Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 52, Issue 2
April
Displaying 1-15 of 15 articles from this issue
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  • Satoko AIKAWA, Takanobu SATO, Tetsuo ONO, Takako KATO, Yukio KATO
    2006 Volume 52 Issue 2 Pages 195-201
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: December 28, 2005
    JOURNAL FREE ACCESS
    Prop-1 acts as an upstream regulator for the Pit-1 gene to induce development of Pit-1 lineage pituitary cell lines, GH-, PRL-, and TSH-producing cells, in the early stage of pituitary organogenesis. Furthermore, Prop-1 is presumed to be involved in the function of FSH/LH-producing cells, gonadotropes, since the defective Prop-1 gene shows hypogonadism. Recently, we reported evidence that Prop-1 directly regulates expression of the porcine FSHβ gene, thus providing a novel advance in understanding the function of Prop-1 in FSH/LH production and hypogonadism. This study was intended to demonstrate the expressions of Prop-1 gene in pituitary tumor-derived cell lines. RT-PCR analyses were conducted of Pit-1, glycoprotein α subunit (αGSU), GnRH receptor, and cyclophilin A (a ubiquitously expressing gene). We observed expression of the Pit-1 gene in αT1-1, TαT1, MtT/S, GH3, and TtT/GF cells, expression of the αGSU gene in αT1-1, αT3-1, LβT2, LβT4, TαT1, and GH3 cells, and expression of GnRH receptor gene in αT3-1, LβT2, LβT4, and GH3 cells, respectively. These expression profiles were in accord with their cell lineages, with only a few exceptions. To accurately measure the expression level of the Prop-1 gene, a quantitative analysis was performed using the real-time PCR method. This analysis demonstrated that the LβT2 and LβT4 gonadotrope cell lines, which express the FSHβ gene, express the Prop-1 gene. Taken together with our previous observation that Prop-1 is present in the adult porcine pituitary gonadotropes, Prop-1 might also be involved in development of gonadotropes and hormone production.
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  • Maria JEDLINSKA-KRAKOWSKA, Grazyna BOMBA, Karol JAKUBOWSKI, Tadeusz RO ...
    2006 Volume 52 Issue 2 Pages 203-209
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: December 28, 2005
    JOURNAL FREE ACCESS
    The aim of the study was to verify whether an increased supply of vitamins E and C prevents the detrimental effects of ozone on the testes. The experiment was performed on 5-month-old rats exposed to ozone (0.5 ppm) for 50 days (5 h daily). Simultaneously, the animals were injected with the vitamins in 5-day intervals and at different doses (0.5, 1.5, 4.5, 5 and 15 mg of vitamin E; 0.5, 3, 9, and 50 mg of vitamin C; or both vitamins together, respectively). Gonad sections were PAS stained. In the ozonized males, depletion of germ cells occurred. In the vitamin E groups, the testes were comparable to the controls, excluding the 0.5-mg-dose vitamin E group in which perivascular fibrosis and intertubular hyalinization were observed. In the vitamin C groups, intertubular hyalinization, partial arrested spermatogenesis, and desquamation of the seminiferous epithelium appeared proportionall to the vitamin dose. Additionally, premature spermiation was found at a vitamin C dose of 50-mg. In the rats injected with both vitamins, hyalinization and fibrosis appeared in addition to partial arrest of spermatogenesis and vacuolar degeneration. In conclusion, vitamin E protects against the detrimental effects of ozone in rat testes irrespective of the dose applied. This was not observed for vitamin C. Moreover, administration of higher doses of vitamin C intensified the damage to the testes caused by ozone.
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  • Yukinori YOSHIMURA, Hiroki OHASHI, Kalpana SUBEDI, Masahide NISHIBORI, ...
    2006 Volume 52 Issue 2 Pages 211-218
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: December 28, 2005
    JOURNAL FREE ACCESS
    Gallinacins (Gal) are antimicrobial peptides that play significant roles in innate immunity in chickens. The aim of this study was to examine whether age of birds and egg-laying activity (laying and non-laying caused by feed-regulation) affect the mRNA expression of Gal-1, -2, and -3 in the vagina of hens, and whether their expressions are changed in response to the stimulation with salmonella enteritidis (SE) and lipopolysaccharide (LPS). White Leghorn hens were divided into groups of young and old laying hens, and groups of laying and non-laying hens after feed-regulation. Vaginal cells were cultured and stimulated with SE or LPS. Expressions of Gal-1, -2, and -3 mRNA in their vaginal mucosa and cultured cells were examined by quantitative real-time RT-PCR. The expressions of Gal-1, -2, and -3 of the vaginal mucosa were significantly greater in old birds than in young birds. Expression of these Gals in the vagina were decreased in the regressed oviduct of non-laying birds compared with laying birds. The expressions of Gal-1, -2, and -3 in the cultured vaginal cells were increased by stimulation with SE or LPS within 24 h. These results suggest that the mRNA expressions of Gal-1, -2 and -3 in the vagina of laying hens increased with age, whereas they decreased in the regressed oviduct during the non-laying phase. Also, synthesis of these antimicrobial peptides in the vagina may increase in response to SE and LPS to eliminate SE bacteria.
