Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 53 , Issue 2
April
Showing 1-32 articles out of 32 articles from the selected issue
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  • Toh-Ichi HIRATA, Toshinori HOSHINA, Shu-Ichi SASAKI, Osamu SASAKI, Tak ...
    2007 Volume 53 Issue 2 Pages 171-177
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 01, 2006
    JOURNALS FREE ACCESS
    We conducted a progesterone-based timed AI protocol after follicular fluid aspiration using the ovum pick-up (OPU) technique to examine its applicability to the suckled beef cow. A total of 19 beef cows were randomly allocated to one of the following three groups based on the number of days postpartum: 13 to 60 days (Group A: suckled; early postpartum period, n=9), 61 to 150 days (Group B: suckled; mid postpartum period, n=6), or 151 to 281 days (Group C: non-suckled; prolonged open period, n=4) postpartum. These cows were treated with follicular fluid aspiration and insertion of a progesterone-releasing intravaginal device (PRID) on day 0. The PRID was removed and 500 μg of cloprostenol was intramuscularly administered on day 7. A dose (100 μg) of fertirelin acetate was injected intramuscularly 48 hours later, and this was followed by a timed AI (TAI) after another 18 hours (day 10). Serum samples were taken on days 0, 7, 9, 10, 12, 17, 24 and 31 for determination of the estradiol-17β (E2) and progesterone concentrations. Pregnancy diagnosis was made by rectal palpation approximately 60 days after TAI. There was no significant difference in the peripheral E2 concentrations among the three groups during the period of the hormonal treatment. The average progesterone concentrations in Group A on day 17 were significantly higher than those in Group B and exceeded 1.0 ng/ml on day 17 and thereafter. There was no significant difference in the numbers of collected immature oocytes among the three groups. The pregnancy rates in Groups A, B, and C were 77.8% (7/9), 83.3% (5/6) and 50.0% (2/4), respectively. In conclusion, this timed AI protocol is applicable to suckled beef cows within the period of 60 days postpartum.
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  • Jae-Ho SHIN, Hyun Ju MOON, Il Hyun KANG, Tae Sung KIM, Su Jung LEE, Ji ...
    2007 Volume 53 Issue 2 Pages 179-188
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 01, 2006
    JOURNALS FREE ACCESS
    Calbindin-D9k (CaBP-9k) is a cytosolic calcium-binding protein that is induced by estrogenic compounds possibly through estrogen receptors. We compared CaBP-9k mRNA expression in the uterus with uterotrophic response in immature rats exposed to methoxychlor (MC), an environmental chemical with estrogenic activity. MC was orally or subcutaneously administered to 3-week-old female Sprague-Dawley rats for 3 days. The weights of the uterus and vagina significantly increased in the oral treatment group at a dose of 50, 100 and 200 mg/kg, but those of the subcutaneous (SC) treatment group only increased at 200 mg/kg. Northern blot analysis showed that CaBP-9k mRNA expression was significantly induced in a dose-dependent manner at doses of 50, 100 and 200 mg/kg/day in the oral treatment group. SC administration of MC induced significant expression at only a dose of 200 mg/kg/day; this was similar to the uterotrophic response. MC has an estrogenic effect on the uterus as shown by the increase in weight and induction of CaBP-9k mRNA expression, which were much greater following exposure via oral gavage than via the SC route. The strong correlation between the results of in vivo uterotrophic assay and CaBP-9k mRNA expression suggests that CaBP-9k mRNA expression in the rat uterus may be used as an early gene marker for detection of the estrogenic effects of putative environmental chemicals.
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  • Masahito WATANABE, Kazuhiro UMEYAMA, Hiro-omi KAWANO, Naoko IZUNO, Hir ...
    2007 Volume 53 Issue 2 Pages 189-200
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 01, 2006
    JOURNALS FREE ACCESS
    A diabetic mouse model was produced using a mutant human hepatocyte nuclear factor-1α gene (HNF1αP291fsinsC) regulated by the porcine insulin promoter. The functionality of two different constructs containing HNF1αP291fsinsC, termed PD1 and PD2 (cytomegalovirus enhancer minus and plus), were examined in transgenic mice. The blood glucose levels and body weights of the PD1 transgenic mice did not differ from their non-transgenic littermates over the period from 3 to 8 weeks of age. Conversely, the PD2 transgenic mice exhibited hyperglycemia and decreased body weight. Western blot analysis demonstrated that mutant HNF-1α protein (HNF1αP291), derived from the PD2 transgene, was expressed in the PD2 mice. Morphometric studies of the pancreas of a PD2 mouse revealed that the number of pancreatic islets present was less than that in the non-transgenic mice, indicating disturbed islet neogenesis. These results suggest that impaired insulin secretion in disrupted islets causes hyperglycemia. In addition, the phenotype of PD2 transgenic mice similar to that of the HNF-1α gene-deficient mouse, which displays growth retardation and impaired viability. These results indicate that HNF1αP291 expression driven by the porcine insulin promoter, together with the cytomegalovirus enhancer, induces a diabetic phenotype in transgenic mice.
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  • Li-yi CAI, Takako KATO, Kazumi ITO, Michie NAKAYAMA, Takao SUSA, Satok ...
    2007 Volume 53 Issue 2 Pages 201-209
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 29, 2006
    JOURNALS FREE ACCESS
    A transgenic rat was established using the construct of porcine FSHβ subunit promoter, the -852/+10 bp region, fused to a Herpes simplex virus thymidine kinase (HSV-TK) gene. Integration of the transgene was confirmed by PCR of tail DNA. RT-PCR of total RNAs of the pituitary, gonad, cerebellum, liver, kidney, adrenal gland, prostate, and uterus revealed that FSHβ was only expressed in the pituitary. Analysis of the expression of reporter gene, HSV-TK, using two specific primer sets revealed that different transcripts were present in the pituitary and testis. The transcript initiated at the transcription initiation site of the porcine FSHβ gene was detected in the pituitary, and another within the TK gene was found in the testis, indicating ectopic testis-specific expression. Immunohistochemistry of the pituitary glands of the transgenic rats for FSH and HSV-TK demonstrated that the FSH-producing cells also produced HSV-TK. The results indicated that the -852/+10 bp region of the FSHβ promoter contains an element(s) that determines the tissue-specific expression. We succeeded in producing FSHβ promoter-driven HSV-TK transgenic rats and were the first time to do so using an animal other than the mouse. The transgenic rats show male infertility that involves abnormal spermatogenesis. We also observed a decrease in the weight of the testis and epididymis, and both motile and living spermatozoa were absent in the epididymis. Consequently, the FSHβ-HSV-TK transgenic rat will provide a useful model for studies on FSH function and male infertility.
