Isao Ishibashi

Developmental ability of porcine ova matured in porcine follicular fluid in vitro and fertilized in vitro

Developmental ability of porcine ova matured in porcine follicular fluid (pFF) with FSH in vitro and fertilized in vitro was examined by culturing in BMOC-2. Forty-eight hours after insemination, 35.6% of ova cleaved normally, and this rate was significantly higher (13.0%) than that of the ova matured in a modified Krebs-Ringer bicarbonate solution. Twenty-four percent (29 120 ) of ova matured in pFF with FSH developed to the four-cell stage and two of them developed to the eight-cell stage 66 h after insemination. Most cleaved embryos stopped developing at the four-cell stage and neither the morula nor blastocyst stage was observed throughout the culture period as reported in the in vivo matured ova. In culture at 37 degrees C, the appearance of two-cell and four-cell embryos was delayed from that of in vivo embryos, but their development was significantly accelerated by culturing at 39 degrees C. These results show that pFF is an excellent maturation medium for porcine oocytes, and the developmental capacity of the ova matured in pFF seems to be similar to that of in vivo matured ova. Culturing at 39 degrees C was found to be more suit-able for the development of ova than 37 degrees C.

Number of follicles and serum levels of steroid hormone in adult rats which mated following treatment with various doses of PMSG and hCG.

高単位のPMSGとhCGを投与した成熟ラットの着床困難の原因を検討するため,卵胞の動態を組織学的に観察すると共に,P,E1,E2値をラジオイムノアッセイ法により測定した。1.無処置ラットの500μm以上の正常卵胞数は,交配後12hの5.2個から60~84hの12.6~13.6個へ増加した。2.PMSGとhCG投与群の500μm以上の正常卵胞数は,hCG投与後24(交配後12)hでは無処置に比較して有意に多く(P<0.05または0.01),48(36)hでは40IU投与群のみが多かった(P<0.01)。3.低単位のホルモン投与群の小型(<499μm)の閉鎖卵胞は,hCG投与後48~72(交配後36~60)hまで増加する傾向を示した後減少した。4.P値は時間の経過に伴って増加し,無処置では交配後108hに58.9ng/ml,10,20IU投与群ではhCG投与後96(84)hに98.3および113.3ng/mlに達し,120(108)hでもほぼその値が維持された。40IU投与群では72(60)hに170.3ng/mlのピークに達した後,急激に減少した。5.E1値は無処置とホルモン投与群の間,および経時的に大きい差違が認められなかった。無処置およびホルモン投与群のE2値は,hCG投与時(-12h)に高い値を示したが,24h後には減少した。以後無処置と10および20IU投与群では大きい変動はなかったが,40IU投与群ではhCG投与後72(60)hに無処置より有意に高かった(P<0.01)。以上の結果から,高単位のPMSGとhCGを投与した成熟ラットの着床困難は,卵管および子宮におけるホルモン環境の異常に起因していると考えられる。そしてそれはhCG投与後72(交配後60)hにおけるE2の増加による卵子下降の促進と,この時期以後におけるPの減少による胚の維持困難に由来している可能性が考えられる。

Effects of aminoglutethimide phosphate administration on embryonic development and implantation and on steroid hormone levels in superovulated rats

The present study was undertaken to determine the relationship between preimplantation development of embryos and hormone levels in superovulated immature rats treated with aminoglutethimide phosphate (AGP), an inhibitor of steroid synthesis. In addition, the effects of AGP on incidence of pregnancy and the number of implantation sites in superovulated rats were studied. Immature or adult rats were pretreated with 20 or 40 IU of pregnant mares serum gonadotropin (PMSG) at 11.00–13.00 h followed by 20 or 40 IU of human chorionic gonadotropin (hCG) to the respective groups 52 to 54 h later. Ten or 20 mg AGP/day was administered once on day 0, 1 or 2; twice during days 0 to 2; or three times on days 0 to 2 of pregnancy. Animals were killed 24 to 120 h after hCG injection (estimated 12 or 108 h after ovulation) or on day 8 of pregnancy. Pregnancy rates in superovulated immature and adult rats given 10 or 20 mg AGP for the first 3 days were 73.3–80.0 or 93.3%, respectively. Injection of 10 or 20 mg AGP to these animals resulted in 10.9–16.2 or 20.2–21.6 implantation sites per mated rat, respectively. Transport of ova in superovulated rats receiving no AGP treatment was faster than in AGP-treated rats by 72 h after hCG injection, and most embryos were lost from the uterus at 120 h in the former group, but not in the latter. Serum progesterone concentrations at 48 and 72 h and estradiol-17β levels at 48 h in AGP-treated rats were significantly lower than those in controls (P < 0.05-0.01). These findings suggest that the diminished incidence of implantations is caused by hypersecretion of estrogen, and this was counteracted by AGP treatment.