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  • Longquan REN, Mohamed S. MEDAN, Mariko OZU, Chunmei LI, Gen WATANABE, ...
    2006 Volume 52 Issue 2 Pages 219-228
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: January 16, 2006
    JOURNAL FREE ACCESS
    The effect of induced cryptorchidism on testicular function and sperm motility was investigated. Bilateral cryptorchidism was created surgically in adult male rats (treated group), and sham-operated rats were used as a control group. Five rats from each group were sacrificed on days 1, 3, 5, and 7 after surgery. The percentage of motile spermatozoa began to decrease 1 day after the operation, followed by an abrupt decline 3 and 5 days later in cryptorchid rats. Furthermore, there were significant decreases in the other sperm motility parameters 5 days after inducement of cryptorchidism. In cryptorchid rats, plasma concentrations of LH, FSH, testosterone, and inhibin B were significantly lower than in the control group 1 day after the operation. Thereafter, plasma concentrations of LH, FSH, and testosterone gradually increased in the cryptorchid rats. On the other hand, plasma concentrations of inhibin B showed a further decline from day 3 after the operation onward. Concentrations of immunoreactive (ir)-inhibin, but not testosterone, in testicular interstitial fluid were remarkably increased until 3 days after surgery in the cryptorchid rats, and declined thereafter. Testicular response to human chorionic gonadotropin (hCG) for testosterone release was decreased in the cryptorchid rats compared with the control rats, indicating that heat stress to testes resulted in a reduction of the activity of Leydig cells and Sertoli cells. These results clearly indicate that heat stress to the testes resulted in a significant reduction of sperm activity within 3 days, and this was followed by changes in testicular endocrine function.
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  • Chul Wook KIM, Yeon Hee HONG, Sung-Il YUN, Sang-Rae LEE, Young Hyun KI ...
    2006 Volume 52 Issue 2 Pages 229-237
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: January 16, 2006
    JOURNAL FREE ACCESS
    To identify genetic markers associated with economic traits in pigs, 157 microsatellite markers were examined in Yorkshire pigs. Thirty eight female Yorkshire pigs were initially examined and six of them were selected as progenitors; half were more than 1.5 standard deviations (SD) above the mean for average daily gain (ADG) and backfat thickness (BFT), and the remaining half were more than 1.5 SD below the mean. These pigs were then mated to male Duroc pigs, and 200 F2 pig offspring were examined for the association of specific alleles with ADG and BFT. To confirm the specific markers identified in the initial analysis, associations of significant markers with economic traits were further examined in 228 additional performance-tested purebred pigs. Twenty-five microsatellite markers were significantly associated with either ADG or BFT, and among these, 17 were associated with both traits. The markers with the highest association to ADG were also associated with BFT. Our study reveals that specific markers could be used to predict economic significance, and confirms several quantitative trait loci (QTL) identified in previous studies. However, further analysis with more closely-spaced microsatellite markers is required to refine predictive values for economic traits and positions of QTL that are reliable for actual phenotypic prediction.
