Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 49, Issue 1
February
Displaying 1-14 of 14 articles from this issue
Japanese Society for Animal Reproduction: Award for Outstanding Research 2002
  • Kazumasa HONDA
    Article type: Japanese society for Animal Reproduction:Award for Outstanding Research 2002
    Subject area: none
    2003 Volume 49 Issue 1 Pages 1-11
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    To explore mechanisms of osmotic control of neurohypophysial hormone release, electrical activity of magnocellular neurons (MCNs) in the hypothalamus, related neurons and efferent renal sympathetic nerve activity (RSNA) were recorded from urethane-anesthetized rats. Local osmotic stimulation applied to organum vasculosum of the lamina terminalis (OVLT) or median preoptic nucleus (MnPO) excited MCNs. Although OVLT neurons projected to MCNs were unresponsive to hyperosmotic stimulation, those projected to MnPO and also receiving excitatory inputs from MCNs, were excited by it. MnPO neurons, which were driven by OVLT stimulation and projected to MCNs, were also osmosensitive. Excitatory connections thus exist from MCNs to OVLT, from OVLT to MnPO and from MnPO to MCNs. Neurons in each of these connections were osmosensitive. This circuit thus appears to constitute an osmoreceptor complex essential for the osmoreception of MCNs. MnPO neurons constituting a part of the osmosensitive circuit were also sensitive to hemodynamic change. Thus this circuit may integrate hemodynamic and osmotic information. Local anesthesia of MnPO diminished activation of RSNA and pressor response induced by third cerebroventricular injection of hypertonic saline. The results suggest that the osmosensitive circuit is involved in body fluid regulation not only by controlling vasopressin secretion but also by modulating sympathetic outflow.
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  • Shinichi HOCHI
    Article type: Japanese society for Animal Reproduction:Award for Outstanding Research 2002
    Subject area: none
    2003 Volume 49 Issue 1 Pages 13-21
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both in vitro and in vivo when the embryos were exposed to vitrification solution in a stepwise manner. The vitrification procedure was also applied to the relatively large expanded blastocysts. (2) Bovine embryos produced in vitro have been considered to be highly sensitive to the process of cryopreservation. To solve this problem, Day-7 blastocysts produced in a serum-free system were cooled at 0.3 C/min rather than 0.6 C/min before being plunged into liquid nitrogen, resulting in no loss of the post-thaw viability. The supplementation of LAA in IVM/IVF media or IVC medium was effective in producing pronuclear-stage zygotes or morula-stage embryos relatively tolerable to freezing, respectively. (3) Transmission electron microscopic observation of immature equine oocytes showed that cellular injury occurred near the sites of gap-junctions between cumulus cells and the oocyte. In cattle, higher fertilization rates of oocytes were obtained when the oocytes were subjected to cryopreservation at an intermediate stage during IVM (GVBD for freezing, Met-I for vitrification). Vitrification of bovine Met-II oocytes in open-pulled glass capillaries, characterized by an ultra-rapid cooling rate (3,000-5,000 C/min), was found to avoid any harmful influence of vitrification and warming.
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  • Masahiko NISHIGAI
    Article type: Japanese society for Animal Reproduction:Award for Outstanding Research 2002
    Subject area: none
    2003 Volume 49 Issue 1 Pages 23-36
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    The conditions of embryo transfer by the stepwise method, in which frozen-thawed embryos are transferred on day 7 (day 0=onset of estrus ) , were investigated with the aim of increasing pregnancy rates in frozen-thawed embryo transfer. The use of a vaginal speculum to prevent bacterial infection when passing an embryo transfer gun through the vagina yielded a pregnancy rate equal to or higher than that with application of a sheath cover to the transfer gun. Administration of a sedative, xylazine, to recipient cattle for preventing movement at the time of embryo transfer improved the pregnancy rate. The influence of the time from thawing of frozen embryos to transfer and of the transportation of the recipient by truck upon pregnancy rate was investigated. Embryo transfer within 60 minutes after aspiration into a straw or transportation of the bovine recipient, 1.5 hours each way before and after transfer, had no influence on pregnancy rate. Relations of the embryonic developmental stage and morphological quality after thawing of frozen embryos to pregnancy rate were investigated in recipients of nulliparous Holstein heifers. The pregnancy rate increased as the embryonic developmental stage advanced from compacted morula, early blastocyst, and blastocyst in that order. The pregnancy rate obtained with blastocyst stage embryos was significantly (P<0.05) higher than that with compacted morula stage embryos, and there was no significant difference in pregnancy rates between excellent morphological quality and good morphological quality for compacted morula stage