Zona-free uncompacted 8-cell (8C) or partially compacted 8-cell (8-M) stage embryos were cocultured with frozen-thawed TT2 embryonic stem (ES) cells at a density of either 5.5 × 105 or 5.5 × 106 cells/ml. When zona-free embryos at the 8C and 8-M stage were cocultured with frozen-thawed ES cells at a density of 5.5 × 105 cells/ml, 5% and 32% of the embryos developed to the blastocyst stage after overnight culture, respectively. At a density of 5.5 × 106 cells/ml, however, developmental rates to the blastocyst stage of 8C and 8-M were only 12% and 15%, respectively. When the 8C and 8-M embryos were cocultured with frozen-thawed ES cells at a density of 5.5 × 105 cells/ml, there was no difference in the number of ES cells attached to either of them. The 8-M showed no significant difference in development to blastocyst stage in spite of the number of attached ES cells. However, the development rate to blastocyst was significantly reduced when the 8C aggregated with high numbers of ES cells. When the blastocysts were transferred into recipients, the percentage of chimeric mice in newborns ranged between 0-25%. In comparison, the percentage of newborn chimeric mice after transfer of the morula to small blastocyst ranged between 13-60%. One cause of this high frequency of chimerism after transferring morula derived from 8C was delayed preimplantation development of the embryos with high numbers of attached ES cells.
The effects of estradiol-17β (E2) on progesterone (P4) and oxytocin (OT) release were examined. Corpora lutea (CL) were obtained from cows at different stages of the estrous cycle (days <5, 5-7, 8-12 and 15-18) and were implanted with an in vitro microdialysis system (MDS). A 3-h infusion with E2 (100 nmol/l, 1 nmol/l, 10 pmol/l and 100 fmol/l) shifted from no effect (days 5-7 and 8-12) to an inhibitory effect (days 15-18) on P4 release during the estrous cycle. E2 at all test doses was most stimulative on the release of OT during the early luteal phase (days 5-7) but continuously inhibited OT release from the middle to late phase of the estrous cycle. Contrary to results obtained from days 5-7, in CL on days <5, a stimulation (100 nmol/l) or inhibition (1 nmol/l and 10 pmol/l) of OT release was observed. In contrast, preexposure to a potent, specific estrogen antagonist (ICI 182, 780; 10 nmol/l, 3 h) during the early luteal phase (days 5-7) showed no effect on P4 release and completely impeded the stimulatory effect of E2 on the OT release. In addition, a 3-h preexposure perfusion of ICI 182, 780 prevented the inhibition of OT secretion by E2 on days 8-12. The results suggest that E2 acts directly and acutely on the secretory function of bovine CL in the MDS during the early luteal stage (days 5-7) of the estrous cycle and may act together with the released OT as a luteotropic factor.
This study was undertaken to determine the release period of EPF-like substance(s) in in vivo fertilized rat ova and in vitro fertilized bovine ova. Using the rosette inhibition test, EPF-like substance(s) were detected in the culture media of in vivo fertilized rat and in vitro fertilized bovine ova a few hours after putative ovulation and insemination, respectively. In conclusion, it is confirmed that EPF-like substance(s) are released by in vivo fertilized rat ova and in vitro fertilized bovine ova a few hours after putative fertilization.
Ten serum constituents in 53 minke whales (Balaenoptera acutorostrata) from the southern Antarctic ocean were measured and compared with sex and three physiological states (pregnant, mature/non-pregnant, and immature) in females. Body length and body weight of immature females (n=5) were 7.2 ± 0.6 m and 4.6 ± 1.0 t, respectively. Both body weight and weight of pregnant (n=20: 8.9 ± 0.1 m and 7.6 ± 0.8 t) and mature/non-pregnant (n=6: 8.8 ± 0.2 m and 7.2 ± 0.4 t) females were similar. All males (n=22) were mature, and the testicular weights were 1493.9 ± 109.4 g and 1435.2 ± 87.7 g for left and right, respectively. There were no significant differences in the serum concentrations of the ten constituents between sexes or among the physiological states. Serum glucose values in males (183.5 ± 13.9 mg/dl) were significantly (P<0.05) correlated with body length (r=-0.49) and body weight (r=-0.46). Body length was positively correlated with serum concentrations of sodium (176.3 ± 6.2 mM/L: r=0.51, P<0.05), chloride (124.8 ± 5.8 mM/L: r=0.56, P<0.01) and calcium (15.2 ± 1.6 mg/dl: r=0.53, P<0.05). In females, body weight was positively correlated with the values of total-protein (6.9 ± 0.4 g/dl: r=0.38, P<0.05) and albumin (4.0 ± 0.2 g/dl: r=0.36, Plt;0.05). Although detailed and clear reasons for the significant correlations were not clarified in the present study, these results provide useful information on the serum chemistry of minks whales and other marine mammals.
To evaluate the correlation between luteolysis and T lymphocytes (T-cells) found in the corpus luteum (CL), peripheral blood lymphocyte (PBL) blastogenesis to mitogens tested for the presense of luteolytic factor(s) in the supernatant culture medium of luteal tissues from various stages of pseudopregnancy (PSP). PBL blastogenesis was measured by 3H-thymidine uptake. The culture medium of luteal tissue from Day 15 PSP caused a significantly high 3H-thymidine uptake in the Con A stimulation group of the PBL blastogenesis test. The present finding suggests the presence of some factors which activate in the regressive CL.
