Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 42, Issue 1
February
Displaying 1-11 of 11 articles from this issue
Original Articles
  • Hiroshi TORIUMI, Shigehisa TSUMAGARI, Yasushi KUWABARA, Kaoru TAKAGI, ...
    1996 Volume 42 Issue 1 Pages 1-6
    Published: 1996
    Released on J-STAGE: November 30, 2001
    JOURNAL FREE ACCESS
    The aim of this study was to diagnose ovarian disorders by evidence of external signs and through repeated rectal palpation in post-weaned sows. Blood samples were collected weekly from 59 sows with ovarian disorders from weeks 1 to 5 following weaning (the first examinations were done 7 to 10 days after weaning and were defined as post-weaned week 1), to determination progesterone (P) and estradiol-17β (E2) levels by radioimmunoassay. The ovarian disorders were devided into the following: ovarian subfunction (OS), small multiple follicular cysts (SMFC), large multiple follicular cysts (LMFC), large multiple luteal cysts (LMLC), and retention of corpora lutea (RCL). The ovaries, uteri, and vulvae were poor in sows with OS. Serum levels of P and E2 in sows with OS were low, and P levels decreased further in post-weaned week 3. In ovaries with SMFC and LMFC, numerous follicles less than 8-mm and 15- to 50-mm in diameter were observed, respectively. Animals with SMFC showed medium to high uterine contraction. There were differences in both follicular size and in the degree of uterine contraction between the sows with SMFC and LMFC. P levels remained low in both the sows with SMFC and LMFC, whereas E2 levels were persistently high. The walls of the LMFC were thin and weak, whereas those of the LMLC were thick, secure and tough. The sows with the latter type of cysts had high P levels, showing different findings from the sows with LMFC. Thus, LMLC and LMFC can be differentiated by the palpation characteristics of cyst walls and by serum P levels. In sows with RCL, the ovarian findings resembled those in the luteal phase of normally cycling sows, whereas LMLC ovaries were large. Therefore, RCL and LMLC could be differentiated by the size of the ovaries. These differential diagnoses will allow for appropriate treatments of sows with these ovarian disorders in the future.
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  • Mutsuro MOTOISHI, Kazufumi GOTO, Keiko TOMITA, Shoji OOKUTSU, Yoshihik ...
    1996 Volume 42 Issue 1 Pages 7-13
    Published: 1996
    Released on J-STAGE: November 30, 2001
    JOURNAL FREE ACCESS
    Various concentrations of dithiothreitol (DTT) and/or heparin were examined for their ability to induce in vitro decondensation of bovine and human sperm nuclei. Bull sperm nuclei decondensed when DTT (10 mM) and heparin (10 μg /ml) were added simultaneously or when heparin (100 μg/ml) was added after the pretreatment with DTT (100 mM). Neither DTT (500 mM) nor heparin (10 mg/ml) alone, even at high concentration, could induce decondensation of bull sperm nuclei. In contrast, human sperm nuclei decondensed when DTT alone (10 mM) or heparin alone (1 mg/ml) was added. Pretreatment of human sperm with DTT (0.1 mM) decreased the concentration of heparin required for decondensation to 10 μg/ml, a concentration incapable of inducing decondensation by itself. Overall, higher concentrations of DTT and heparin were required for bovine than for human sperm for nuclear decondensation to occur. The difference in nuclear decondensation between bull and human sperm in response to DTT and heparin is likely related to the differences between the two species in the DNA-protamine complex of sperm nuclei.
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  • Hiroyuki SUZUKI, Miho MORIGUCHI, Ryoko KIDA, Yuetsu MORO
    1996 Volume 42 Issue 1 Pages 15-22
    Published: 1996
    Released on J-STAGE: November 30, 2001
    JOURNAL FREE ACCESS
    Ova were collected from young (9-12-week old) and aged (43-53-week old) golden hamsters at various times before and after fertilization to decide the timing of ovulation, sperm penetration and pronuclear formation. Ovulation was delayed 1 h in the aged animals, although overall ovulation rate did not differ statistically between young and aged animals. Sperm penetration also showed another 1 h delay in aged hamsters. The types of abnormalities during early fertilization were asynchronous development of male and female pronuclei, polyspermy, polygyny and pycnosis. Asynchronous development of the male and female pronuclei, mainly a delay in the development of the male pronucleus, was found more often in the aged females (8.2%), contrasting 3.1% (P<0.01) in the young females. Polyspermy accounted for a small proportion of the abnormalities in both groups (0.5 and 0.4% in the young and aged groups, respectively), whereas abnormalities observed only in the aged group were polygyny (5.5%) and pycnosis (0.4%). It is suggested that increased abnormalities of fertilization observed in the aged hamsters (approximately 15% of the total ova) may be directly related to delayed ovulation and fertilization attributed to defective ova and deteriorated reproductive ability of the aged females. These delays in ovulation and fertilization may be the main factors causing the preimplantation wastage of embryos in the aged hamsters.