embryos. When correlation of luteal function and pregnancy rate was investigated in bovine recipients, pregnancy rate showed a tendency to increase with increasing blood progesterone (P) concentration on the day before (on day 6 after estrus) and the day of embryo transfer. The pregnancy rate in bovine recipients, which showed a blood P concentration of ≥2.5 ng/ml on the day before embryo transfer, was significantly (P<0.05) higher than that in those with a blood P concentration of <2.5 ng/ml. Pregnancy rate showed a tendency to increase with decreasing blood estradiol-17β (E 2) concentration on the day of embryo transfer. Activation of luteal function by administration of human chorionic gonadotropin (hCG) in cycling cattle was investigated for its effect on increasing pregnancy rate in bovine recipients. A follicle coexisting with cyclic CL ovulated and induced CL formed after injection of hCG 1,500 IU 5 days after ovulation. The blood P concentration was significantly (P<0.05) higher in the administration group than in the control group, and the blood E2 concentration rapidly decreased, showing a lower concentration than in the control group. These results suggest the possibility that the pregnancy rate could be improved by administration of hCG. Pregnancy rate following intramuscular injection hCG 1,500 IU was comparatively investigated in parous Japanese Black beef cattle receiving frozen-thawed embryos 7 days after estrus. Pregnancy rate was 67.5% in the group in which hCG was administered on day 6 after estrus, and was significantly (P<0.05) higher than that in the control group (45.0%) and the group in which hCG was administered on day 1 after estrus (42.5%), revealing that hCG administration facilitated pregnancy. Transfer of frozen-thawed embryos in the blastocyst stage within 60 minutes after the aspiration into a straw, with a vaginal speculum after administration of xylazine is suggested as a way of improving pregnancy rate in bovine recipients with favorable luteal function and in those with luteal function activated by administration of hCG on the day before embryo transfer.
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Original Article
  • Sachiko IKUMI, Masatsugu ASADA, Ken SAWAI, Yutaka FUKUI
    Article type: Original Article
    Subject area: none
    2003 Volume 49 Issue 1 Pages 37-43
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    A principal nuclear transfer procedure is to inject a donor cell into the perivitelline space in an enucleated oocyte and then electric fusion is performed (cell fusion method). The effects of activation methods in reconstructed oocytes for the serum-starved somatic cell cloning procedure were investigated in this study by means of intracytoplasmic injection (ICI). Bovine oocytes were enucleated at 18-22 h for in vitro maturation, and subsequently the nucleus of cumulus cell collected from Japanese Black Bulls (JBCC) after 5-7 days of starved culture was injected into the recipient cytoplast with a piezo-micromanipulator. At 1 h after ICI, reconstructed oocytes were stimulated with ethanol (ET) or calcium ionophore (CaI) as the first activation treatment, followed by cycloheximide (CHX) or 6-dimethylaminopurin (DMAP) treatment as the second activation. In the experiment on the first activation method, the proportion of reconstructed oocytes developing to the blastocyst stage was significantly (p<0.01) higher in the ET activation method than that with CaI (10.5% and 4.7%, respectively). And the experiment on the second activation method after ET treatment showed similar proportions of blastocyst development in both CHX and DMAP treatments (5.9% and 2.8%, respectively). The present results indicated that combined activation treatment with ET and CHX was efficient for reconstructed bovine oocytes by ICI.
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  • Kazuyoshi HASHIZUME, Toru TAKAHASHI, Manabu SHIMIZU, Junichi TODOROKI, ...
    Article type: Original Article
    Subject area: none
    2003 Volume 49 Issue 1 Pages 45-53
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    In vitro cell culture is a convenient tool for studying cellular mechanisms. In the present study, production of matrix-metalloproteinases (MMPs) in bovine endometrial (containing both epithelial and stromal cells) monolayer cells was examined. Blastocysts attached to the endometrial cells in a monolayer culture were examined for their effects on MMP-2 production. Initial attachment of blastocysts to the monolayer inhibited MMP-2 production by endometrial cells. But once trophoblast cells began to migrate into the endometrial cell layer, MMP-2 production increased, and at the same time MMP-9 production also became evident in the medium. In order to understand how blastocysts affected MMP-2 production, we examined the effect of progesterone, estradiol, insulin-like growth factors (IGFs), tumor necrosis factors (TNFs), and interferon-tau (IFN-τ) supplementation. It was IFN-τ that inhibited the production of MMP-2. In addition, progesterone at a lower dose appeared to inhibit MMP-2 production. Both TNF-α and TNF-β strongly stimulated the production of MMP-2 and MMP-9, whereas IGFs had no effect. Based on these findings, it appears that conceptus has the capacity to inhibit MMP activity.
