Nucleocytoplasmic interactions in controlling mouse embryonic development were tested by transplanting nuclei into different cytoplasmic environments. The nucleocytoplasmic ratios in 2-and 4-cell stage embryo were altered by removing one nucleus from a fused 2-cell and three nuclei from a fused 4-cell embryo. Such embryos containing relatively increased amounts of cytoplasm develop normally but at a slower rate than control embryos.
The effects of transplanting 4-cell donor nuclei to the cytoplasm of an enucleated fused 2-cell embryo led to successful in vitro development and also to term in six out of 67 reconstituted embryos. When the donor nucleus was from an 8-cell embryo, successful development in vitro was less frequent and none were carried to term. Cytoplasms from the 2-cell and 4-cell embryos were very similar in affecting further development, but cytoplasms of the oocyte failed to provide a suitable environment for further development. Most of the reconstituted embryos containing a nucleus from an 8-cell embryo and cytoplasms of 2 or 4-cell embryos compacted at the subsequent 4 cell or even at the 2-cell stage of development. Thus, an 8-cell nucleus produced compaction in cytoplasms from an earlier stage.
To test for reprogramming of nuclei transferred into the cytoplasms of an earlier stage of development, the nuclei from 4-cell stage reconstituted embryos were transferred into the cytoplasms of 2-cell embryos. Of the 53 recloned embryos 36(67.9%), 15(28.3%), and 1(1.7%) developed to the 2-, 4-, and 8-cell stages, respectively. The percent cleavage of recloned 4-cell stage nuclei was much lower than in the original reconstituted embryos. However no significant difference in frequency of cleavage was found between recloned embryos and reconstituted embryos with nuclei obtained from the normal morula stage. Forty-one of the recloned embryos were transferred to the oviducts of recipient mice. Of these, 17 were recovered from the oviducts after 72 h and five then continued to develop in vitro to the morula stage. These results suggest that embryonic nuclei are not reprogrammed, even by retransfer into less differentiated cytoplasms obtained from enucleated, fused 2-cell embryos.

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Urinary component level was examined by proton nuclear magnetic resonance (¹H-NMR) spectroscopy 2-3 days before mating, on day 17-19 of pregnancy and on day 9-11 of lactation in SHN female mice. Urinary levels of taurine and betaine, both of which were found in milk, continued to decline through pregnancy and lactation. Creatinine and creatine decreased and increased, respectively, in the middle of lactation. Allantoin also increased at the end of pregnancy. There was little difference in the levels of urea and lactic acid at any stage examined. Concurrent pregnancy affected little any component level in the middle of lactation. Among correlations between reproductive parameters and urinary component levels, litter size on the day of parturition and total pup weight on day 12 of lactation had the positive and the negative correlations with urinary levels of lactic acid and taurine, respectively. The significance of these findings was considered and the possibility that the measurement of urinary components by ¹H-NMR is a useful method to estimate the metabolic change of small experimental animals during reproduction was provided.

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The present investigation was performed to elucidate the mechanism of the suppression of ovarian follicular development and maturation in lactating rats. Administration of bromocriptine (CB-154) to lactating rats did not increase basal levels of LH, FSH, inhibin and estradiol-17β in the plasma, nor did it initiate follicular development and maturation, though plasma levels of prolactin and progesterone were markedly decreased by CB-154. The effects of high levels of plasma prolactin and progesterone on secretion of gonadotropins and ovarian hormones as well as follicular development and maturation were examined using postpartum non-lactating pituitary grafted rats. Plasma levels of LH, estradiol-17 β and inhibin as well as the number of antral follicles increased significantly in postpartum non-lactating pituitary grafted rats when compared with those in control lactating rats, though high levels of plasma prolactin and progesterone were maintained in both groups. These results indicate that high levels of prolactin and progesterone do not suppress follicular development nor do they suppress secretion of LH in the postpartum rat. The suckling stimulus suppresses follicular development and maturation probably through severe inhibition of LH secretion from the pituitary gland.