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  • Qi-En YANG, Yun-Peng HOU, Guang-Bin ZHOU, Zhong-Qiang YANG, Shi-En ZHU
    2007 Volume 53 Issue 2 Pages 211-218
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 29, 2006
    JOURNALS FREE ACCESS
    In the present study, mouse blastocysts were employed to investigate the feasibility and efficiency of stepwise in-straw dilution and direct transfer using the open pulled straw (OPS) method. In experiment I, the effects of various vitrification solutions (VS) on embryo survival were examined. After thawing, the expanded blastocyst rates (97.59 and 95.05%) and hatching rates (80.48 and 78.95%) achieved in the EDFS30 [15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll, and sucrose] and EFS40 [40% EG, Ficoll, and sucrose] groups were no different from those (96.15% and 83.33%) of the control group. However, the rates in the EFS30 [30% EG, Ficoll, and sucrose] (87.80 and 55.43%) and EDFS40 [20% EG, 20% DMSO, Ficoll, and sucrose] (95.69 and 70.97%) groups were significantly lower than those (96.15 and 83.33%) of the control group (P<0.05). In the experiment II, the effects of the volume of VS in the OPS on the survival of embryos after in-straw thawing were investigated. When the length of the VS in the column was less than 1 cm, the in vitro viability of embryos thawed by stepwise in-straw dilution was no different among the experimental and control groups. The embryos could be successfully thawed by immersing the OPS in 0.5 M sucrose for 3 min and then 0.25M sucrose for 2 min. In experiment III, the effect of immersion time of the OPS in diluent (PBS) on the viability of vitrified embryos was investigated. After in-straw thawing, OPSs were immersed immediately in 1 ml PBS for 0 to 30 min. When the immersion time of the OPSs in PBS was less than 12 min, in vitro development of the in-straw thawed embryos was no different from that of the controls. In experiment IV, in-straw thawed blastocysts were directly transferred to pseudopregnant mice to examine their in vivo developmental viability. The pregnancy (91.67%) and birth rates (42.42%) of embryos in-straw thawed and directly transferred were no different from those of the unvitrified controls (90.90 and 40%) and embryos thawed by the conventional method (84.61 and 46.94%). These results demonstrate that mouse embryos vitrified with OPS could be successfully thawed by stepwise in-straw dilution and transferred directly to a recipient and that this method might be a model for field manipulation of vitrified embryos in farm animals.
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  • Chang-Long NAN, Zi-Li LEI, Zhen-Jun ZHAO, Li-Hong SHI, Ying-Chun OUYAN ...
    2007 Volume 53 Issue 2 Pages 219-228
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 29, 2006
    JOURNALS FREE ACCESS
    Somatic cell nuclei can be dedifferentiated in ooplasm from another species, and interspecies cloned embryos can be implanted into the uteri of surrogates. However, no full pregnancies have been achieved through interspecific mammalian cloning. Rat blastocysts transferred into mouse uteri provide a unique model for studying the causes of interspecific pregnancy failure. In this study, intraspecific pregnancy (mouse-mouse) and interspecific pregnancy (rat-mouse) models were established. On Day 9 of pregnancy, the fetoplacental units were separated from the uterine implantation sites and the expression of messenger (m)RNA was quantitated by real-time PCR. We compared the mRNA expression levels of type-1 T helper (Th1) and type-2 T helper (Th2) cytokines, interferon-gamma (IFN-γ), and interleukin-4 (IL-4) in fetoplacental units between intraspecific and interspecific pregnancy groups. The mRNA expression of IFN-γ in the fetoplacental units of the interspecific pregnancy group was significantly higher than that of the intraspecific pregnancy group (P<0.05). The mRNA expression of IL-4 in the interspecific pregnancy group was significantly lower than that in the intraspecific pregnancy group (P<0.05). We also analyzed the ratio of IFN-γ/IL-4 mRNA, and an increased IFN-γ/IL-4 mRNA ratio was observed in the interspecific pregnancy compared with that in the intraspecific pregnancy group. The IFN-γ and IL-4 mRNA expressions indicate that there is a Th1/Th2 imbalance in the feto-maternal interface of interspecific pregnancies. Bias of Th1 cytokine dominance may be a barrier to reproductive success between species.
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  • Chunhua MENG, Fangxiong SHI, Zhenqi ZHOU, Ruihua HUANG, Gentao LIU, Ge ...
    2007 Volume 53 Issue 2 Pages 229-236
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 29, 2006
    JOURNALS FREE ACCESS
    Experiments were conducted to examine the cellular localization of inhibin α-subunit, protein kinase B (PKB/Akt), and FoxO3a proteins in the ovaries of minipigs, Chinese Xiang pigs, by immunohistochemistry. The results indicated that inhibin α-subunits were localized in the granulosa cells of follicles at all stages but were not localized in corpora lutea. PKB was localized in the granulosa cells of primordial follicles and in the basal layers of the granulosa cells of preantral and antral follicles, but were not localized in atretic follicles and corpora lutea. FoxO3a was localized in the granulosa cells of follicles at all stages and was extensively localized in the cytoplasma of the luteinized granulosa cells of corpora lutea. Together, the stage- and cell-specific expression patterns of inhibin α-subunit, FoxO3a, and PKB suggest that these proteins might play potential roles in follicular development, atresia, and luteinization in the minipig.
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  • Kei MIYAMOTO, Yoichiro HOSHINO, Naojiro MINAMI, Masayasu YAMADA, Hiros ...
    2007 Volume 53 Issue 2 Pages 237-246
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 29, 2006
    JOURNALS FREE ACCESS
    The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.
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  • Chiho KAWASHIMA, Katsuya KIDA, Ken-Go HAYASHI, Carlos AMAYA MONTOYA, E ...