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  • Tomomi TANAKA, Tsuyoshi SHIINA, Shinji HAYASHI, Hiroaki OKAMURA, Hideo ...
    Article type: Original Article
    Subject area: none
    2003 Volume 49 Issue 1 Pages 55-60
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    The distribution and morphology of immunoreactive estrogen receptor α (ERα) - containing cells were examined in the cattle brain. The brains were collected from two cows and a male calf. One of the cows was sacrificed one week after parturition. ERα expressions in the preoptic area of the rostral forebrain and the medial basal hypothalamic area were evaluated immunohistochemically using a mouse monoclonal antibody. In all animals, the signals that indicate ERα were detected in the medial preoptic area, the level of the organum vasculosum of the lamina terminalis and the bed nucleus of the stria terminalis. In the caudal hypothalamic region, they were detected in the arcuate nucleus, the periventricular and ventromedial hypothalamic nuclei. ERα-containing cells were characterized by a dense nuclear reaction product. In all the subjects examined, ERα signals were clearly detected in the cytoplasm as well. The density of ERα expression was different among the three cattle; the number of ERα containing cells in the postpartum cow was much lower than that in the other cow and the male calf. The present findings suggest that ERα expression in the forebrain is influenced by the reproductive status in the cattle.
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  • Shu HASHIMOTO, Kouji KIMURA, Hisataka IWATA, Ryo TAKAKURA
    Article type: Original Article
    Subject area: none
    2003 Volume 49 Issue 1 Pages 61-66
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    The effects of the medium (TCM 199 or SOFaa) and temperature (20 or 39 C) during meiotic arrest by cycloheximide (CHX) under air on the developmental competence of bovine oocytes after in vitro maturation (IVM) and fertilization (IVF) were investigated. Oocytes were maintained in meiotic arrest by 10 μg/ml CHX in a 50-μl droplet of 25-mM HEPES-buffered TCM 199 (H199) at 39 C or synthetic oviduct fluid (HSOFaa) at 20 or 39 C in air for 24 h. After release from the arrest, the oocytes was matured and fertilized in vitro and their developmental competence was examined. The developmental rate of oocytes arrested in HSOFaa at 20 C to the blastocyst stage was similar to that of non-arrested oocytes but was significantly higher (P<0.05) than that of oocytes arrested at 39 C in H199 or in HSOFaa. In consideration of oocyte transport conditions, we also investigated the meiotic arrest of oocytes maintained in a 0.25-ml straw by CHX individually with 10 μl HSOFaa or as a group (40-50 oocytes) with 170-200 μl HSOFaa at 20 C in air for 24 h. After release from meiotic arrest, the developmental competence of these oocytes was assessed similarly. The developmental rate of oocytes treated with CHX individually was similar to that of those treated with CHX in 50-μl droplet of HSOFaa at 20 C. However, the developmental rate of oocytes treated with CHX as a group was lower than that of oocytes treated with CHX in a 50-μl droplet. Five blastocysts developed from oocytes maintained in meiotic arrest in a plastic straw were transferred to five recipient heifers. Consequently, three recipients became pregnant and 2 calves were delivered. The results of the present study indicate that bovine oocytes treated with CHX in HSOFaa at 20 C under air retain the same developmental competence as non-arrested oocytes.
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  • Shinji TSUKAHARA, Korehito YAMANOUCHI
    Article type: Original Article
    Subject area: none
    2003 Volume 49 Issue 1 Pages 67-77
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    Neurons in the lateral septum (LS) and preoptic area (POA) are known to play an inhibitory role in feminine sexual behavior regulation in male rats. In this study, the distribution of neurons containing glutamic acid decarboxylase (GAD) and of the peptidergic neurotransmitters neurotensin (NT), enkephalin (ENK), neuropeptide Y (NPY), and cholecystokinin (CCK), was examined immunohistochemically in the LS and POA of castrated male rats subcutaneously implanted with estrogen-containing Silastic tubes. Colchicine was injected into the lateral ventricle of the animals. The forebrain sections were immunostained for each substance. A large number of GAD-immunoreactive (ir) cells were found in the LS. Many NT-ir cells were seen in the intermediate and ventral parts of the LS at the rostral and middle levels. A considerable number of ENK-ir cells were scattered over the LS at the rostral and middle levels and were observed in the ventral part of the caudal LS. There were only a few NPY-ir cells in the LS. No CCK-ir cells were observed in the LS. In the POA, GAD-ir cells were observed in abundance. Many NT-ir cells were seen, especially in the medial preoptic nucleus. Some ENK-ir cells and a few NPY-ir cells were found in the medial POA. CCK-ir cells of the POA were restricted to the periventricular and paraventricular hypothalamic nuclei.