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  • Katsuhiko OHNUMA, Kazuei ITO, Yho-Ichi MIYAKE, Jutaro TAKAHASHI, Yasuh ...
    1996 Volume 42 Issue 1 Pages 23-28
    Published: 1996
    Released on J-STAGE: November 30, 2001
    JOURNAL FREE ACCESS
    Early Pregnancy Factor (EPF) has been detected in the pregnant sera of almost all the species examined. Yet no EPF of mare sera had been reported prior to this study, because the method of examination had not yet been established. The present study was carried out to examine various factors which influence the rosette inhibition test of mares and to establish a reliable assay system for Rosette Inhibition Titre (RIT). The following items were found optimal for the detection of RIT in mare sera: human erythrocytes (red blood cells) showed the best reactivity among human, sheep, and rat erythrocytes; the ratio of lymphocytes to erythrocytes of 1:20 was proven to show better results; and Guinea Pig Complement (GPC) was not necessary. When the rosette inhibition test was performed on 10 pregnant and 9 nonpregnant sera, the RIT values ranged from 6 to 8 (7.2 ± 1.0) in nonpregnant sera, and from 9 to 12 (9.5 ± 1.0) in pregnant sera, with a significant difference (p<0.01). These results suggested the availability of the RIT for pregnant mare sera as an index of early detection of pregnancy in mare.
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  • Kazushi ARAKI, Rika SUZUKI, Minesuke YOKOYAMA, Kunihiko NAITO, Eimei S ...
    1996 Volume 42 Issue 1 Pages 29-33
    Published: 1996
    Released on J-STAGE: November 30, 2001
    JOURNAL FREE ACCESS
    The fertilizability of mouse oocytes matured in serum-free medium was extremely lower than those matured with fetal calf serum (FCS). The addition of FCS into maturation medium, however, complicates the analytical studies on the oocyte maturation. In the present study, a chemically defined medium supplemented with fetuin, a major protein in FCS to prevent zona hardening, and one of pregnant mare's serum gonadotrophin (PMSG), follicle stimulating hormone (FSH) or epidermal growth factor (EGF) was used to examine the effect on oocyte maturation and fertilizability of the oocytes. The fertilization rates of the oocytes matured with bovine serum albumin (BSA) alone or fetuin alone were significantly lower than that matured with FCS (35.3%, 44.3% and 95.5%, respectively). On the other hand, the addition of both fetuin and one of PMSG (10 iu/ml), FSH (0.1 armour unit/ml) and EGF (10 ng/ml) induced clear cumulus-expansion and improved the fertilization rates (84.1%, 88.4%, and 78.3%, respectively) to the comparable level as FCS supplementation. The removal of cumulus cells just before insemination abolished the effects of PMSG completely. These results suggest that the effects of FCS maintaining high fertilizability of in vitro matured oocytes work on both zona pellucida and cumulus-cells, and are fully replaced by fetuin and one of PMSG, FSH or EGF. This serum-free maturation system would be useful for the analysis of the factors and mechanisms regulating oocyte maturation.