    2007 Volume 53 Issue 2 Pages 247-254
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 29, 2006
    JOURNALS FREE ACCESS
    The aim of the present study was to investigate the changing profiles of plasma metabolic hormones during the ovarian cycles of beef and dairy cattle. We used 16 non-pregnant, non-lactating Japanese Black beef cattle (6 heifers and 10 cows; parity=2.3 ± 0.8) and 12 multiparous Holstein dairy cows (parity=3.0 ± 0.3). Blood samples for hormonal analysis (growth hormone, GH; insulin-like growth factor-I, IGF-1; insulin; and progesterone, P4) were obtained twice weekly for 40 days before artificial insemination for Japanese Black cattle and from 50 to 100 days postpartum for Holstein cows. Luteal phases were considered normal if the P4 concentrations for at least 3 time points over the course of 7 days remained above 1 ng/ml and at least 2 of the time points were above 2 ng/ml. The patterns of the ovarian cycles were classified into two types (normal or abnormal, such as having prolonged luteal phase and cessation of cyclicity) on the basis of the plasma P4 profiles. The plasma concentrations of IGF-1 in both breeds increased transiently during the preovulatory period when the P4 levels were low and decreased to lower levels during the luteal phase when the P4 levels were high. The plasma concentrations of insulin in the 3rd week of normal ovarian cycles when the plasma P4 concentration dropped to less than 1 ng/ml were higher than those at other time points in the Japanese Black cattle, but not in the Holstein cows. The plasma concentrations of GH did not change during the ovarian cycle in either breed. In conclusion, the present study indicates that the plasma IGF-1 concentration increases during the follicular phase (low P4 levels) and decreases during the luteal phase (high P4 levels) in non-lactating Japanese Black and lactating Holstein cattle. The results suggest that ovarian steroids, rather than nutrient status, may be related to the cyclic changes in IGF-1 secretion from the liver in cattle.
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  • Misuzu YAMASHITA, Kazuo YAMAGATA, Keiko TSUMURA, Tomoko NAKANISHI, Tad ...
    2007 Volume 53 Issue 2 Pages 255-262
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 30, 2006
    JOURNALS FREE ACCESS
    To improve assessment of the acrosome reaction of mouse epididymal sperm, we employed anti-Izumo1 antibody instead of antibodies against acrosomal proteins. The acrosomal states among acrosome-intact, spontaneously acrosome-reacted, truly acrosome-reacted, and probably dead and/or membrane-damaged sperm were clearly distinguished by combined application of anti-Izumo1 antibody, DNA dye Hoechst 33342, and monoclonal antibody MN7 to paraformaldehyde-fixed sperm. When the acrosome reaction of capacitated epididymal sperm on the oocyte zona pellucida was examined using anti-Izumo1 antibody, approximately 20% of sperm bound onto the zona pellucida were acrosome-reacted 30 min after insemination. We also observed the moment of the acrosome reaction of live sperm on the zona pellucida by time-lapse monitoring using fluorescein isothiocyanate-conjugated anti-Izumo1 antibody.
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  • Masaki YOKOO, Takashi SHIMIZU, Naoko KIMURA, Woro Anindito Sri TUNJUNG ...
    2007 Volume 53 Issue 2 Pages 263-270
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 30, 2006
    JOURNALS FREE ACCESS
    Previous our studies have shown that CD44, the principal receptor for hyaluronan, is present on cumulus cells during oocyte maturation. Although hyaluronan-CD44 interaction has been implicated in cumulus expansion and/or oocyte maturation, the full significance of CD44 remains unknown. The objective of the present study was to further investigate the role of CD44 in cumulus expansion and oocyte maturation in pigs. We demonstrate here in that CD44 has a key role in oocyte maturation but not in cumulus expansion. Previous studies have reported the physiological significance of cumulus expansion in oocyte maturation. However, our results suggest that cumulus expansion is a necessary condition for oocyte maturation, but that it is not sufficient on its own. Furthermore, western blot analysis demonstrated that the CD44 of the in vitro-matured cumulus-oocyte complexes (COCs) had a larger molecular weight and more terminal sialic acid, which has been proven to inhibit the hyaluronan-binding ability of the receptor, than the CD44 of the in vivo-matured COCs, indicating that the hyaluronan-CD44 interactions during in vitro maturation might be insufficient compared with those in vivo. The insufficient interactions of hyaluronan-CD44 during in vitro maturation may cause the inferior capacity of fertilization and development of oocytes matured in vitro.
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  • Hideaki NAOI, Takeshige OTOI, Takano SHIMAMURA, Ni Wayan Kurniani KARJ ...
    2007 Volume 53 Issue 2 Pages 271-277
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 01, 2006
    JOURNALS FREE ACCESS
    We conducted two experiments to investigate the effects of storage temperature and period for cat ovaries on the meiotic and developmental competence of oocytes collected from the ovaries. In Experiment 1, ovaries were stored in physiological saline for 24 h at 4 C, 23-25 C, or 38 C (cold, room, and incubator temperature groups, respectively), and then oocytes were collected from the ovaries in each group. Morphologically intact oocytes were then selected and cultured in maturation medium for 24 h. Significantly more oocytes reached metaphase II (MII) in the cold temperature group (53.4%) than in the room and incubator temperature groups (20.0 and 2.4%, respectively). In Experiment 2, ovaries were stored in physiological saline at room temperature for 0, 6, 12 or 18 h, and then they were stored at 4 C (cold storage) until reaching a total storage period of 24 h. After storage of the ovaries, morphologically intact oocytes were matured, fertilized with frozen-thawed spermatozoa, and cultured in vitro. The rates of morphologically intact oocytes obtained from the ovaries stored at room temperature for 0, 6, 12 or 18 h were 35.3, 30.0, 26.4 and 14.7%, respectively, and the rates of intact oocytes that reached MII were 63.2, 36.4, 26.5 and 11.9%, respectively. The results suggested that the numbers of morphologically intact oocytes and intact oocytes that reached MII after in vitro maturation decrease gradually as the period of storage at room temperature before cold storage increases. Only oocytes from ovaries stored for 6 h developed to the blastocyst stage after in vitro maturation and fertilization when ovaries were stored at room temperature before cold storage. These results indicate that 24 h storage of ovaries at high temperatures (>23 C) decreases the meiotic competence of oocytes. The quality and developmental competence of oocytes are influenced by the length of storage at room temperature before cold storage.