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  • Hiromi MIKI, Narumi OGONUKI, Kimiko INOUE, Tadashi BABA, Atsuo OGURA
    2006 Volume 52 Issue 2 Pages 239-248
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: January 16, 2006
    JOURNAL FREE ACCESS
    In micromanipulation experiments using immature oocytes, final ooplasmic maturation is often compromised because the oocytes are usually first freed from their nurturing cumulus cells. This study was undertaken to determine whether cumulus-free in vitro maturation (IVM) in mice could be improved by modifying IVM medium having defined components. Cumulus-free germinal vesicle (GV) stage oocytes were subjected to IVM in either αMEM medium, TYH medium, or a 1:1 mixture of the two (termed TaM). TYH medium produced a better maturation rate (181/196; 92.3%) than αMEM (184/257; 71.6%). However, αMEM supported better embryo development to the morula/blastocyst stage than TYH following in vitro fertilization (93.3% vs. 76.5%) or parthenogenetic activation (82.4% vs. 60.4%). Mitochondrial distribution in MII oocytes was diffuse following IVM in αMEM, but was aggregated with TYH. The maturation promoting factor (MPF) activity in MII oocytes was significantly higher in TYH than in αMEM (P<0.05). Oocytes cultured in TaM had intermediate characteristics and essentially resembled in vivo matured oocytes, with the mitochondrial distribution pattern being most typical of that condition. The highest rate of development from GV oocytes to full-term fetuses following in vitro fertilization and embryo transfer to foster mothers (23.8%) was obtained using TaM. When this IVM system was applied to MI oocytes injected with spermatocytes, offspring were first obtained without cytoplasmic replacement at MII. Thus, optimization of the culture medium can considerably improve the quality of cumulus-free oocyte IVM in mice.
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  • Klaus-Peter BRÜSSOW, Helmut TORNER, Jozsef RÁTKY, Noboru M ...
    2006 Volume 52 Issue 2 Pages 249-257
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: January 20, 2006
    JOURNAL FREE ACCESS
    In the pig, a temporal relationship is suggested between sperm release from the sperm reservoir (SR) and ovulation, but the mechanism(s) is still under discussion. In two experiments, the influence of transferred ova on the release of SR-spermatozoa at ovulation and the effect of supplementation with non-sulfated glycosaminglycan hyaluronan (HA) on embryo development and the number of accessory spermatozoa, respectively, were examined. PMSG/hCG primed ovectomized gilts that had previously received endoscopic low-dose insemination into the cranial uterine horn were used as an experimental model. After salpingectomy, tubal segments (ampulla, cranial, and caudal isthmus) were flushed and sperm numbers or respective accessory spermatozoa were counted. In Experiment 1, the distribution of the sperm population was altered in the presence of cumulus-oocyte-complexes (COCs). A higher proportion of spermatozoa was found after transfer of COCs into one oviduct in the ampulla and cranial isthmus segments compared with the controls (17.5 vs. 4.9%, p<0.05). In Experiment 2, the quality of the transferred ova and treatment influenced the presence of accessory spermatozoa. Transfer of COCs together with HA increased (p<0.05) the number of accessory spermatozoa compared with the other treatment groups and was similar to those in the "undisturbed" controls. No modifications were obtained regarding mean blastomere numbers (2.6 ± 0.2 to 3.1 ± 0.2). In summary, this study was demonstrated that cumulus-oocyte-complexes may be involved in triggering sperm release from the pig oviductal SR and that HA might be related to sperm release.
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  • Maksym KREMENSKOY, Yuliya KREMENSKA, Masako SUZUKI, Kei IMAI, Seiya TA ...
    2006 Volume 52 Issue 2 Pages 259-266
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: February 10, 2006
    JOURNAL FREE ACCESS
    Methylation of DNA in CpG islands plays an important role during fetal development and differentiation because CpG islands are preferentially located in upstream regions of mammalian genomic DNA, including the transcription start site of housekeeping genes and are also associated with tissue-specific genes. Somatic nuclear transfer (NT) technology has been used to generate live clones in numerous mammalian species, but only a low percentage of nuclear transferred animals develop to term. Abnormal epigenetic changes in the CpG islands of donor nuclei after nuclear transfer could contribute to a high rate of abortion during early gestation and increase perinatal death. These changes have yet to be explored. Thus, we investigated the genome-wide DNA methylation profiles of CpG islands in nuclei donor cells and NT animals. Using Restriction Landmark Genomic Scanning (RLGS), we showed, for the first time, the epigenetic profile formation of tissues from NT bovine fetuses produced from cumulus cells. From approximately 2600 unmethylated NotI sites visualized on the RLGS profile, at least 35 NotI sites showed different methylation statuses. Moreover, we proved that fetal and placental tissues from artificially inseminated and cloned cattle have tissue-specific differences in the genome-wide methylation profiles of the CpG islands. We also found that possible abnormalities occurred in the fetal brain and placental tissues of cloned animals.
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  • Yukihiro FUJINO, Yoshiyuki NAKAMURA, Hiroshi KOBAYASHI, Kazuhiro KIKUC ...
