid enzyme immunoassay (EIA) system for urinary Estrone-3-glucuronide (E1G) using commercial reagents. The developed EIA system took only around 3 hours, including all procedures to obtain a result. It indicated good reproducibility (intra-assay CV of 5.16%, interassay CV of 15.4%) and sensitivity (lowest standard concentration was 0.0156 ng/ml) for measurement of the urinary concentrations of E1G in the giant panda. There was a positive correlation (r=0.934) with the data for estrone (E1) in the same samples, as measured by radioimmunoassay (RIA) performed in a commercial laboratory. The changes in the E1G concentrations were almost synchronous with the changes in E1 assayed by RIA in urine collected during 4 consecutive estrous seasons. The dynamics of urinary E1G measured by this system highly correlated with the occurrence of the presenting estrous behavior in the giant panda. The above results indicate that this assay system may be normally, rapidly and practically used for measurement of the urinary concentration of E1G in the giant panda.
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Technical Note
  • Yutaka FUKUI, Hirohide KOHNO, Tetsuro TOGARI, Mami HIWASA, Kentaro OKA ...
    Article type: -Technical Note-
    2008 Volume 54 Issue 4 Pages 286-289
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: April 14, 2008
    JOURNAL FREE ACCESS
    The present study aimed to compare the fertility of ewes intrauterinally inseminated with frozen-thawed semen using a soybean-based semen extender (AndroMed) with those of ewes intrauterinally inseminated with frozen-thawed semen using a Tris-based extender containing either egg yolk or BSA. Suffolk ewes (n=104) were treated with an intravaginal sponge containing 40 mg fluoroprogesterone acetate (FGA) for 12 days and an intramuscular injection of 500 IU equine chorionic gonadotropin to induce estrus and ovulation during the non-breeding season (July, 2007). Intrauterine insemination was carried out 40-46 h after removal of the FGA sponge (n=90), regardless of the incidence of estrus. The pregnancy rates were not significantly different among the semen extenders containing egg yolk (64.5%) or BSA (58.6%) and AndroMed extender (56.7%). The lambing rates (64.5, 55.2 and 56.7% for the semen extenders containing egg yolk, BSA and AndroMed, respectively) and prolificacy (1.59 to 1.75) were also not significantly different. The present results indicate that an egg yolk-containing semen extender can be replaced with the non-animal derived extender AndroMed, which could be used for intrauterine insemination using frozen-thawed ram semen without reducing fertility.
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  • Yasuyuki ABE, Dong-Soo LEE, Hikaru SANO, Koji AKIYAMA, Yoshiko YANAGIM ...
    Article type: -Technical Note-
    2008 Volume 54 Issue 4 Pages 290-294
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: May 13, 2008
    JOURNAL FREE ACCESS
    Due to the recent outbreak of avian influenza, transportation of frozen canine semen with egg yolk has been sharply restricted. Thus, there is urgent need to develop a novel egg yolk-free extender for freezing canine spermatozoa. In the present study, the effect of using skim milk/glucose (SG)-based extender without egg yolk on the motility and fertilizing capacity of canine spermatozoa frozen-thawed in the presence of glycerol was examined. There was a tendency for the proportion of motile spermatozoa exposed to SG-based extender for 3 h to be higher than that exposed for 1 h, but the difference was not significant. The motility and other viability parameters of canine spermatozoa after thawing were similar to those obtained with an egg yolk-based extender. When spermatozoa frozen with SG-based extender containing glycerol after 3 h exposure were transcervically inseminated into 2 recipient bitches, a total of 6 pups were obtained. These results suggest that a simple extender composed of skim milk, glucose and glycerol is useful for cryopreservation of canine spermatozoa, which may contribute to improved exchange of genetic material and efficient production of companion and working dogs, such as guide dogs for the blind.
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  • Hiroshi OHTA, Yuko SAKAIDE, Teruhiko WAKAYAMA
    Article type: -Technical Note-
    2008 Volume 54 Issue 4 Pages 295-298
    Published: 2008
    Released on J-STAGE: August 21, 2008
    Advance online publication: May 13, 2008
    JOURNAL FREE ACCESS
    We previously demonstrated that testicular spermatozoa can be preserved as frozen testicular sections, allowing us to preserve male gametes in less space than conventional methods. However, it remains unclear whether the testicular spermatozoa can be preserved for a long period using this procedure. In this study, we examined the function of testicular spermatozoa preserved as frozen testicular sections for l year at -30 or -80 C. Testicular spermatozoa were successfully retrieved from frozen testicular sections preserved at either -30 or -80 C, and their function was assessed using intracytoplasmic sperm injection (ICSI). Over 90% of the oocytes injected with long-term preserved testicular spermatozoa formed pronuclei, which was a frequency similar to that obtained with spermatozoa preserved for a short term, indicating that the testicular spermatozoa retained oocyte activation factor(s). Approximately 70% of the fertilized oocytes developed to 2-cell stage embryos, and 9.3 to 12.8% of the embryos developed to term after transfer into pseudopregnant females, regardless of the preservation temperatures examined. These results indicate that the birthrates of progeny did not differ between the preservation temperatures examined. They also indicate that male gametes can be preserved in testicular frozen sections for at least 1 year without loss of function.
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Erratum
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