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  • Kapil Deo SHAH, Toshihiko NAKAO, Hirokazu KUBOTA, Teruo MAEDA
    2007 Volume 53 Issue 2 Pages 279-288
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 30, 2006
    JOURNALS FREE ACCESS
    The aim of this study was to evaluate the changes in plasma concentrations of estrone sulfate (E1S) and estradiol-17β (E2β) during the peripartum period (from day 10 prepartum to day 1 postpartum) associated with and without retention of fetal membranes (RFM) in Holstein-Friesian cattle (n=42). Plasma samples were analyzed for E1S and E2β by ELISA. All parturitions were spontaneous and normal. Of 38 cattle delivering singletons, 29 had no RFM (singleton-normal group) and nine had RFM for more than 12 h (singleton-RFM group). Four cows gave birth to twins, and each twin had its own fetal membrane (FM). Two twinning cows expelled both FMs normally within 12 h (twin-normal group). In the remaining 2 twinning cows (twin-RFM group), the FM was expelled normally for one twin (first), while the FM of the other (second) was retained. There were no significant differences in the E1S concentrations or their increments from the concentrations on the preceding day between the normal and RFM groups of singleton cows on any peripartum day. The mean plasma E2β concentrations on each day from day 10 to day 3 prepartum were significantly lower (P<0.05) in the singleton-RFM group compared with the singleton-normal group; however, on days 2 and 1 prepartum, the increments in the E2β concentrations from the concentrations on the preceding days were significantly higher (P<0.05) in the singleton-RFM group than in the singleton-normal group. Thus, the plasma E1S concentrations just before parturition may not be associated with RFM. In the cows with RFM, the lower plasma E2β concentrations that were found prior to day 2 prepartum may have been associated with immature placentomes, and the rapid rise in plasma E2 β within 1 to 2 days prior to calving may have produced asynchrony of placental and/or fetal maturation in relation to calving, thus resulting in RFM.
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  • Ewa Lucja GREGORASZCZUK, Anna PTAK, Tatiana WOJCIECHOWICZ, Krzysztof N ...
    2007 Volume 53 Issue 2 Pages 289-295
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 01, 2006
    JOURNALS FREE ACCESS
    Leptin is an important satiety hormone and reproductive regulator found, along with its receptors, throughout the ovary. To date, the changes in ovarian expression of leptin receptor during the prepubertal period and throughout the estrous cycle in the pig have not been studied. In this study, the long form of the leptin receptor was a detectable level in immature pig ovarian follicles when assayed using semiquantitative reverse transcription-polimerase chain reaction (RT-PCR). Moreover, its level was increased in growing follicles during follicular phase of the estrous cycle (6-fold in early antral [EAF] and antral [AF] follicles) and was highest in newly formed corpora lutea. Its changes paralleled those in steroid secretion and especially progesterone (P4) secretion. Additionally, we showed that insulin-like growth factor (IGF)-I had stimulatory effect on leptin receptor expression using a tissue culture model of follicular and luteal cells, with a 12-fold increase in prepubertal ovaries and a 1.5- to 2-fold increase in growing follicles and newly formed corpora lutea (CL). These results suggest that ovarian expression of leptin receptor is regulated throughout the estrous cycle by ovarian steroids, with peak expression at ovulation, indicating a possible involvement in follicular development and corpus luteum formation. Moreover, this data points to an important role of IGF-I in leptin receptor expression during the entire estrous cycle, with a special role during the prepubertal period.
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  • Shuichi CHIBA, Masatoshi SUZUKI, Keitaro YAMANOUCHI, Masugi NISHIHARA
    2007 Volume 53 Issue 2 Pages 297-307
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 20, 2006
    JOURNALS FREE ACCESS
    Recent studies have demonstrated the presence of neurogenesis in the adult mammalian hippocampus, and it has been suggested that estrogen and various growth factors influence the processes of adult neurogenesis. The present study assessed cell proliferation in the dentate gyrus and the mRNA expression levels of granulin, insulin-like growth factor-I (IGF-I), and brain-derived neurotrophic factor (BDNF) in the hippocampus 4 h after treatment with estradiol benzoate (EB) in 3- and 12-month old ovariectomized rats. At 3 months of age, mRNA expression of granulin precursor and cell proliferation were increased by EB treatment, although the mRNA expressions of IGF-I and BDNF remained unchanged. At 12 months of age, however, neither mRNA expression of the three genes nor cell proliferation in the dentate gyrus were affected by EB treatment. In addition, 17β-estradiol enhanced the proliferation of neural progenitor cells derived from hippocampal tissue of 3-month-old female rats in vitro; this was inhibited by neutralization of granulin with specific antibody. These results suggest that estrogen induces granulin gene expression in the hippocampus and that the product of this gene is involved in the mitogenic effects of estrogen in the dentate gyrus, although the responses to estrogen decline with age.
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  • Tatjana I. KUZMINA, Hannelore ALM, Vitaly DENISENKO, Armin TUCHSCHERER ...
    2007 Volume 53 Issue 2 Pages 309-316
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 01, 2006
    JOURNALS FREE ACCESS
    The objectives of this study were to evaluate the effects of recombinant bovine somatotropin (rbST) on the nuclear and cytoplasmic maturation of bovine oocytes and their further developmental competence to blastocysts in vitro. We analyzed the mitochondrial activity and concentration of intracellular stored calcium ([Ca2+]is) in matured oocytes and the morphology and chromatin status of produced embryos after in vitro fertilization. Cumulus-oocyte complexes were incubated in TCM 199 containing 10% fetal calf serum (control medium 1: CM 1) or 10% estrus cow serum (control medium 2: CM 2). The culture medium of the treatment groups was modified by supplementation of the control medium with 10 ng/ml rbST (CM 1A and CM 2A), 106/ml granulosa cells (CM 1B and CM 2B), or 10 ng/ml rbST plus 106/ml granulosa cells (CM 1C and CM 2C). No differences were observed in the percentages of oocytes reaching metaphase II between the groups. However, the proportion of blastocysts was highest in treatment groups CM 1C and CM 2C (P<0.05). The type of serum did not alter the positive effect of rbST on the developmental competence of embryos. The fluorescence intensity of metabolically active mitochondria measured by intensity per oocyte (Em 570) after MitoTracker CMTM Ros Orange labeling was significantly increased in oocytes matured in the presence of 10 ng/ml rbST and granulosa cells (309.21 vs. 119.97 μA; P<0.01). In parallel, the concentration of [Ca2+]is in oocytes, determined using fluorophore chlortetracycline, was significantly decreased (0.85 ± 0.02 vs. 0.97 ± 0.03 AU; P<0.05). Based on these results, we concluded that rbST, in interaction with granulosa cells stimulates the oxidative activity of ooplasmic mitochondria and decreases the content of [Ca2+]is in oocytes. These facts support the hypothesis that somatotropin influences the developmental competence of bovine oocytes during maturation in vitro, and this effect can be modulated by granulosa cells.