The experiments reported here take advantage of the large number of in vitro matured and in vitro fertilized (IVM/IVF) bovine oocytes which can be produced, permitting the design of controlled experiments to establish a simple defined medium for the study of early embryo requirements and to further test this medium as a component of co-culture. A total of 1386 IVM/IVF oocytes were used to compare a simple medium (KSOM) with complex culture conditions used successfully for culture of bovine embryos. All experiments were extensively replicated factorials. In Experiment 1, KSOM was equivalent to the complex Menezo B2 medium in producing blastocysts from IVM/IVF produced embryos and was superior to Menezo B2 medium when both were used with buffalo rat liver cells (BRLC), yielding 25% vs 8% blastocysts, respectively (P<0.05). In Experiment 2 a, KSOM was tested with 0 or 1 ng/ml of platelet derived growth factor (PDGF) and in Experiment 2 b, 0, 1 and 5 ng/ml of PDGF were tested. In Experiment 2 a blastocyst formation was higher (P<0.05) when PDGF was added to the co-culture and also was higher (P<0.05) for morulae plus blastocysts in Experiment 2 b when PDGF treatments were combined and compared to no PDGF. The results with the simple KSOM medium are sufficiently promising to indicate that growth factors, amino acids and other specific requirements of the embryo may be examined in future studies with KSOM as a base. Additionally, KSOM appears to be superior to Menezo B2 medium often used commercially for co-culturing bovine embryos with BRLC.
Diffusion into the ovarian artery of prostaglandin F2α (PGF2α) infused into the uterine vein of a cow was studied to demonstrate the existence of the so-called “utero-ovarian counter current mechanism” which was responsible for luteal regression in sheep and guinea pigs. In the first part of Exp. 1, PGF2α was infused in the uterine vein through an indwelling cannula. The plasma PGF2α concentration in the ovarian artery was increased to more than 300 times that in the jugular vein 1 min after the start of injection. In the second part which was begun 15 min after the completion of final blood sampling in the first part the plasma PGF2α concentration in the ovarian artery was increased to 2.7 times that in the jugular vein one minute after the start of injection. Though the difference between the PGF2α concentrations in the arterial and venous blood was smaller in the second part of Exp. 1, these results clearly show the existence of the counter current mechanism in the cow. In Experiment 2, PGF2α (15 mg in 3 ml solvent) was injected into the gluteal muscle 30 min after the completion of the final blood sampling to examine the changes in plasma concentrations of PGF2α in the ovarian artery, jugular vein and uterine vein after systemic injection of PGF2α. The PGF2α concentration in uterine venous blood prior to intra-gluteal injection was extremely high (60000 pg/ml). The concentrations in the ovarian artery and jugular vein were slightly increased with time after injection. Although the value was somewhat higher in the ovarian artery, almost similar patterns of change were seen in both the ovarian artery and the jugular vein.
The effect of a temporary dietary restriction (10 g/day) duirng gestation or lactation on the subsequent maturation of female offspring was examined. Mother rats were divided into 6 groups according to the period of restriction: namely, Group A (between days 0 and 7 of gestation), Group B (between days 8-14 of gestation), Group C (between days 15-21 of gestation), Group D (between days 1-7 of lactation), Groupt E (between days 8-14 of lactation), Group F (no restriction). In Group E, the mother's body weight decreased by 17% compared to that on day 0 of gestation. Female pups in this group showed retardation in growth rate concurrent with a delay in vaginal opening. No significant effects were found in any other groups. These results suggest that adequate nutrition during midlactation is critical for optimal growth of female pups and subsequent opening of the vagina.
To investigate whether preincubation of spermatozoa was a prerequisite procedure for successful in vitro fertilization (IVF) in the pig, we inseminated in vitro-maturing oocytes with either Percoll-washed or control-washed (diluted-and-pelleted) boar spermatozoa without preincubation. Both forms of washing were carried out in Tyrode's-based medium, and IVF was performed in modified Tyrode's solution containing 2 mM caffeine, 4.6 mM calcium and 5 mg BSA/ml. Percoll-washed and control-washed spermatozoa penetrated 94-100% and 95-97% of GVBD (germinal vesicle breakdown) oocytes, respectively, and the speed of penetration was similar for the 2 preparations. These results show that boar spermatozoa can penetrate oocytes in vitro without preincubation, and there was no difference in the oocyte penetration rate between those inseminated with Percoll-washed and those inseminated with control-washed (diluted-and-pelleted) spermatozoa.
Changes in plasma progesterone and estradiol-17β concentrations in 3 non-pregnant and 2 pregnant cheetahs were determined by radioimmunoassays. Progesterone concentrations in the non-pregnant cheetahs varied between 0.78 and 6.20 ng/ml and exhibited elevations at intervals of 10 to 12 weeks. There was one cheetah (2-year-old) in which plasma estradiol-17β concentrations elevated 1 to 2 weeks prior to the peak plasma progesterone concentrations. In the pregnant cheetahs, plasma progesterone concentrations increased at Day 3, rose to a peak (13 ng/ml) at Day 27, and decreased gradually until the time of birth. Plasma estradiol-17β concentrations in the pregnant cheetah attained a peak value (430 pg/ml) at Day 4 (before copulation), decreased gradually until Day 60 and increased again at Day 66 (409 pg/ml). In an aborted cheetah, the estradiol-17β level showed only a slight increase. These data provide the first assessment of reproductive functions in the female cheetah as monitored by plasma steroid profiles.