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  • Keiichiro TOMINAGA, Kazuhiro YONEDA, Kyozo UTSUMI
    1996 Volume 42 Issue 1 Pages 35-40
    Published: 1996
    Released on J-STAGE: November 30, 2001
    JOURNAL FREE ACCESS
    This experiment was undertaken to determine if developmental rate to the blastocyst stage and the blastocyst production capacity of donor cows affects embryo sex ratio, and to clarify the relationship between individual donor cows and male sex predominance of embryos that reach blastocyst stage relatively early. In vitro matured (IVM), in vitro fertilized (IVF) and in vitro cultured (IVC) embryos produced separately from donor cows were used. The sex of the embryos at the blastocyst stage was determined by PCR analysis. Although there was no significant difference in the sex ratio of embryos in relation to days after insemination, a higher proportion of male blastocysts was obtained on days 6.5 and 7.0 (59.3% and 58.0%, respectively) than on days 7.5 and 8.0 (50.7% and 49.3%, respectively). No relationship between male predominance and the developmental rate of embryos was found. Nor was there any evidence of a characteristic relationship between the developmental rate and the sex of embryos produced from individual donor cows. However, when donor cows were classified according to the period required for their embryos to reach blastocyst stage, a higher percentage of cows showed a higher male percentage in their embryos on the least number of days after insemination than on later days. This difference also was not significant. Therefore, there is little relationship between embryonic sex and the developmental rate of embryos in cows.
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  • Yoshimi IKEMIZU, Satoru YAMAZAKI, Jutaro TAKAHASHI, Yasuhisa YASUDA
    1996 Volume 42 Issue 1 Pages 41-45
    Published: 1996
    Released on J-STAGE: November 30, 2001
    JOURNAL FREE ACCESS
    Early pregnancy factor (EPF) has been detected in pregnancy sera of many animal species. Isolation and purification of the EPF in pregnancy rat serum (Day 4) was attempted by using ammonium sulfate fractionation, ion-exchange chromatography, affinity chromatography and Immunoblotting. As a result, rat EPF was detected in 40% ammonium sulfate supernatant, CM-Sepharose Fast Flow 20 mM and 30 mM NaCl elution fraction, DEAE-Sepharose Fast Flow unbound fraction and Affinity chromatography unbound fraction. By the result of immunoblotting, there were three bands (22 KD, 24 KD and 28 KD) in the purified rat EPF fraction.
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  • Kazuyoshi HASHIZUME, Hirotada TSUJII, Yuji TAKAGI, Makoto ROKUTANDA
    1996 Volume 42 Issue 1 Pages 47-53
    Published: 1996
    Released on J-STAGE: November 30, 2001
    JOURNAL FREE ACCESS
    The process of spermatogenesis was studied in mutant hypogonadal (hpg) male mice treated with equine chorionic gonadotropin (eCG) or gonadotropin releasing hormone (GnRH) for 60 days. Untreated adult hpg male mice had very small gonads (5.7 ± 0.6, n=7, mean ± SEM) and no mature spermatozoa due to a genetic deficiency of GnRH. Testicular weights increased to 55.7 ± 5.6 mg in eCG (5 IU/day) injected mice (n=7) and 30.0 ± 3.8 mg in GnRH (1 μg/day) injected mice after 60 days. Similar increases were seen in seminal vesicles. In eCG-administered mice, matured spermatozoa appeared in epididymides after 45 days of treatment. A lower dose of eCG (2.5 IU/day) or GnRH (1 μg/day) were less effective for stimulation of spermatogenesis. Although the propotion of hpg mouse sperm fertilized embryos that developed to the blastocyst stage (18%) was lower than the proportion of normal mouse sperm fertilized embryos (43%), at least some of the hpg mouse sperm fertilized embryos produced viable offspring through embryo transfer. The present study proved that chronic eCG or GnRH administration could stimulate spermatogenesis in hpg males, and cauda epididymal spermatozoa were fertile when obtained from eCG-injected mutant mice. These results intimated that hpg male mice provide valuable information for clarifying the endocrine and paracrine control processes of spermatogenesis.
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  • Makoto KIKUCHI, Yutaka SENDAI, Shoko YAMASHITA, Takeshi SATOH, Hiroyos ...