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  • Maho ISHIDA, Keiji HIRABAYASHI, Masatoshi SUZUKI, Keitaro YAMANOUCHI, ...
    Article type: Original Article
    Subject area: none
    2003 Volume 49 Issue 1 Pages 79-85
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    20α-hydroxysteroid dehydrogenase (20α-HSD), a member of the aldo-keto reductase (AKR) superfamily, metabolizes progesterone to its inactive form, 20α-dihydroprogesterone (20α-OHP). 20α-HSD is associated with functional luteolysis and plays a significant role in the reproductive system of rodents. Here we report cloning and determination of the chromosomal location of the mouse 20α-HSD gene. The mouse 20α-HSD gene cloned using 129 SvJ mouse genomic library spanned approximately 18 kb from exon 1 to exon 9. A single transcription start site was identified by 5'-rapid amplification of cDNA ends (RACE) at the site of 50 nucleotides upstream from the ATG translation initiation codon. The exon-intron organization of mouse and human 20α-HSD genes were similar. Using 17.5 kb of genomic clone as a probe, we determined the chromosomal location by fluorescence in situ hybridization (FISH). The chromosome in which the mouse 20α-HSD gene was detected was identified as chromosome (Chr) 13 according to simultaneous G- and R-banding. Because type 5 17β-HSD gene, a member of the AKR superfamily, is also located on Chr 13, the present result supports the syntenic relationship between mouse Chr 13 and human Chr 10, which was previously suggested at the chromosomal loci of a gene cluster of the AKR superfamily.
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  • Satoko KAWAZU, Hisashi KISHI, Erina SAITA, Wanzhu JIN, Akira K. SUZUKI ...
    Article type: Original Article
    Subject area: none
    2003 Volume 49 Issue 1 Pages 87-97
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    Gonadal function in the male golden hamster (Mesocricetus auratus) was investigated during exposure to a short photoperiod condition. Within 3 weeks of exposure to the short photoperiod condition, FSH and testosterone in the plasma significantly decreased, and subsequently immunoreactive (ir)-inhibin significantly decreased. Testicular contents of ir-inhibin and testosterone, and pituitary contents of LH and FSH also significantly decreased by 3 weeks with regression of weight of testes, epididymis and seminal vesicles and sperm head count. Circulating LH varied but not significantly. Thereafter, all reproductive parameters and secretion of LH, FSH, ir-inhibin and testosterone gradually recovered after 17 weeks of exposure even though animals continued to be subjected to the short photoperiod condition. Plasma concentrations of inhibin B and inhibin pro-αC were detectable and were significantly decreased after 15 weeks of exposure to the short photoperiod, but their levels were still detectable. Immunopositive reaction of inhibin α and βB subunits was found in Sertoli cells and Leydig cells in the regressed testes of animals subjected to short photoperiod as was also seen in animals before exposure to the short photoperiod. Although the spermatogenic cycle was suppressed like prepubertal animals, the present study showed that the testicular recovery, so-called refractoriness, is functionally different from the developing stage of immature animals, especially with regard to inhibin secretion. The present results showed that changes in FSH preceded changes in inhibin during the regression and recovery phases, indicating that FSH is a major regulatory factor of inhibin secretion in male golden hamsters. The present study also demonstrated that regressed testes still secrete a small amount of bioactive inhibin during exposure to a short-photoperiod condition.