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  • Gholamali JELODAR, Nematollah RAZMI, Vahid GHOLAMPOUR
    2007 Volume 53 Issue 2 Pages 317-321
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 20, 2006
    JOURNALS FREE ACCESS
    This study was conducted to evaluate possible alteration in the activity of arginase, an important enzyme of cell proliferation and vascular smooth muscle contraction regulator in diabetics, that may be correlated with low fertility in diabetic patients. In this investigation, 6 apparently healthy adult male dogs were selected and divided in two groups, diabetics and non-diabetics. Diabetes mellitus was induced in one group by intravenous (IV) injection of alloxan (100 mg/kg). Dogs with a fasting blood glucose (FBS) of more than 200 mg/dl were considered to be diabetic. Four weeks following induction of diabetes mellitus, the animals in both groups were anesthetized by an IV injection of sodium thiopental. Livers and whole reproductive systems, including the testes, penis, urethra, and prostate, were dissected. The epididymides, corpus cavernosum, corpus spongiosum, penile urethra, and vas deferens were also dissected and removed from the reproductive system. Arginase activity and total protein were measured by the urea and Lowry's methods respectively in above mentioned sections. Plasma testosterone was determined by the radioimmunoassay method. The results showed significantly (P<0.05) increased arginase specific activity (ASA) in the liver, epididymis, prostate, corpus cavernosum and corpus spongiosum of the diabetic dogs. In the reproductive system of the diabetic dog, the maximum and minimum ASA was seen in the corpus cavernosum and testes, respectively (105.12 ± 8.76 vs. 25.0 ± 0.55). No such variation was observed in the ASA of normal dogs (39.0 ± 5.47 vs. 25.0 ± 5.47). There was no significant difference in plasma testosterone level between the groups. In conclusion, diabetes increased the ASA in liver, prostate, epididymis, corpora cavernosa, and corpora spongiosum of the male dogs and may contribute to erectile dysfunction or low fertility in diabetics.
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  • Hitomi FUJIOKA, Keitaro YAMANOUCHI, Tatsuo AKEMA, Masugi NISHIHARA
    2007 Volume 53 Issue 2 Pages 323-331
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 20, 2006
    JOURNALS FREE ACCESS
    Gonadotropin-releasing hormone (GnRH) neurons arise in the olfactory placode, migrate into the preoptic area (POA), and then extend axons to the median eminence during embryogenesis. Little information is available concerning the properties of GnRH neurons during the late gestational period when GnRH neurons reach the POA and form neuronal networks, although many studies have examined such properties during earlier developmental stages or the postnatal period. The present study was performed to elucidate the involvement of γ-aminobutyric acid (GABA), one of the major neurotransmitters modifying GnRH neural activity, in regulation of GnRH gene expression on embryonic day 18.5 (E18.5) using transgenic rats expressing enhanced green fluorescence protein (EGFP) under the control of GnRH promoter. First, using RT-PCR, the mRNA of two isoforms of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), GAD65 and GAD67 was detected in E18.5 embryonic POA-containing tissues. GAD67-positive cells were also demonstrated in close vicinity to GnRH-positive cells by immunohistochemistry, and immunoreactivity for both the GABA-A and GABA-B receptor subunits was detected in GnRH neurons. Next, primary cultures derived from anterior hypothalamic tissue of E18.5 embryos were prepared, and the effects of GABA and its agonists on GnRH promoter activity were evaluated using EGFP expression as a marker. GABA and the GABA-A receptor agonist muscimol, but not the GABA-B receptor agonist baclofen, significantly increased the EGFP-positive/GnRH-positive cell ratio. These results suggest that GABA plays a role in stimulating GnRH gene expression through GABA-A receptors in embryonic GnRH neurons in late gestational stages.
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  • Hyun Ju MOON, Soon Young HAN, Jae-Ho SHIN, IL Hyun KANG, Tae Sung KIM, ...
    2007 Volume 53 Issue 2 Pages 333-344
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 27, 2006
    JOURNALS FREE ACCESS
    This study examined whether or not exposure to 4-nonylphenol (NP) during late gestation affects reproductive and mammary development in the offspring of female rats. Time pregnant Long Evans rats were gavaged with NP (10 or 100 mg/kg), atrazine (ATR, 100 mg/kg), or corn oil on gestation days 15-19. The uterus weights of the NP (100 mg/kg/d)-exposed pups were higher than those of the controls but the weights of the other organs were unchanged. Delayed mammary gland (MG) development was detected in the ATR pups on PND 4 and persisted through to PND 66. The high dose NP pups had advanced lobular development of their MG on PND 22, while the glands from the low dose NP pups were no different morphologically from the controls. Immunohistochemical comparisons of the mammary sections from PND 41 demonstrated low levels of estrogen receptor (ER) staining in the control gland stroma and epithelium but higher levels in the tissue of the pups exposed to NP and ATR. ATR also elevated ER in the stroma surrounding the epithelial layer of the terminal end buds. The level of progesterone receptor (PR) staining was markedly lower in the epithelium of the 100 mg/kg NP glands vs. the control glands. However, PR was present at high levels in the epithelium of the 10 mg/kg NP glands and was even more prominent in the ATR-exposed ductal epithelium and fat cell nuclei. The level of prolactin staining was only elevated in glands containing lobule areas (NP-exposed) compared with the control levels. These results suggest that NP and ATR have opposite effects on the development of MG after gestational exposure. Exposure to them during the critical period of epithelial outgrowth altered the receptor levels of mammary progesterone and prolactin and might contribute to the differences in the mammary morphology at PND 41.
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  • Shiro KURUSU, Shizuka ISHII, Mitsumori KAWAMINAMI
    2007 Volume 53 Issue 2 Pages 345-350
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 20, 2006
    JOURNALS FREE ACCESS
    Our previous study revealed that a fall in plasma progesterone (P4) level was associated with a transient increase in cytosolic phospholipase A2 (PLA2) activity and prostaglandin F2α level in the rat uterus and cervix during natural parturition. This study determined the changes in the PLA2 activities during modulated occurrence of delivery by P4 antagonist or agonist late in pregnancy. In rats undergoing P4 antagonist-induced preterm delivery, the PLA2 activities of both uterine and cervical cytosol significantly decreased 12 h after the challenge and tended to be attenuated within 72 h. The plasma P4 level altered in a similar pattern. Blockade of delivery by chronic treatment with P4 agonist was not associated with changes in uterine PLA2 activity compared with that in normally delivering rats, although there was a persistent rise in cervical PLA2 activity. The obtained data indicates that the PLA2 activities in rat uterine and cervical cytosol are not regulated solely by P4 and that delivery can occur without activation of this enzyme.
