Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 51, Issue 3
June
Displaying 1-15 of 15 articles from this issue
Original Article
  • Qiang WENG, Mohamed S. MEDAN, Gen WATANABE, Toshio TSUBOTA, Yoshikuni ...
    2005 Volume 51 Issue 3 Pages 299-304
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: February 22, 2005
    JOURNAL FREE ACCESS
    The objective of this study was to investigate immunolocalization of steroidogenic enzymes in Göttingen miniature (GM) pig testes. Testes of 6 adult GM pigs were obtained in September 1996 (n=2), February (n=2) and June (n=2), 1997. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3β-hydroxysteroid dehydrogenase (3βHSD), porcine testicular 17α-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were observed in all testes throughout the year. Moreover, P450scc, 3βHSD, P450c17and P450arom were identified in Leydig cells but not in Sertoli cells of all testes. These results suggested that adult GM pig testes have the ability to produce germ cells throughout the year, and the synthesis of progestin, androgen and estrogen occurs in the Leydig cells of GM pig testes.
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  • Ayako INOUE, Sakae TAKEUCHI, Sumio TAKAHASHI
    2005 Volume 51 Issue 3 Pages 305-313
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: February 22, 2005
    JOURNAL FREE ACCESS
    Much evidence has suggested that sex steroid hormone-induced growth of uterine cells is mediated by polypeptide growth factors synthesized in uterine tissues. The present study aimed to clarify the effect of insulin-like growth factor-I (IGF-I) on the proliferation of mouse endometrial stromal cells obtained from immature mice. IGF-I and IGF-I receptor (type I) mRNAs were detected in the endometrial stromal cells. IGF-I increased bromodeoxyuridine (BrdU) uptake in the endometrial stromal cells, indicating an increase in DNA replication. E2 increased IGF-I mRNA levels in the endometrial stromal cells. IGF-I receptor is a tyrosine kinase receptor, and treatment with genistein, a tyrosine kinase inhibitor, reduced IGF-I-induced BrdU-uptake in the endometrial stromal cells. IGF-I signaling pathways involve mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 kinase (PI-3 kinase). Treatment with 10-7 M of the MAP kinase inhibitor PD098059 and 10-5 M of the PI-3 kinase inhibitor LY294002 decreased IGF-I-induced BrdU-uptake in the endometrial stromal cells. However, LY294002 (10-5 M) also decreased the BrdU-uptake in the absence of IGF-I treatment. These results suggest that endometrial IGF-I is involved in the proliferation of endometrial stromal cells in a paracrine or autocrine manner, and that the MAP kinase pathway is involved in DNA replication of endometrial stromal cells.
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  • Hanako NISHIZAWA, Maki MORITA, Miki SUGIMOTO, Satoshi IMANISHI, Noboru ...
    2005 Volume 51 Issue 3 Pages 315-324
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: February 28, 2005
    JOURNAL FREE ACCESS
    To evaluate the effects of bisphenol A (BPA), a candidate endocrine disruptor (ED), on embryonic development, we examined the mRNA expression levels of the arylhydrocarbon receptor (AhR), which binds with many EDs and plays crucial roles in xenobiotic metabolism, and of the retinoic acid receptor (RAR) α and retinoid X receptor (RXR) α, key factors in nuclear receptor-dependent retinoid signal transduction, in murine embryos exposed in utero to BPA (0.02, 2, 200, and 20,000 μg/kg/day) at 6.5-13.5 or 6.5-17.5 days post coitum (dpc), using the real-time reverse transcription-polymerase chain reaction (RT-PCR) method. Extremely low-dose BPA (0.02 μg/kg/day; 1/100 the dose of environmental exposure) remarkably increased AhR mRNA expression in the cerebra, cerebella, and gonads (testes and ovaries) of male and female 14.5- and 18.5-dpc-embryos. In utero exposure to BPA at 2, 200, and 20,000 μg/kg/day also increased levels of AhR mRNA. In gonads of 14.5-dpc-embryos, AhR mRNA levels were elevated and showed diphasic (U) dose-response curves following exposure to BPA, but inverted U dose-response curves were obtained for 18.5-dpc-embryos. Exposure to BPA increased expression levels of RARα and RXRα mRNAs in the cerebra, cerebella, and gonads of male and female 14.5- and 18.5-dpc-embryos. Extremely low-dose BPA (0.02 μg/kg/day) increased RARα mRNA expression in the cerebella of male and female 14.5- and 18.5-dpc-embryos and in the gonads of female 14.5-dpc-embryos, and significantly increased RXRα mRNA expression in the cerebra and cerebella of male and female 14.5-dpc-embryos. The present findings confirm that in utero exposure to an extremely low dose of BPA up-regulates the mRNA expression of AhR, RARα, and RXRα in murine embryos and disrupts the receptor-dependent signal transducing systems, and will contribute to the assessment of the toxic effects of BPA on xenobiotic metabolism and retinoid signals in embryogenesis.