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The reliability of DNA fingerprinting in evaluating the parent/offspring relationship (pedigree) of 5 related hybrid dogs (parents: 2; offspring: 3) and 2 non-related Beagle breeds was investigated with the Myo probe. DNA was isolated from leucocyte, digested with Hinf I before subjecting to agarose gel electrophoresis and transferred to a nylon membrane eventually. The nylon membrane was hybridized with ³²P-labeled Myo probe, and the respective bands were detected after autoradiography. Bands derived from the 3 offspring resembled that of either the father or mother. This clearly indicates that DNA fingerprinting can be effectively employed for pedigree evaluation, and may serve as a positive proof of paternity as well.

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A sequential electron microscopic study of XY synaptonemal complexes of male Turkish hamster sprematocytes by 2 methods was carried out. The results revealed that 1 or 2 chromatin fibril-like strands were commonly extending and connecting the XY axes either around the border of the differential and non-differential segments or near the attachment plaques. The existence of such strands between 2 axes, and the number and position they presented were found to be highly correlating to stages/sub-stages of the spermatocytes. A possibility of visual evidence for chiasma formation and terminalization in the sex bivalent in the present species is discussed.

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Antiserum to prostaglandin (PG) F₂ and indirect immunofluorescence were used immuno-histochemically to demonstrate the presence of PGE₂ in unfertilized and fertilized bovine eggs matured in vitro, and embryos from the 2-cell to hatched blastocyst stage developed in vitro. Fluorescence showing the presence of PGE₂ was found in the cytoplasm of all the eggs and embryos, though its intensity was weak in unfertilized eggs to morulae, and was strong in blastocysts. No difference in the intensity was observed between unfertilized and fertilized eggs, among embryos at the cleaving stages, and among those at the early, expanded and hatched blastocyst stages.

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To elucidate what kind of medium would be desirable for mammalian embryo culture, we evaluated the effect of heavy metal ions on mouse in vitro embryonic development. Pronuclear stage embryos recovered from ICR mice were cultured in Ham's F-10 and α-MEM and the developmental ability was compared between them. The major difference of these 2 media is that only Ham's F-10 contains heavy metal ions such as Zn²⁺, Fe²⁺ and Cu²⁺, and hypoxanthine. When pronuclear stage embryos were cultured in α-MEM, the rates of embryos reaching the 4-cell, blastocyst and hatched blastocyst stages were 96.5%, 75.4% and 66.7%, respectively. These values were significantly (P<0.01) higher than the rates of embryos cultured in Ham's F-10; 61.8%, 5.5% and 3.6% respectively. The deletion of CuSO₄, ZnSO₄, FeSO₄ and hypoxanthine from Ham's F-10 significantly increased the rates of embryos reaching the 4-cell, blastocyst and hatched blastocyst stages to the extent comparable to those in α-MEM. In contrast, the addition of all or one of CuSO₄, ZnSO₄, FeSO₄ and/or hypoxanthine to α-MEM significantly decreased the in vitro embryonic development. The strongest inhibition was observed when all of them were added. The developmental ability in α-MEM to which all of them were added was as low as that in Ham's F-10. These results suggest that the low developmental ability in Ham's F-10 may be mainly due to the deleterious effect of heavy metal ions and hypoxanthine. The toxic effect of heavy metal ions and hypoxanthine might be interpreted as the damage on embryos by an increased generation of oxygen radicals and the medium without constituents which may enhance the production of oxygen radicals seems to be desirable for the culture of mammalian embryos.

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The possible influence of a β-adrenergic agonist, Clenbuterol, on GH, IGF-1, LH and cortisol secretion was investigated in young female calves. Clenbuterol (5 μg/kg liveweight) was fed twice daily for 3 weeks. Blood samples were taken twice daily; on days -5, 1, 3 and 5 of treatment, series of frequent samplings (10 h, 30 min intervals) were conducted to evaluate GH, LH and cortisol secretion. IGF-1 plasma concentrations were measured in the daily samples throughout the experiment. To register metabolic changes, nonesterified fatty acid (NEFA) concentrations were also recorded. In contrast to cortisol and LH secretion, that were not affected by Clenbuterol treatment, GH secretion was suppressed during the first day of treatment. On days 3 and 7 the differences between control and treated animals diminished. In the Clenbuterol treated calves, the age related increase of IGF-1 plasma concentrations slowed down during days 6-15 of treatment. Then this divergence leveled off. Postprandial NEFA concentrations were increased in the treated calves (P≤0.05). These results do not support an indirect mode of action of Clenbuterol through the somatotropic axis since the observed changes were not persistent and rather related to direct metabolic changes induced by Clenbuterol.

