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K. Ogawa and K. Goto

Summary. LH and progesterone added to cultures of the follicle wall of hens increased the total activity of neutral and acid proteases and collagenase and the increase was greater for follicle tissues from the stigma region. Adenohypohysectomy of hens resulted in the decreased activities of neutral and acid proteases and collagenase in the first and second largest follicles.

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K. Ogawa, K. Goto, and T. Tokunaga

Summary. Ovine LH and ovine FSH stimulated progesterone production in granulosa cells isolated from the F1, F2 and F3 follicles of hypophysectomized and control (sham-operation) hens when they were collected 6 h after operation, but the steroidogenic response to LH was greater for granulosa cells from hypophysectomized hens. At 15 h after operation progesterone production by granulosa cells was stimulated by LH in all 3 follicle types of control hens, but only in the F1 follicles of hypophysectomized hens. The response to FSH at 15 h was similar for control and hypophysectomized hens. The time after hypophysectomy therefore appears to affect the LH-stimulated progesterone production by granulosa cells of the F2 and F3 follicles.

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H. Tojo, M. Fujii, and K. Ogawa

Summary. There were no significant changes in the activities of collagenase, acid and neutral protease up to ovulation. Neutral protease activity increased significantly in the post-ovulatory follicle obtained immediately after ovulation. Acid protease and collagenase showed an increasing activity with time after ovulation. The progesterone concentration of the follicle wall rose 3 h before ovulation, peaked 1 h before ovulation and remained high until ovulation. Plasma progesterone values were high but decreased before those in the follicle. Follicular progesterone concentrations decreased markedly soon after rupture, but oestradiol values then increased.

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K. Ogawa, H. Tojo, and Y. Kajimoto


Administration of PMSG or FSH to hypophysectomized hens prevented the multiple ovulation (2-3 ova) induced by an OIH. Inhibition of multiple ovulation did not occur when FSH was injected ½-2 hr before the OIH injection. The results are believed to support the theory that withdrawal of FSH stimulation sensitizes a follicle to the OIH in the normal hen.

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K. Ogawa, M. Kurohmaru, H. Sugino, and Y. Hayashi

Cyclic changes in follistatin localization and the role of the primary gonadotrophin surge in regulating follistatin expression were studied in rat ovaries by immunohistochemistry. Two different polyclonal antisera were raised against synthetic peptides corresponding to amino acids 123–134 and 300–315 of human follistatin. Immunoreactive follistatin was detected in granulosa cells of secondary and mature follicles. Although immunoreactions with anti-follistatin (300–315) occurred in preovulatory follicles until immediately before ovulation (23:00 h on the day of pro-oestrus), follistatin was not immunodetected in newly formed corpora lutea (11:00 h on the day of oestrus). Granule-like immunoreactions with anti-follistatin (123–134) serum in preovulatory follicles markedly decreased in intensity on the evening of pro-oestrus, indicating the loss of follistatin production. Blocking the primary gonadotrophin surge by a pentobarbitone injection (40 mg kg−1) at 13:30 h on the day of pro-oestrus sustained the immunoreactivity with anti-follistatin (123–134) in preovulatory follicles up to 23:00 h on the day of pro-oestrus. Injection of pentobarbitone-treated animals with exogenous LH (100 μg kg−1) or FSH (50 μg kg−1) at 15:30 h on the day of pro-oestrus eliminated the immunoreactions. These results indicate that the expression of follistatin in preovulatory follicles is suppressed by the primary gonadotrophin surge during pro-oestrus. Hence, it is conceivable that the cessation of follistatin production in preovulatory follicles may occur before ovulation, and that it may be caused by the primary gonadotrophin surge.

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K. Goto, Y. Kajihara, S. Kosaka, M. Koba, Y. Nakanishi, and K Ogawa

Summary. Bovine follicular oocytes surrounded by cumulus cells for more than one-third of their surface were matured, fertilized and developed in vitro utilizing a co-culture system with bovine cumulus cells. Embryos developed into blastocysts were non-surgically transferred to the uteri of cows at Day 6, 7 or 8 (Day 0 = oestrus). Out of 6 recipient cows (19 blastocysts transferred), 3 became pregnant. One of the 3 pregnant cows carried twins.

The results of this study demonstrated the viability of embryos obtained from in-vitro maturation of bovine oocytes followed by in-vitro fertilization and culture to the blastocyst stage in vitro.

Keywords: in-vitro; maturation; ferilization; cattle

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N. Tada, M. Sato, J. Yamanoi, T. Mizorogi, K. Kasai, and S. Ogawa

Summary. When mouse epididymal spermatozoa were rapidly frozen in two steps (37 to −70°C for solid CO2 and −70 to −196°C for liquid nitrogen) as pellets, 18% raffinose provided the greatest protection to ICR mouse spermatozoa against cold-shock; sperm motility and fertilizing ability were 43% and 22·4%, respectively. A small proportion of spermatozoa frozen with 10% sucrose was motile but incapable of fertilizing ovulated oocytes. Glycerol and dimethylsulphoxide were less effective at any concentration examined. However, the fertilizing ability of frozen–thawed ICR spermatozoa was significantly improved (35·5%) by addition of glycerol (1·75% final concentration) to medium containing 18% raffinose. Spermatozoa from one outbred (ddY) and 5 inbred (C57BL/6N, C3H/HeN, DBA/2N, BALB/c and kk) strains of mice were successfully frozen in the presence of 18% raffinose and 1·75% glycerol, although the fertilization rates of frozen–thawed spermatozoa varied among strains (13% for C57BL/6N to 64% for DBA/2N). A small fraction of mouse eggs resulting from fertilization by frozen–thawed spermatozoa developed normally in vitro (37% in C57BL/6N to 71% in ICR) to the blastocyst stage and in vivo (19% for C57BL/6N spermatozoa and ddY oocytes) to Day 18 of gestation.

Keywords: mouse; spermatozoa; cryopreservation; pellet method; cryoprotectant; raffinose