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  • Hataitip TRISOMBOON, Gen WATANABE, Phongphat WETCHASIT, Kazuyoshi TAYA
    2007 Volume 53 Issue 2 Pages 351-356
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 20, 2006
    JOURNALS FREE ACCESS
    The aim of the present study was to investigate the testosterone-like effect of Kaempferia parviflora (KP). Castrated immature rats were randomized and divided into two groups (control and KP-treatment groups). The rats (n=7-8) were treated daily for 5 days by oral route with water in the control group and 1,000 mg/kg of KP in the treatment group. All rats were decapitated 24 h after their last dose and then blood samples were collected for assay of serum FSH, LH, testosterone, progesterone and corticosterone levels. The seminal vesicles plus coagulating glands, ventral prostate, levator ani muscle plus bulbocavernosus muscle, glans penis, kidneys and the adrenal glands were collected and weighed for organ wet weight. Body weight and weight of food intake were recorded throughout the study period. The results show that relative body weight gain in the KP-treatment group was significantly increased 24 and 48 h after the first dose (P<0.05) and then was indistinguishable from the control group. There were no significant differences in the relative reproductive and non-reproductive organ weights between the groups, although all organ weights, except for the glans penis, tended to increase in the KP-treatment group. The serum testosterone levels were significantly increased in the KP-treatment group. There were no significant differences in the serum FSH, LH, progesterone, or corticosterone levels between the groups, even though the serum progesterone level tended to increase and serum LH level tended to decrease in the KP-treatment group. The present study indicates that KP has no testosterone-like effect on reproduction in male rats.
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  • Akinori MITSUI, Midori YOSHIZAWA
    2007 Volume 53 Issue 2 Pages 357-366
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 20, 2006
    JOURNALS FREE ACCESS
    An electrofusion methodology for transferring meiosis-II chromosomes (M-II-t) has not been completely established. The present study compared the use of two temperatures (fusion at 37 C for Group A and 25 C for Group B) during an electrofusion procedure for mouse oocyte M-II-t and investigated the cytogenetic normality and developmental competence of embryos derived from in vitro fertilization using oocytes reconstructed by M-II-t. The M-II-t oocytes were fertilized in vitro and cultured to the blastocyst stage; the resultant embryos were analyzed cytogenetically. Subsequently, chromosomal normality of the resultant embryos at the prometaphase stage of first cleavage division and the integrity of the meiosis-II spindles of the reconstructed oocytes were analyzed. The success rate of electrofusion in Group B was 92.1%, which was significantly different from that in Group A (49.2%) (P<0.05). The fertilization rates (A, 80.7%; B, 77.2%) and development rates (A, 70.9%; B, 65.5%) in the M-II-t groups were significantly lower than those in the control group (95.0 and 92.2%, respectively) (P<0.05). The incidence of chromosomal abnormalities in the Group A embryos (20.5%) at the blastocyst stage was significantly higher than that in the control group embryos (8.5%) (P<0.05), but the incidence of chromosomal abnormalities in Group B (12.5%) was not significantly different compared with the other groups. A temperature of 25 C during the electrofusion procedure for M-II-t resulted in a good fusion rate, good development rate, and efficient production of chromosomally normal blastocysts. Furthermore, the incidence of chromosomal abnormalities in the first cleavage embryos at the prometaphase stage in Group B (9.6%) did not differ significantly from that in the control group (6.6%). The spindle morphology of the M-II-t oocytes in Group B was normal.
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  • Sachika ADACHI, Shunji YAMADA, Yoshihiro TAKATSU, Hisanori MATSUI, Mik ...
    2007 Volume 53 Issue 2 Pages 367-378
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: January 10, 2007
    JOURNALS FREE ACCESS
    Metastin/kisspeptin, the KiSS-1 gene product, has been identified as an endogenous ligand of GPR54 that reportedly regulates GnRH/LH surges and estrous cyclicity in female rats. The aim of the present study was to determine if metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges. We demonstrated that preoptic area (POA) infusion of the anti-rat metastin/kisspeptin monoclonal antibody blocked the estrogen-induced LH surge, indicating that endogenous metastin/kisspeptin released around the POA mediates the estrogen positive feedback effect on GnRH/LH release. Metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) may be responsible for mediating the feedback effect because the percentage of c-Fos-expressing KiSS-1 mRNA-positive cells to total KiSS-1 mRNA-positive cells was significantly higher in the afternoon than in the morning in the anteroventral periventricular nucleus (AVPV) of high estradiol (E2)-treated females. The percentage of c-Fos-expressing metastin/kisspeptin neurons was not different between the afternoon and morning in the arcuate nucleus (ARC). Most of the KiSS-1 mRNA expressing cells contain ERα immunoreactivity in the AVPV and ARC. In addition, AVPV KiSS-1 mRNA expressions were highest in the proestrous afternoon and lowest in the diestrus 1 in females and were increased by estrogen treatment in ovariectomized animals. On the other hand, the ARC KiSS-1 mRNA expressions were highest at diestrus 2 and lowest at proestrous afternoon and were increased by ovariectomy and decreased by high estrogen treatment. Males lacking the surge mode of GnRH/LH release showed no obvious cluster of metastin/kisspeptin-immunoreactive neurons in the AVPV when compared with high E2-treated females, which showed a much greater density of these neurons. Taken together, the present study demonstrates that the AVPV metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges in female rats.
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  • Shu HASHIMOTO, Kanako OHSUMI, Yoko TSUJI, Naoko HARAUMA, Yuko MIYATA, ...
    2007 Volume 53 Issue 2 Pages 379-384
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 20, 2006
    JOURNALS FREE ACCESS
    The aim of this study was to establish a culture system to improve the meiotic competence of porcine oocyte-granulosa cell complexes (OGCs) obtained from preantral or early antral follicles. Porcine OGCs were recovered from follicles with diameters of 230-300 (preantral follicles), 300-500, and 500-700 μm (early antral follicles) using scalpels. The OGCs were cultured for 2 weeks in culture medium. We examined the effects of the sizes of the follicles from which OGCs were recovered, the concentrations of polyvinylpyrrolidone (PVP, 0-8%) in the culture medium, and 2 types of culture dish (Falcon 3002 vs 1007) on formation of the antrum of OGCs. After culture, the oocytes were matured for 44 h to assess their meiotic competence. OGCs recovered from small follicles (230-500 μm) required longer (P<0.05) than larger follicles to form the antrum structure. The percentage of OGCs forming the antrum structure that were cultured in 2% PVP (31%) was higher (P<0.05) than for those cultured in other PVP concentrations (0-11%). The percentages of antrum-structure formation for OGCs cultured on Falcon 3002 (83% for 2% PVP and 60% for 4% PVP) were higher (P<0.05) than those cultured on Falcon 1007 (47% for 2% PVP and 9% for 4% PVP). Furthermore, all of the intact oocytes that were obtained from culture of OGCs and that formed an antrum were in the GV stage (n=28). When these immature oocytes were cultured for 44 h, the percentage of oocytes that reached the metaphase II stage (25%, n=68) was higher (P<0.0001) than that of oocytes matured without culture (0.7%, n=137). The results of the present study show that porcine OGCs obtained from preantral or early antral follicles acquire meiotic competence in vitro.