    2006 Volume 52 Issue 2 Pages 267-275
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: February 01, 2006
    JOURNAL FREE ACCESS
    We examined the relationship between the time elapsed after human chorionic gonadotropin (hCG) administration and developmental stage of porcine embryos after collection. Prepubertal gilts, 7 to 8 months old, were given 1500 IU equine chorionic gonadotropin (eCG) intramuscularly, followed by 500 IU hCG 72 h later. The treated gilts were inseminated artificially on Day 1 (Day 0=the day of hCG administration) and on Day 2. Embryos were collected surgically on Day 6 (140, 144, and 147 h after hCG administration) or on Day 7 (164, 168, and 171 h), and the developmental stages of the collected embryos were examined. From 75.2% (276/367) of the prepubertal gilts treated with hormones, we collected an average of 20.7 embryos per gilt with normal morphology. At 140 h after hCG administration, morulae (54.4%) could be collected. At 144 h, morulae and early blastocysts (57.7% and 28.9%, respectively) were collected. By 147 h, the proportion of embryos at the blastocyst to expanded blastocyst stages had increased (10.0%). From 164 h to 171 h, expanding or expanded blastocysts of more than 200 μm in diameter and hatched blastocysts could be collected. The proportion of hatched blastocysts increased from 3.2% (164 h) to 41.0% (171 h). These results suggests that although the number of ovulations differed among gilts, porcine embryos at the appropriate stages can be collected efficiently by controlling the time elapsed between hCG administration and embryo collection.
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  • Maksym KREMENSKOY, Yuliya KREMENSKA, Masako SUZUKI, Kei IMAI, Seiya TA ...
    2006 Volume 52 Issue 2 Pages 277-285
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: February 10, 2006
    JOURNAL FREE ACCESS
    Abnormal development and fetal loss during postimplantation period are concerns for production of nuclear transferred animals. Aberrant DNA methylation is one of the reasons for poor survival of cloned animals. In mammalian genome DNA, CpG islands are preferentially located at the start of transcription of housekeeping genes and are associated with tissue-specific genes. The correct and consecutive mechanisms of DNA methylation in the CpG islands are necessary for selective gene expressions that determine the properties of individual cells, tissues, and organs. In this study, we investigated the methylation status of the CpG islands of the bovine Leptin and POU5F1 genes in fetal and placental tissues from fetuses produced by artificial insemination (AI) and nuclear transfer (NT) at days 48 and 59 of pregnancy. Altered DNA methylation was observed in the normal and cloned fetal, placental, and endometrial tissues using bisulfite sequencing and pyrosequencing. Different tissue-specific methylated regions in the bovine Leptin and POU5F1 genes show a variable methylation status in NT fetuses compared to AI control.
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Research Note
  • Takashi SHIMIZU, Hirotada AKIYAMA, Yasuyuki ABE, Hiroshi SASADA, Eimei ...
    2006 Volume 52 Issue 2 Pages 287-291
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: December 28, 2005
    JOURNAL FREE ACCESS
    Protein phosphorylation on certain serine or threonine residues preceding proline (Ser/Thr-Pro) is a pivotal signaling mechanism in diverse cellular processes. Pin1 is a highly conserved enzyme that isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Although much protein is phosphorylated in the ovary, the role of Pin1 in the ovary is still unknown. The purpose of this study is to investigate the effects of gonadotropins on protein and mRNA expression of Pin1 in mice ovaries. Quantitative PCR analysis showed that the expression of Pin1 mRNA significantly increased in the ovaries of equine chorionic gonadotropin (eCG)-treated mice compared with those of untreated mice (P<0.05). However, human chorionic gonadotropin (hCG) attenuated the expression of Pin1 mRNA increased by eCG. The protein level of Pin1 showed the same tendency as the expression of mRNA. The mRNA expression of E2F transcription factor, which controlled the expression of Pin1, was significantly decreased in the eCG-treated ovaries compared with the controls (P<0.05). These observations suggest that gonadotropins may regulate the expression of Pin1 without E2F transcription factor, indicating that Pin1 might be an important factor for protein signal transduction during follicular development.
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  • Takako ISHIJIMA, Yoshiyasu KOBAYASHI, Dong-Soo LEE, Yoshiko Yanagimoto ...