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  • Johannes KAUFFOLD, Hussein A. H. AMER, Uwe BERGFELD, Wolfgang WEBER, A ...
    2005 Volume 51 Issue 3 Pages 325-332
    Published: 2005
    Released on J-STAGE: July 06, 2005
    JOURNAL FREE ACCESS
    This study investigated the relationship between follicle size (FS) and developmental competence of calf oocytes. Cumulus-oocyte-complexes (COCs) from follicles >8 (L-COCs; n=19), 4-8 (M-COCs; n=54), and 2-3 mm (S-COCs; n=155) were recovered from non-stimulated 1-4 months old dairy calves post mortem and ex vivo (laparoscopy), and in parallel from slaughtered adult cows from follicles of identical size categories [> 8 (n=91); 4-8 (n=138); 2-3 mm (n=193)]. Morphologically intact COCs were subjected to in vitro maturation, fertilization, and embryo culture. Cleavage rate (CR; 46 h post-insemination=p.i.), rate of morulae/blastocysts (M/Bl; day 7 p.i.), and blastocysts (Bl; day 9 p.i.) were recorded. FS had no effect on the CR in calves. However, calf L-COCs yielded the highest rates of M/Bl and Bl compared with the two other size categories (P<0.05). In contrast, calf S- and M-COCs gave similar rates of M/Bl, whereas the proportion of Bl was lowest for S-COCs (P<0.05). This was almost identical to findings in cows, except that the CR was highest for L-COCs and M/Bl yields were lowest for S-COCs (P<0.05). There were no differences between calf and cows with regard to CR for the respective FS categories. L-COCs from calves and cows yielded similar rates of M/Bl and Bl, whereas calf S- and M-COCs yielded lower rates of Bl than S- and M-COCs from cows and a lower rate of M/Bl when S-and M-COCs were analyzed as one group (P<0.05). Whereas the CR was similar in calves and cows, calf COCs yielded lower rates of M/Bl and Bl (P<0.05). In conclusion, the results show that the developmental competence of calf oocytes is higher in those derived from follicles larger than 8 mm, and thus are almost equally as competent as cow oocytes derived from follicles of identical size. This suggests that calf oocytes acquire developmental competence within the large follicle, potentially due to a process similar to prematuration of the oocyte in the adult cow. It is proposed that procedures that facilitate prematuration, such as "coasting" following a preceding superstimulation, might increase the developmental competence of calf oocytes.
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  • Ken KUSAKABE, Yoshinori OTSUKI, Yasuo KISO
    2005 Volume 51 Issue 3 Pages 333-340
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: March 18, 2005
    JOURNAL FREE ACCESS
    Uterine natural killer (uNK) cells in the pregnant uterus are known to be associated with the normal development of the placenta. In the mouse pregnant uterus, many uNK cells exist during mid pregnancy, although they show a sudden decrease during late pregnancy and almost disappear before delivery. Our previous study indicated that uNK cells showed clear apoptotic morphology during late pregnancy. Therefore, the present study was carried out to define the involvement of Fas ligand (FasL) and Fas in apoptosis induction of uNK cells. Immunohistochemical analyses revealed that uNK cells expressed FasL in the cytoplasmic granules and Fas on the cell membrane during late pregnancy. In lpr/lpr mice, which genetically lack Fas, many uNK cells were clearly observed during late pregnancy compared with wild-type mice, and moreover uNK cells still existed at day-18 of pregnancy, although there were few in wild-type mice during the same period. In the experiment of in vitro culture, uNK cells derived from wild-type placenta showed chromatin condensation and DNA fragmentation frequently following the anti-Fas antibody treatment, as compared with the control. From these results, it is suggested that FasL and Fas-dependent apoptosis regulates cell appearance of uNK cells in the mouse pregnant uterus.
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  • Toshiro TAKEDOMI, Hisashi KISHI, Mohamed S. MEDAN, Yoshito AOYAGI, Mas ...