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Bovine cumulus-oocyte complexes were inseminated in fertilization medium, mBO medium including caffeine (5 mM), heparin (10 μg/ml) and bovine serum albumin (BSA; 10 mg/ml) with or without glucose (13.9 mM). Oocytes were freed from cumulus cells 8 h post-insemination and transferred into culture medium, mTCM-199 supplemented with BSA (3 mg/ml) and with or without glucose (5.56 mM). When oocytes were examined 24 and 72 h post-insemination, high proportions of oocytes were penetrated (88-92%) and cleaved to more than the 2-cell stage (68-71%), respectively, regardless of the presence or absence of glucose during fertilization and/or further culture without cumulus cells. At 144 and 192 h post-insemination, however, the proportions of oocytes developed to the morula and blastocyst stages, respectively, were significantly (P<0.01) higher in culture medium without than with glucose regardless of the presence of glucose during fertilization. In addition, significantly (P<0.05) higher proportions of oocytes developed to the morula (23 vs. 40%) and blastocyst (5 vs. 12%) stages when they were cultured in glucose-free medium after fertilization in medium without than with glucose. When glucose was added in culture medium 120 or 144 h post-insemination, 23 to 24% of inseminated oocytes developed to the early or expanded blastocyst stage 192 h post-insemination.

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Mouse oocytes were induced to mature by injecting with pregnant mare serum gonadotropin and human chorionic gonadotropin, and the c-fos protein accumulated in the oocytes was detected by immunohistochemical method. Intense/very intense staining was observed with monoclonal antibodies against the c-fos protein in maturing oocytes and cumulus cells in Graafian follicles, but slight/moderate staining in oocytes with germinal vesicle in antral follicles; the most intensive signal was observed in oocytes which induced germinal vesicle breakdown in Graafian follicles at 6 h after hCG administration. Immunoreactions associated with cellular compartments of the oocyte cytoplasm was identified under the electron microscope. The c-fos protein was also detected in the matured oocytes in the oviduct, although the intensity decreased gradually after ovulation. Positive immunostaining was not detected in granulosa and theca layers, and interstitial connective tissue, confirming that the expression of the c-fos protein is elevated particularly in the maturing oocytes in the ovary.

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A simplified procedure for bisection of bovine morulae and blastocysts involving a fine microsurgical blade and Dulbecco's phosphate-buffered saline (PBS) supplemented with 0.3 M sucrose and 0.1 mM ethylene diamine tetra acetic acid (EDTA) was employed to examine the developmental ability of the bisected demi-embryos. This procedure did not require a holding pipet to restrain the embryo and only a single micromanipulator was needed. Bisected embryos were transferred to recipient heifers either as pairs of demi-embryos or as single demi-embryo to compare maintenance of pregnancy and fetal survival as to the incidence of identical twins. Blastocyst formation rate was significantly (P<0.05) higher (78.3% vs 60.5%) in embryos bisected in medium containing sucrose as compared to controls when early or mid-blastocyst stage embryos were bisected. Bilateral transfer of bovine demi-embryos generated more fetuses per original embryo than unilateral transfer of a single demi-embryo (105% vs 93%). Embryos producing identical twin pregnancies from sets of demi-embryos (bisected-twins) represented 30.7% of the total while 39.8% of embryos produced one pregnancy and the remaining 29.5% failed to result in pregnancy. The results show that bovine embryos can be bisected by a simplified method and lead to normal pregnancies with a high incidence of identical twins.

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