    2006 Volume 52 Issue 2 Pages 293-299
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: December 28, 2005
    JOURNAL FREE ACCESS
    The cryopreservation of ovarian tissues is a technology with significant potential for the preservation of the genetic resource materials of working dogs, including guide dogs for the blind. However, no attempt has been reported on cryopreservation of the canine ovary. Thus, we evaluated a vitrification method for cryopreservation of canine ovaries and determined the potential functionality of vitrified-warmed canine ovaries by means of transplantation into non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice. All ovarian tissues cryopreserved by vitrification were morphologically normal in terms of histology. Cryopreserved ovaries were transplanted into the ovarian bursa of the NOD-SCID mice, and the xenografts were recovered from 23 of 23 mice (100%) 4 weeks after the operation. The transplanted canine tissue was tightly adhered to the mouse ovary. Although antral follicle formation did not occur after grafting, proliferating cell nuclear antigen immunoreactivity was detectable in many of the granulosa cells in the primary follicles of the grafts. These results indicate that cryopreservation of the canine ovary by vitrification appears to have the potential to restore endocrine function and ovulation potential.
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  • Akira OKANO, Hisashi KISHI, Hitomi TAKAHASHI, Masashi TAKAHASHI
    2006 Volume 52 Issue 2 Pages 301-306
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: January 17, 2006
    JOURNAL FREE ACCESS
    Some studies in mammalian species recently demonstrated that tumor necrosis factor (TNF)-αplays an important role for corpus luteum (CL) function by way of apoptosis during the estrous cycle. The objectives of this study were to clarify the induction of apoptosis in cultured porcine luteal cells by TNF-α treatment. Luteal cells prepared from porcine ovaries collected from crossbred mature gilts on Days 10-14 of the estrous cycle were isolated and examined as follows: 1) Flow cytometric analysis was carried out to determine apoptosis in cultured luteal cells. 2) Single-cell gel electrophoresis (comet assay) was performed to investigate apoptotic DNA fragmentation in luteal cells. The results of the flow cytometric analysis and comet assay demonstrated coincidentally that TNF-α induces DNA fragmentation in luteal cells causing apoptosis. These results revealed that TNF-α is an inducing factor of apoptosis in luteal cells.
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  • Bajram BERISHA, Harald WELTER, Takashi SHIMIZU, Akio MIYAMOTO, Heinric ...
    2006 Volume 52 Issue 2 Pages 307-313
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: January 16, 2006
    JOURNAL FREE ACCESS
    The aim of this study was to evaluate the expression pattern of mRNA for fibroblast growth factor 1 (FGF1), FGF7, and their receptor variants (FGFR2IIIb) in time-defined follicle classes before LH surge, between LH surge and ovulation, and in the early corpus luteum (CL) in the cow. The ovaries were collected by transvaginal ovariectomy (n=5 cows/group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (before LH surge); 3-5 h after GnRH (during LH surge); 10 h after GnRH; 20 h after GnRH; 25 h after GnRH (periovulation), and early CL (Days 2-3). The mRNA expression was analyzed by quantitative real-time PCR (RotorGene 3000). The mRNA expression of FGF1 showed no significant differences in the follicle groups examined, but increased significantly at the early CL phase. A transient increase in FGF7 mRNA expression was observed 3-5 h after GnRH and again in the early CL phase. In contrast, the expression of FGFR2IIIb was constant throughout the period from the final growth of the follicle to early CL formation. The results of this study suggest that FGF1 and FGF7 may be involved differently in the process of follicle maturation and CL formation, which is strongly dependent on angiogenesis.
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  • Naoko NUKUMI, Mami SEKI, Tokuko IWAMORI, Tetsushi YADA, Kunihiko NAITO ...
    2006 Volume 52 Issue 2 Pages 315-320
    Published: 2006
    Released on J-STAGE: April 29, 2006
    Advance online publication: February 01, 2006
    JOURNAL FREE ACCESS
    Although whey acidic protein (WAP) has been identified in the milk of a range of species, it has been predicted that WAP is not secreted into human milk as a result of critical point mutations within the coding region. In the present study, we first investigated computationally the promoter region of mutated human WAP genes by comparing with those of other known WAP genes. Computational database analyses showed that the human WAP promoter region was highly conserved, as in other species with milk WAP. Next, we evaluated the activity of the human WAP promoter (2.6 kb) using a reporter gene assay. MCF-7 cells were stably transfected with the hWAP/hGH (human growth hormone) fusion gene, cultured on Matrigel, and treated with lactogenic hormones. Radioimmunoassay detected hGH in the culture medium, indicating that the human WAP promoter was responsible for the lactogenic hormones. The human WAP promoter was significantly more active in MCF-7 cells than the mouse WAP promoter (2.4 kb). The present results provide us with important information on the molecular evolution of milk protein genes.
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