    2005 Volume 51 Issue 3 Pages 341-346
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: March 11, 2005
    JOURNAL FREE ACCESS
    The effect of active immunization against inhibin on the response to superovulatory treatment by porcine FSH (pFSH) was investigated in cattle. Japanese black cows were sc injected with 1 mg of porcine inhibin α-subunit fragment (1-26) conjugated with rabbit serum albumin (inhibin-immunized group; n=14) or rabbit serum albumin alone (control group; n=12) in Freund's complete adjuvant. Booster injections (half the amount of the primary injection) were given 35 and 70 days after the primary injection. All cows were superovulated three times with pFSH. Three days after each injection of the antigen, a progesterone-releasing intravaginal device (CIDR-B) was inserted vaginally into all animals and left in place for 10 days. Forty-eight hours before CIDR-B removal, all animals were sc injected with 30 mg pFSH dissolved in 40% polyvinylpyrrolidone, and im injected with 750 μg of PGF2α at CIDR-B removal. Cows were artificially inseminated twice during estrus, and ova or embryos were collected 7 or 8 days after estrus. The number of corpora lutea, the number of ova or embryos and the number of transferable embryos in inhibin-immunized cows (12.1 ± 1.2, 11.1 ± 1.3 and 6.2 ± 1.0, respectively) were significantly greater than those in the controls (8.2 ± 1.0, 5.7 ± 1.1 and 3.1 ± 0.7, respectively). These results indicate that active immunization against inhibin enhanced ovarian response to the usual superovulatory treatment in cattle. Therefore, immunization against inhibin may be a useful approach for improving the response to superovulation in cattle.
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  • Mohamed Elshabrawy GHANEM, Masahide NISHIBORI, Toshihiko NAKAO, Masaha ...
    2005 Volume 51 Issue 3 Pages 347-352
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: March 23, 2005
    JOURNAL FREE ACCESS
    Genomic DNA extracted from bovine mummified tissue is valuable material for detection of some genes that may contribute to fetal abnormalities. In this study bovine genomic DNA was extracted from the hardened tissue samples of ten bovine mummified fetuses. The amount of genomic DNA extracted from 2 g of the mummified tissues by the phenol/chloroform-ethanol method was low (less than 4 μg/ml) for all samples. The extracted DNA was then amplified by the GenomiPhiTM DNA amplification system. After amplification, the amount of DNA was increased to more than 100 μg/ml for all samples. This amplification system was shown to be a good tool for amplifying the genomic DNA of the mummified fetuses. The amplified genomic DNA was used for testing the mummies for Factor XI gene deficiency, an autosomal recessive deficiency involved in the early stages of the intrinsic blood coagulation pathway. Exon 12 of the Factor XI gene of the mummies was amplified by PCR. Two of the ten mummified fetuses were heterozygous for the Factor XI gene as indicated by the presence of two amplified DNA fragments of 320 bp and 244 bp. Factor XI deficiency has already been described in Holstein cattle. However, no report is available for bovine fetus. In this study, DNA was extracted and amplified from the bovine mummified fetuses, and the samples were successfully tested for Factor XI gene deficiency in the mummies.
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  • PeiXin YANG, Mohamed S. MEDAN, Koji Y. ARAI, Gen WATANABE, Kazuyoshi T ...
    2005 Volume 51 Issue 3 Pages 353-358
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: March 11, 2005
    JOURNAL FREE ACCESS
    The present study was undertaken to determine changes in circulating levels of immunoreactive (ir)-inhibin, FSH, LH, estradiol-17β, progesterone, and testosterone during the ovulatory cycle of Shao ducks. Serial blood samples were taken from two groups of laying ducks for measurement of ir-inhibin, gonadotropins, and steroid hormones at 2 h intervals for 24 h. Plasma concentrations of ir-inhibin did not change significantly during the ovulatory cycle. The highest level of plasma ir-inhibin was observed 6 h prior to ovulation, which coincided with a decreased level of plasma FSH. One FSH surge was found 12 h after ovulation. Estradiol-17β, progesterone, and testosterone were also determined during the ovulatory cycle. Two peak values were detected for estradiol-17β 8 h before ovulation and 4 h after ovulation, while progesterone started to increase 4 h before ovulation and reached a peak at ovulation. The highest level of plasma testosterone was detected around the time of ovulation. These results suggest that inhibin may be involved in the control of FSH secretion during the ovulatory cycle. In addition, both LH and progesterone are of importance in the ovulation process of Shao ducks.