    1996 Volume 42 Issue 1 Pages 55-60
    Published: 1996
    Released on J-STAGE: November 30, 2001
    JOURNAL FREE ACCESS
    The matrix metalloproteinases and their inhibitors are thought to play an important role in such biological processes as ovulation, embryo development, implantation, angiogenesis and tumor cell metastasis. Because recent findings indicated that the tissue inhibitor of metalloproteinase-1 (TIMP-1) produced by granulosa cells improved bovine embryo development in vitro, the patterns of gene expression have been determined for TIMP-1 and collagenase during bovine preimplantation development by reverse transcription-polymerase chain reaction (RT-PCR). mRNA transcripts for bovine TIMP-1 and collagenase were detectable in the morula and blastocyst stages of bovine embryos. This data suggested that TIMP-1 and collagenase may be involved in the growth and development of bovine embryos in an autocrine fashion as well as in a paracrine manner.
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  • Hiroshi TAKANO, Keisuke KOYAMA, Chiaki KOZAI, Satoru SHIMIZU, Yoko KAT ...
    1996 Volume 42 Issue 1 Pages 61-65
    Published: 1996
    Released on J-STAGE: November 30, 2001
    JOURNAL FREE ACCESS
    In vitro development and changes in the nuclei of reconstituted oocytes following fusion were investigated to determine the effects of cell cycle stage of donor nuclei on bovine nuclear transplantation. Enucleated oocytes, cultured for 44 to 46 h following maturation or preactivated for 9 h before fusion, were used as the source of recipient cytoplasm. Donor embryos were obtained from embryos matured, fertilized and cultured in vitro. Blastomeres of the embryos which had developed to the 4-cell stage at 39 h after insemination were separated. Each blastomere was cultured with aphidicolin for 2 to 4 h. These blastomeres, shortly after the next division (G1/S stage of the cell cycle), or those cultured for 6 h without aphidicolin after division (S stage of cell cycle), were fused with non-activated (Groups 1 and 3) or activated (Groups 2 and 4) oocytes. The proportion of embryos which developed into blastocysts were higher in Groups 2 (16%) and 4 (17%) than in Groups 1 (10%) and 3 (6%). The diameter of the nuclei after fusion with the recipient cytoplasm increased, but to a lesser extent in activated than in the non-activated cytoplasm. Some nuclei which had fused with non-activated cytoplasm showed nuclear envelop breakdown and premature chromosome condensation (PCC, 21%). No donor nuclei showed PCC when fused with activated cytoplasm.
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  • Toshiyuki KOJIMA, Taizo IWASAKI, Yumie ZENIYA, Junryo YOSHINO, Kiyosh ...
    1996 Volume 42 Issue 1 Pages 67-72
    Published: 1996
    Released on J-STAGE: November 30, 2001
    JOURNAL FREE ACCESS
    The study on cryopreservation of porcine embryos were conducted to determine an effect of dietary intake of polyunsaturated fatty acid by donor gilts on embryo survival after freezing and thawing. Linoleic acid and a-tocopherol were supplemented to a conventional concentrated diet at the rate of 3.75% and 600 ppm, respectively. The diet was given to 5 pubertal gilts with an initial age of 6 months old for approximately 4 months (2 to 3 kg/head/day). From at least 70 days after starting of feeding of the experimental diet, embryo collections were performed. The control embryos were obtained from pubertal gilts given only the conventional concentrated diet. Embryos were surgically collected by flushing both uteri of donors on day 6 (day 1=the last day of estrus). Expanded blastocysts were used immediately for experiment, after measuring the diameter by an ocular micrometer. The younger-stage embryos were cultured in vitro in Ham's F-12 supplemented with 12 mg/ml BSA fraction V in 5% CO2 in air at 38.5 C up to expanded blastocyst stage with more than 200 μm in diameter. The embryos were equilibrated with 10% ethylene glycol solution containing 0.2 M trehalose for 5 min at room temperature. The embryos loaded into 0.25-ml plastic straws were immersed in alcohol bath at -7 C, seeded at -7 C, cooled to -30 C at the rate of 0.4 C/min and plunged directly into liquid nitrogen. After being thawed in air, the embryos were subjected to count the viable nuclei following a two-step dilution of the cryoprotectant with 0.5 M trehalose solution. As a result of counting the viable nuclei of frozen-thawed embryos, the mean number of viable nuclei of the embryos derived from gilts given the linoleic-acid supplemented diet was larger (P=0.0553) than that of the embryos derived from gilts given the control diet. The result of the present study suggests that dietary linoleic acid intake by embryo donor gilts is likely to have a potential effect on enhancement of freezing tolerance in porcine embryos.
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