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  • Alexander V. SIROTKIN, Roland. GROSSMANN
    Article type: Original Article
    Subject area: none
    2003 Volume 49 Issue 1 Pages 99-106
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    The aim of our study was to examine the involvement of IGF-II, tyrosine kinases (TK)- and MAP kinases (MAPK)-dependent intracellular mechanisms in the control of ovarian functions in the domestic fowl, as well as the role of these kinases in mediating the IGF-II effect on this process. For this purpose, we studied the influence of IGF-II (0,1,10 or 100 ng/ml), inhibitors of TK (AG1024, 1 μg/ml), MAPK (PD98059, 5 μg/ml), and their combinations, on proliferation (expression of proliferation-related substances PCNA), apoptosis (apoptosis-associated protein bax), TK (phosphotyrosine), MAPK (ERK1,2), cyclin-dependent protein kinase 2 (p34/cdc2) and transcription factor CREB-1, as well as on the release of progesterone (P), testosterone (T), estradiol (E) and arginine-vasotocin (AVT) in cultured fragments of ovarian follicles. The presence of substances within ovarian cells was evaluated by SDS PAGE-Western immunobloting, and release of the substances was measured by using RIA/EIA of ovarian fragments-conditioned medium. It was found, that the addition of IGF-II to the culture medium (1-100 ng/ml) substantially increased expression of PCNA, MAPK and CREB, and decreased the level of p34/cdc2 and bax, but not TK. Furthermore, exogenous IGF-II inhibited P (at a concentration of 100 ng IGF-II/ml medium), and stimulated T (1,10,100 ng/ml), E (10,100 ng/ml) and AVT (1 ng/ml) release by cultured ovarian cells. Inhibitor of TK, when given alone, increased MAPK and E, inhibited p34/cdc2 and AVT, and did not affect accumulation of TK, P or T. Furthermore, TK blocker prevented effects of IGF-II on T, E and AVT, but not on TK, MAPK, p34/cdc2 and P. MAPK blocker augmented PCNA, MAPK, T and AVT expression, but not P or E, and suppressed expression of p34/cdc2 and bax. Furthermore, MAPK inhibitor, given together with IGF-II, prevented or even reversed the action of IGF-II on PCNA, P, T and AVT, but not on MAPK, p34/cdc2, CREB, bax or E. These observations suggest the involvement of IGF-II, TK and MAPK in the control of proliferation, apoptosis, steroid and peptide hormones by avian ovarian cells, as well as of the involvement of these kinases in mediation of some IGF-II effects on ovarian cells.
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Research Note
  • Shin SUGIURA, Shin-ichi KASHIWABARA, Shigeki IWASE, Tadashi BABA
    Article type: Research Note
    Subject area: none
    2003 Volume 49 Issue 1 Pages 107-111
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    The gene encoding TATA-binding protein-related factor 2 (TRF2/TLF/TLP/TRP), essential for the progress of spermiogenesis, is abundantly expressed in mammalian testis. A sequence database search revealed that mouse TRF2 is encoded by two mRNAs containing the same protein-coding region and different 5'-untranslated regions. Northern blot analysis using DNA probes specific for the 5'-untranslated regions demonstrated that these two mRNAs are distinguished from each other by the expression patterns: ubiquitous and testis-specific expression. The ubiquitously expressed form of TRF2 mRNA was present at a very low level throughout testicular development, whereas expression of the testis-specific form was first detectable in the 14-day-old testis, and the mRNA level abundantly increased at the later stages of testicular development. Western blot analysis indicated that the TRF2 level increases during testicular development, which is consistent with the expression pattern of the testicular form of TRF2 mRNA. Thus, the presence of the testis-specific form of TRF2 mRNA may account for overexpression of the TRF2 gene in the testis.
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  • Tomomi TANAKA, Toshihiko YAMAGUCHI, Hideo KAMOMAE, Yoshihiro KANEDA
    Article type: Research Note
    Subject area: none
    2003 Volume 49 Issue 1 Pages 113-119
    Published: 2003
    Released on J-STAGE: February 26, 2003
    JOURNAL FREE ACCESS
    Four female Shiba goats were used to determine the influence of body weight loss by dietary restriction on estrous cyclicity. The dietary restriction was started on the day following ovulation. The goats were fed hay cube and straw at an amount of 30% of energy requirement based on weekly body weight measurement. The ovaries were monitored daily by transrectal ultrasonography and blood samples were collected daily by jugular venipuncture for ovarian steroids analysis. After the start of food restriction, all animals lost body weight and entered ovarian quiescence. Intervals to the onset of ovarian quiescence tended to depend on the body weight of each animal at the start of food restriction. The mean concentration of progesterone during the mid-luteal phase (from 7 to 13 days after ovulation) in the last estrous cycle before ovarian quiescence was significantly lower than that in normal estrous cycle of the control period (19.7 ± 2.8 vs 12.3 ± 2.2 ng/ml, P<0.05), whereas there was no significant difference in the length of the luteal phase, determined as the period when corpora lutea existed and concentrations of progesterone were equal to or greater than 1 ng/ml (15.8 ± 1.5 vs 15.0 ± 2.8 days, P>0.1). A rise of estradiol concentration and follicular growth in the follicular phase following a decline of progesterone level after luteal regression tended to be suppressed at the onset of ovarian quiescence. It seems that the present results are consistent with previous findings that nutritionally induced body weight loss influences the secretion of ovarian steroids and eventually induces ovarian quiescence.
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Erratum
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