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  • Tadahiro IKEMOTO, Min Kyun PARK
    2005 Volume 51 Issue 3 Pages 359-377
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: April 05, 2005
    JOURNAL FREE ACCESS
    RFamide-related peptides (RFRPs) regulate the release of various pituitary hormones in vertebrates. It is completely unknown how the functions of RFRPs vary among animal classes and whether vertebrate RFRPs are orthologous to each other and belong to the same peptide family. This report concerns identification of avian RFRP (gonadotropin-inhibitory hormone, GnIH) from the chicken. Chromosome-wide synteny conservation demonstrated the orthologous relationships among vertebrate RFRPs. The consensus motif for RFRP was modified to Pro-Xaa-Arg-Phe-NH2. We also describe the first identification of two distinct types of receptors for non-mammalian RFRP (RFRPR and NPFFR) from the chicken. Amino acid comparison revealed substantial differences in both termini of receptors among classes of vertebrates. The 5'-flanking regions of chicken RFRPR and NPFFR suggested their expressions in the pituitary gland, and this was confirmed by the RT-PCR analysis. Localizations of both chicken RFRP and its receptors were distinct from those of mammals. These results indicated that avian RFRP, unlike the mammalian one, directly acts on the pituitary gland via receptors to regulate gonadotropin release. It was also suggested that functional differences, especially between avian and mammalian RFRPs, are substantially due to divergences in the structures and expression sites of their receptors.
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  • Adam J. ZIECIK, Malgorzata BIALLOWICZ, Monika KACZMAREK, Wieslaw DEMIA ...
    2005 Volume 51 Issue 3 Pages 379-384
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: April 13, 2005
    JOURNAL FREE ACCESS
    Synchronization and superovulation are commonly used to obtain large numbers of embryos for experimental and practical purposes. This study compared the number, quality, and in vitro development of embryos recovered from gilts following single or double estrus synchronization and superovulation. Prepubertal gilts from the single synchronization group were injected with 1500 I.U. PMSG and 1000 I.U. hCG 72 h later. The double synchronized group of gilts was treated with 750 I.U. PMSG and 500 I.U. hCG 72 h later. After 17 days, 1500 I.U. PMSG followed by 1000 I.U. hCG was administered. Five days after insemination embryos were recovered and cultured for 6 days. Both single and double hormonal stimulation schedules resulted in recovery of elevated numbers of embryos (28.4 and 23.4 vs. 11.3; p<0.01and p≤0.05, respectively) with a higher percentage of embryos classified as degenerated (39.2% and 43.1%, respectively) compared to the non-stimulated, control group (5.1%). The number of embryos destined for culture did not differ between the single and double synchronized groups. The highest percentage of hatched embryos was observed in the control group. In conclusion, the single synchronization and superovulation schedule is sufficient to obtain high numbers of embryos, however, both synchronization methods resulted in the recovery of considerable numbers of degenerated embryos. A higher number and percentage of hatched embryos after culture was found among embryos from the control group compared to gonadotropin-stimulated gilts.
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  • Rena J CLARK, Michael A. FURLAN, P. Jorge CHEDRESE
    2005 Volume 51 Issue 3 Pages 385-392
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: April 05, 2005
    JOURNAL FREE ACCESS
    We report the elk (Cervus elaphus) thyroid stimulating hormone (TSH) β-subunit cDNA cloning, nucleotide and deduced amino acid sequences. The TSH β-subunit cDNA was obtained by RT-PCR of polyadenylated pituitary RNA. The deduced elk TSH β-subunit peptide chain shares between 93 to 99% sequence similarities with the reported TSH β-subunit of a sub-set of related species. The TSH β-subunit gene is expressed in the elk pituitary gland as a mature transcript of approximately 600 bases, which corresponds to the size of the mRNA expressed in the sheep pituitary gland. Seasonal expression of the pituitary gonadotropin genes was investigated by Northern blot analyses. Samples of elk pituitary glands collected during the breeding season showed elevated steady state levels of common α-subunit and FSH and LH β-subunit gene expression, consistent with the seasonal reproductive cycling of this species. Samples collected before the breeding season demonstrated decreased expression of the gonadotropin genes. TSH, which is not directly tied to reproduction, had similar levels of expression, regardless of the animal's reproductive status.
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Research Note
  • Takuya HORI, Thicomeporn YAMSAARD, Yoshiko Yanagimoto UETA, Shinji HAR ...