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  • Wichai CHERDSHEWASART, Yosaporn KITSAMAI, Suchinda MALAIVIJITNOND
    2007 Volume 53 Issue 2 Pages 385-393
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: January 17, 2007
    JOURNALS FREE ACCESS
    The aim of this study was to evaluate the estrogenic activity of tuberous samples of phytoestrogen-rich Pueraria mirifica collected from 25 of 76 provinces in Thailand by vaginal cornification assay. Tuberous powders were prepared and administered to ovariectomized rats for 14 consecutive days at dosages of 10, 100 and 1,000 mg/kg BW respectively, and were compared with a daily treatment with 2 mg/kg BW 17β-estradiol (E2). Rats treated with 10 mg/kg BW Pueraria mirifica showed no vaginal cornification. Treatment with 100 mg/kg BW Pueraria mirifica from 13 out of 25 plant samples resulted in development of vaginal cornification. The cell count percentages of the vaginal smeared cells for the treatment with the 2 plant samples that exhibited the fastest vaginal cornification revealed large variation in their estrogenic activities. Treatment with 1,000 mg/kg BW Pueraria mirifica from all plant samples produced vaginal cornification with the mean value for the period (day) of first appearance of cornified cells being 4.08 days compared to 2 days with 2 mg/kg BW E2. The overall appearance period (day) of cornified cells during the treatment and post-treatment period with 1,000 mg/kg BW per day Pueraria mirifica was shorter than treatment with 2 mg/kg BW E2. The results demonstrate that the plant population shows differential estrogenic activity as evaluated by vaginal cornification assay.
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  • Hataitip TRISOMBOON, Suchinda MALAIVIJITNOND, Wichai CHERDSHEWASART, G ...
    2007 Volume 53 Issue 2 Pages 395-403
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 29, 2006
    JOURNALS FREE ACCESS
    This study investigated the changes in the urinary hormone levels of female monkeys (Macaca fascicularis) after single-dose and long-term treatments with Pueraria mirifica (PM). The monkeys were separated into 3 groups (n=3) and orally treated with 10, 100, or 1,000 mg of PM in each group. Two series of experiments were performed. In the first series of experiments, the monkeys were orally treated with a single dose of PM. The experimental schedule was divided into a one menstrual cycle pretreatment period and a two menstrual cycle post-treatment period. In the second series of experiments, the monkeys were orally treated daily with PM for 90 days. The experiment schedule was divided into a one menstrual cycle pretreatment period, a three menstrual cycle treatment period, and a two menstrual cycle post-treatment period. Urinary samples were collected daily and assayed for the FSH, LH, estradiol, and progesterone levels. The results showed that there were no changes in the FSH, LH, estradiol, and progesterone levels after treatment with a single dose of 10, 100, or 1,000 mg of PM or after daily treatment with 10 mg of PM for 90 days compared with the levels observed during the pretreatment period. Daily treatment with 100 mg and 1,000 mg of PM for 90 days only produced a clear reduction in the urinary FSH levels. This suggests that changes of urinary FSH levels can be considered an indicator for study of estrogenic effects on hormonal levels in female monkeys.
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  • Xiang-Shun CUI, Xing-Yu LI, Xi-Jun YIN, IL Keun KONG, Jason-Jongho KAN ...
    2007 Volume 53 Issue 2 Pages 405-418
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 20, 2006
    JOURNALS FREE ACCESS
    Maternal gene expression is an important biological process in oocyte maturation and early cleavage. To gain insights into oocyte maturation and early embryo development, we used microarray analysis to compare the gene expression profiles of germinal vesicle (GV)- and metaphase II (MII)-stage oocytes. The differences in spot intensities were normalized and grouped using the Avadis Prophetic software platform. Of the 12164 genes examined, we found 1682 genes with more highly expression in GV-stage oocytes than in MII-stage oocytes, while 1936 genes were more highly expressed in MII-stage oocytes (P<0.05). The genes were grouped on the basis of the Panther classification system according to their involvement in particular biological processes. The genes that were up-regulated in GV oocytes were more likely to be involved in protein metabolism and modification, the mitotic cell cycle, electron transport, or fertilization or belong to the microtubule/cytoskeletal protein family. The genes specifically upregulated in the MII oocytes were more likely to be involved in DNA replication, amino acid metabolism, or expression of G protein-coupled receptors and signaling molecules. Identification of genes that are preferentially expressed at particular oocyte maturation stages provides insights into the complex gene regulatory networks that drive oocyte maturation and fertilization.
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Research Note
  • Marta WASIELAK, Marek BOGACKI
    2007 Volume 53 Issue 2 Pages 419-426
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 20, 2006
    JOURNALS FREE ACCESS
    Recently, significant progress has been achieved in improving the yield of good quality embryos in vitro. However, efforts are still required to recognize the factors and understand the mechanisms of oocyte maturation, which are essential for subsequent embryo development. The aims of the present study were to determine the frequency of apoptosis in oocytes recovered from slaughterhouse ovaries and to investigate whether insulin-like growth factor (IGF)-I action during oocyte maturation in vitro may withhold apoptosis and improve oocyte quality. Only oocytes of proper morphology with homogenous ooplasm and compact cumulus cells were selected for this study. All oocytes recovered from the slaughterhouse ovaries were divided into two groups. One group of oocytes, chosen for apoptosis detection, was examined immediately after recovery. The other group of oocytes was maturated in vitro. Oocytes were maturated with IGF-I supplementation (100 ng/ml). Oocytes without supplementation were used as a control. Apoptosis in oocytes was determined by positive results of TUNEL assay and active caspase labeling. The percentage of apoptotic oocytes detected by TUNEL fell to zero when the maturation medium was supplemented with IGF-I in comparison to the control matured oocytes (0 vs. 9.87%; P<0.05). However, active caspase labeling was only slightly decreased in the IGF-I matured oocytes compared with the control matured oocytes (1.13 vs. 2.08%; P<0.05). The results indicate that IGF-I may serve as an anti-apoptotic factor during oocyte maturation. We suggest that IGF-I may inhibit apoptosis in oocytes at the stage of caspase activation and may prevent further advancement of oocyte apoptosis.