In vitro developmental capacity of mouse follicular oocytes recovered at various times after hCG injection and matured in vitro

Abstract The study was designed to examine in vitro developmental capacity of mouse follicular oocytes recovered at various times after hCG injection and matured in vitro. Jc1/ICR mice were treated with 5 IU PMSG and 5 IU hCG at 48-h intervals. Oocytes were recovered from follicles at 0, 4, 8 and 12 h after hCG injection and from the oviduct at 12 and 16 h after that (F-0, F-4, F-8, F-12, T-12 and T-16 oocytes, respectively). They were cultured in modified Krebs-Ringer solution (TYH) with or without 5% fetal calf serum (FCS) for the remaining periods to total 16 h before insemination. These matured oocytes were transferred into modified Whittens medium and cultured for up to 120 h after insemination. The TYH medium with FCS increased the proportion of development into blastocysts in F-4 to F-12 oocytes to 80 to 86%. Development of F-8 and F-12 oocytes to blastocysts without FCS was 83 and 89%. These rates were not significantly different from those of T-12 (83%) and T-16 (87%) oocytes. The maturation stages of most F-4, F-8 and F-12 oocytes at recovery were prometaphase-I, metaphase-I plus anaphase-I, and metaphase-II. The process of oocyte maturation in vitro was similar to in vivo. These results suggest that mouse oocytes which resumed meiotic division until prometaphase-I or further stages in vivo, and were cultured in TYH with or without FCS, have a developmental capacity as high as that of ovulated oocytes. In addition, FCS seemed to have most beneficial effect on the fertilization ability.

Effects of aminoglutethimide phosphate administration on the incidence of pregnancy and number of live fetuses in superovulated rats

This study examined the effects of aminoglutethimide phosphate (AGP) administration on the incidence of pregnancy and number of live fetuses in superovulated rats. Immature (31- to 33-day-old) young (8- to 9-week-old) and (14- to 16-week-old) rats were treated with 20 or 40 IU of PMSG and the same dosses of hCG at 52 to 54 hour-intervals (GTH rats). One half of the GTH rats were injected with 10 mg AGP twice a day for the first 3 days of pregnancy (AGP rats). Pregnancy rates of all AGP rats (95 to 100%) were much higher than the GTH rats (12.7). Young and adult AGP rats treated with 20 IU of GTH had 14.7 and 15.3 live fetuses per dam, respectively. These numbers of live fetuses increased to 17.8 and 18.4 after treatment with 40 IU of GTH. These values were significantly greater than those of the control rats (12.3 to 12.8; P<0.01), but the fetal weights were lower. The results show that AGP administration improves pregnancy rates and the number of live fetuses in young and adult superovulated rats.

The stimulating effect of copulation on ovulation in adult rats treated with PMSG alone.

Wistar-Imamichi female rats were treated with 10, 20, or 40 IU of PMSG. The females were placed with a male in order to determine the effect of copulation on PMSG-in-duced ovulation. At 76 to 78 h after PMSG injection, they were killed and the number of ova were counted. The percentage of rats ovulating and the number of ova shed in the non-cop-ulated group decreased markedly with increasing dose of PMSG, but in the copulated ones, the percentage of rats that ovulated decreased only slightly and the number of ova shed increased with increasing dose of PMSG. The ovulatory responses of the copulated group were signifi-cantly different from that of the non-copulated, except the proportion of rats ovulating after treatment with 10 IU of PMSG. When females were placed with a male for 1 to 11/2h, the stimuli with single copulation and mounting had a little or no effect on PMSG-induced ovula-tion. Exposure to a male without copulation overnight had no effect. The percentage of ova fertilized in rats placed with a male overnight decreased significantly with increasing dose of PMSG. These results suggest that the favourable effect of copulation on PMSG-induced ovul-ation in adult rats may caused by the stimulus of multiple intromission.

Effects of aminoglutethimide phosphate on litter size and birth weight in superovulated rats

In this study, litter sizes, birth weights and survival rates of neonates in superovulated rats treated with aminoglutethimide phosphate (AGP) were studied. Young (8- to 9-week old) and adult (14- to 16-week old) rats were treated with 20 or 40 IU PMSG between 1100 and 1300 hours on the day of early diestrus, followed by identical doses of hCG 52 to 54 hours later. These rats received 10 mg AGP on Days 0 to 2 of pregnancy (AGP rats). The duration of pregnancy in the AGP-treated and control rats was about the same. Mean live litter sizes in young and in adult AGP rats treated with 20 IU PMSG and 20 IU hCG were significantly higher (15.5 and 15.4; P<0.01) than in the control rats (11.5 and 12.6). When AGP was given to young and adult rats pretreated with 40 IU PMSG and 40 IU hCG, they produced means of 17.7 and 17.3 live neonates, respectively. The birth weights for young (5.55 to 5.88 g) and adult (5.89 to 5.96 g) AGP rats were smaller than those of respective control rats (6.44 and 6.40 g; P < 0.05-0.01). When the number of neonates was adjusted to 8 per dam for nursing and rearing, all of them were alive at 5 weeks after birth and showed normal growth. However, body weight gains of the progeny of AGP dams were lower than those of controls. These results indicate that rats treated with large doses of gonadotropins and AGP produce larger litter sizes than nontreated controls.