    2005 Volume 51 Issue 3 Pages 393-397
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: March 11, 2005
    JOURNAL FREE ACCESS
    It is well-known that there are considerable strain differences in the relative copulation rates between male and superovulated female mice. In particular, the C57BL/6J strain of mice has a lower rate of successful copulation. We examined the effect of exposure to an electric field on sexual behavior in C57BL/6J male mice. When C57BL/6J males were exposed to a 50 Hz, 45 kV/m electric field for 30 min per day for 11 days and placed in a cage with a superovulated female of the same strain, the successful copulation rates of males was significantly improved compared with unexposed males (P<0.05). These results suggest that the exposure of C57BL/6J male mice to an electric field improves their sub-fertility activity in mating with superovulated females.
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  • LongQuan REN, Mohamed S. MEDAN, Qiang WENG, Wanzhu JIN, ChunMei LI, Ge ...
    2005 Volume 51 Issue 3 Pages 399-404
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: February 28, 2005
    JOURNAL FREE ACCESS
    The objective of this study was to determine the immunolocalization of NGF and its receptors (TrkA and p75LNGFR) in the reproductive tract of the Japanese Shiba goats. Five adult goats were used in this study and sections of ovaries, uteri and oviducts were immunostained by the avidin-biotin-peroxidase complex method (ABC). The results showed that NGF and its receptors (TrkA and p75LNGFR) were expressed in granulosa cells, theca cells, interstitial cells and lutein cells in ovaries. Immunoreactions for NGF, TrkA and p75LNGFR were also detectable in epithelial cells and muscle cells of the ampulla and isthmus of the oviduct, and in epithelial cells and uterine glands of the uterus. These results strongly suggest autocrine and paracrine regulation of reproductive function by NGF in the reproductive tract of female Shiba goats.
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  • Satoshi UENO, Mayuko KUROME, Hideto UEDA, Ryo TOMII, Katsumi HIRUMA, H ...
    2005 Volume 51 Issue 3 Pages 405-410
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: April 05, 2005
    JOURNAL FREE ACCESS
    Incomplete cytoplasmic maturation of in vitro matured (IVM) oocytes has been known to cause microtubule and microfilament alterations, which may result in abnormal pronuclear formation and failed embryonic development. We examined the influences of maturation conditions on meiotic spindle morphology at metaphase of meiosis II (MII) in porcine oocytes. Porcine oocytes were matured under various conditions, i.e., in vitro or in vivo, with different amounts of cumulus cells, with or without hormonal supplements, and with various exposure durations to the hormones, to examine the effects on spindle morphology in MII oocytes by immunofluorescence under confocal laser microscopy. Interpolar spindle length (μm) and spindle area (μm2) were compared among these maturation conditions. The spindle length was significantly shorter in IVM oocytes compared to those matured in vivo. Oocytes collected from cumulus oocyte complexes (COCs), which were poor in cumulus cells, showed smaller spindle areas than those from cumulus-rich COCs. The spindle length and area were both significantly reduced in oocytes grown without hormonal supplements. When oocytes were grown with hormonal supplements for either 6 or 22 hours for the first half of culture, there was no difference in the spindle morphology between these oocytes. These results suggested that maturation conditions significantly influence morphogenesis of MII spindles in porcine oocytes. Oocytes matured in poor conditions were more likely to have a shorter spindle length (long axis) and smaller spindle areas.
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  • Toshiyuki KUDO, Shizuyo SUTOU
    2005 Volume 51 Issue 3 Pages 411-417
    Published: 2005
    Released on J-STAGE: July 06, 2005
    Advance online publication: April 05, 2005
    JOURNAL FREE ACCESS
    Gene silencing with short interfering RNA (siRNA) expression vectors is a powerful method for the analysis of gene functions. For the expression of siRNA in mammalian cells, mammalian U6 small nuclear RNA (snRNA) promoters are widely used. However, the mammalian U6 promoter might not function well in other species. In this study, we cloned four putative chicken U6 promoters by PCR and analyzed their functions. First, we screened the chicken genomic database using the human U6 snRNA gene and identified four candidate sequences. The sequences contained some control elements in their promoter regions, but as we could not rule out that they were pseudogenes, we amplified these sequences and used them as promoters for short hairpin RNA (shRNA) expression. Using the firefly luciferase (Luc) gene as a target, transient expression assays were performed with chicken ovary-derived cells. All four putative chicken U6 promoters exhibited suppressive activity toward Luc, and so could act as a promoter for expression of the snRNA gene in the chicken genome. The promoter activity was not as strong as that of a commercially available siRNA expression vector. This probably reflects artificial sequences between the promoters and synthetic DNA encoding shRNA.
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