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  • Naoko INOUE, Fuko MATSUDA-MINEHATA, Yasufumi GOTO, Kazuhiro SAKAMAKI, ...
    2007 Volume 53 Issue 2 Pages 427-436
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 20, 2006
    JOURNALS FREE ACCESS
    To reveal the intracellular signal transduction molecules involved in granulosa cell apoptosis in porcine ovarian follicles, we cloned the porcine Fas-associated death domain (FADD), an adaptor protein for the cell death receptor, and procaspase-8, an initiator caspase. Porcine FADD (pFADD) was 636 bp (211 amino acids: aa) long and showed 74.0 and 65.4% homology with human and murine FADD, respectively. Porcine procaspase-8 (pprocaspase-8) was 1,431 bp (476 aa) long and 70.6 and 63.4% homologous with human and murine procaspase-8, respectively. To confirm the apoptosis-inducing abilities, we constructed pFADD and pprocaspase-8 cDNA expression vectors with enhanced green fluorescence protein (EGFP) and then transfected them into human uterine cervix tumor (HeLa-K), human granulosa cell-derived (KGN), murine granulosa-derived tumor (KK1), and porcine granulosa cell-derived (JC410) cells. When pFADD and pprocaspase-8 were overexpressed, cell death was induced in these transfected cells. However when caspase-inhibitor p35 was cotransfected, cell death was inhibited. The pFADD and pprocaspase-8 genes are well conserved, as are the physiological functions of their products.
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Technical Note
  • Luca TODINI, Alessandro MALFATTI, Olimpia BARBATO, Silva COSTARELLI, A ...
    2007 Volume 53 Issue 2 Pages 437-441
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 30, 2006
    JOURNALS FREE ACCESS
    The aim of this trial was to evaluate the effectiveness (fertility and lambing) of priming with a single injection of progesterone plus PMSG in anovulatory lactating Sarda ewes subjected to the ram effect (RE) in spring. Thirty ewes (P4 group) were i.m. injected with 30 mg progesterone and 500 IU PMSG 36 h before ram introduction (d 0). This treatment was compared to a 12-day treatment with fluorogestone acetate intravaginal sponges that was followed by injections of 350 IU PMSG upon sponge withdrawal (FGA group, n=30). All ewes responded to RE, showing plasma progestrone concentrations >1 ng/mL between d 6 and 12 (FGA) or 6 and 9 (P4). Eighty-nine percent of the P4 ewes conceived at first ovulation, and 11% conceived following a short estrus cycle. Lambings occurred on d 150.4 ± 3.9, and the lambing rate was 100%. The fertility of the FGA ewes was 83% for the induced ovulation and was 7% for the second ovulation after a normal cycle. The FGA ewes lambed on d 149.8 ± 4.4, and the lambing rate was 83%. Two abortions were recorded for the FGA ewes, which had higher prolificacy than the P4 group (2.2 ± 0.8 vs. 1.8 ± 0.4, respectively; P<0.05). Both fertility and the lambing rate were high in both groups, with a high degree of estrus synchronization, and there were no significant differences between the groups. We concluded that priming of lactating Sarda ewes in spring with P4+PMSG before RE is an effective and competitive method (cheaper and more practical than FGA+PMSG) of inducing fertile ovulations in these ewes.
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  • Keiichiro TOMINAGA, Fumiyuki IWAKI, Shinichi HOCHI
    2007 Volume 53 Issue 2 Pages 443-447
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: November 30, 2006
    JOURNALS FREE ACCESS
    The purpose of this study was to develop a practical cryopreservation method for in vitro-produced (IVP) and sex-predetermined bovine blastocysts that will be applicable to direct transfer of the post-thaw embryos. Blastocysts were harvested 7 days after IVF and allocated to either an intact or biopsy group. The cryoprotective solution contained 0.7 M glycerol and 0, 0.05 or 0.1 M sucrose. Slow cooling at a rate of -0.5 C/min was terminated at -25, -30, or -35 C, and rapid cooling in liquid nitrogen was followed. After one-step thawing and dilution, the IVP blastocysts were cultured for 3 days to assess their survival. The post-thaw survival rate of intact blastocysts after termination of slow cooling at -30 C in 0.7 M glycerol plus 0.1 M sucrose (96.2%) was significantly higher than that at -25 C in 0.7 M glycerol alone (44.4%). The post-thaw survival rate of biopsied bovine blastocysts after termination of slow cooling at -25 C in 0.7 M glycerol alone (53.8%) tended to be lower than that at -25 C in 0.7 M glycerol plus 0.05 M sucrose (91.3%) or -30 C in 0.7 M glycerol plus 0.1 M sucrose (92.3%). Thus, addition of a small amount of sucrose to 0.7 M glycerol cryoprotective solution shortened the process of slow cooling for both the intact and biopsied bovine embryos. Judged from the survival levels in vitro after thawing and one-step dilution of embryos (>80%), this is an improved method of cryopreservation for subsequent direct transfer of IVP and biopsied bovine blastocysts.
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  • Juan CHEN, Yun QIAN, Ting FENG, Ling-Bo CAI, Jia-Yin LIU, Yan ZHANG, W ...
    2007 Volume 53 Issue 2 Pages 449-453
    Published: 2007
    Released: May 12, 2007
    [Advance publication] Released: December 01, 2006
    JOURNALS FREE ACCESS
    The present report describes the birth of a healthy infant after cryopreservation of embryos produced from in vitro-matured oocytes retrieved from a woman at risk of developing ovarian hyperstimulation syndrome (OHSS) during conventional in vitro fertilization (IVF) cycles. A conventional long protocol including gonadotropin-releasing hormone agonist (GnRHa) and gonadotropins induced a risk of OHSS. Oocyte retrieval was performed on day 11 of the cycle, and 27 immature oocytes were obtained. Following incubation for 24 h in maturation medium, 74.1% (20/27) of the oocytes were at the metaphase II stage. Fourteen oocytes (14/20, 70.0%) were fertilized after intracytoplasmic sperm injection (ICSI) with her husband's spermatozoa and cultured for 3 days. On day 4 following oocyte retrieval, three embryos at the 8-16 cell stage were transferred into the woman's uterus, and five spare embryos were frozen. Since the fresh embryo transfer failed to result in pregnancy, three post-thaw embryos were transferred into the woman three months later. Transfer of the frozen embryos resulted in pregnancy with delivery of a healthy infant girl.
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