The effect of the prostaglandin E₂, F₂α and indomethacin on the incorporation of ³H-Methionine in rat blastocyst development was studied by the method of liquid scintillation technique. The eggs of intact pregnant rats when they were on the morula, early blastocyst, blastocyst, and late blastocyst stages were collected. Blastocysts of delayed implantation rats were also collected on the day 6 and 8. The rate of implantation of ³H-Methinine in delayed blastocysts was smaller than in normal blastocysts. Addition of prostaglandin E₂ or F₂α had accelerated the incorporation of ³H-Methionine in blastocysts at preimplantation and during delayed implantation (P<0.05). But addition of indomethacin inhibited the incorporation of ³H-Methionine in blastocyst and delayed implantation on the day 6 and 8 (P<0.05).

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This study was conducted to evaluate the effects of gonadotrophins (PMSG, hCG), fetal bovine serum (FBS), estradiol-17β (E2) and porcine follicular fluid (pFF) on the timing of nuclear maturation and the expulsion of first polar body in cumulus-enclosed (CC+) or denuded (CC-) porcine oocytes. CC+ and CC- oocytes were cultured in mTALP-PVA medium supplemented with the above stimulants alone or in combination for 24, 32 and 46 h. PMSG at all doses significantly (p<0.05) promoted the meiotic resumption (GVBD) in CC+ oocytes, compared to those in control. When CC+ were stimulated by hCG (15 IU/ml), FBS (15%) and E2 (10 μg) for 32 h, the percentage of GVBD was higher than that of control (p<0.05) and for 24 h culture oocytes (p<0.05). After 46 h of culture, PMSG, hCG and E2 significantly (p<0.05) promoted the expulsion of first polar body (Pb1) in CC+ than CC- oocytes. At the same time, pFF in the presence of E2 or FBS significantly (p<0.05) increased the expulsion of Pb1, compared to the stimulation by respective single stimulant. These results suggest that PMSG, hCG and E2 acted through the cumulus cells and stimulated the meiotic maturation; but FBS and pFF directly stimulate, and that pFF in combination with FBS or E2 showed a important role in promoting the expulsion of Pb1.

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This study was conducted to evaluate the effects of supplementation of human or murine leukemia inhibitory factor (hLIF or mLIF) to synthetic oviduct fluid medium (SOFM) containing BSA and to determine their effective dosage on in vitro development of bovine morulae cultured singly or in a group. Morulae were produced by culture of in vitro matured (IVM), fertilized (IVF) and cleaved (2- to 4-cell) embryos in SOFM+BSA for 124 h after IVF, and then cultured singly (1 embryo/30 μl drop) or in a group (4-5 embryos/30 μl drop) in SOFM+BSA supplemented with 0 (control), 500, 1,000, 2,000, 4,000 and 6,000 U/ml of each hLIF or mLIF at 39°C in an atmosphere of 5% CO₂, 7% O₂, 88% N₂ and ≥95% humidity. Culture of embryos in a group improved (p<0.01) embryo development to hatched blastocysts compared to a single culture. Both hLIF and mLIF significantly (p<0.05) increased development to hatched blastocysts when morulae were cultured singly, but not when cultured in a group. The most effective dosage of mLIF was 1,000 U/ml (p<0.05), but there was no significant difference in the addition of hLIF with different doses (500 to 6,000 U/ml) tested. This results indicate that a group culture may be more effective on embryo development than a single culture and supplementation of hLIF or mLIF to SOFM+BSA improve in vitro development into hatched blastocysts of bovine morulae derived from in vitro maturation and fertilization when cultured singly.

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The pattern of nuclear degeneration of oocytes and the effect of follicle stimulating hormone (FSH) on the profile of nuclear degeneration of oocytes in the follicles at the stage of Type 4, 5, 6 or 7 was studied. Mouse ovaries were examined by serial histological sections. Nuclear degeneration of oocytes was found among follicles at Type 4-7 stage of development and the pattern of nuclear degeneration was strongly correlated with the developmental stage of follicule or oocyte. Nuclear degeneration of oocytes was observed in 52.2% of Type 4-5 (preantral) follicles and 39.2% of Type 6-7 (antral) follicles. In the Type 4-5 follicles, degeneration, such as pycnotic changes and disappearance of chromosomes, was dominant. In Type 6-7 follicles, however, about 80% of oocytes induced pseudo-maturation division during the degenerating processes. Pseudo-maturation was observed in the oocytes more than 51 mm in diameter. The administration of FSH significantly increased the number of Type 6-7 follicles, but not Type 4-5 follicles, and decreased the number of both Type 4-5 and Type 6-7 follicles with degenerating oocytes. A large number of macrophages were identified immunohistochemically in the interstitial tissue around the follicles. Macrophages make a cluster around the atretic follicles, but were not identified inside of the follicles containing degenerating oocytes. These results suggest that the pattern of oocyte degeneration depends on the developmental stage of follicles and oocytes, and that FSH accelerates the process of degeneration of oocytes, and that macrophages are involved in the process of removal of degenerating oocytes.

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[Purpose] Different six types of assisted hatching (AHA) were applied to mouse embryo and their effect on hatching was investigated. [Methods] The zona pellucida was treated as follows. (a) Piercingness (PIC): Pierce the zona with a very fine needle. (b) Partial zona dissection-small (PZD-small): Proceed the same as with PZD in microinsemination and make an incision about 1/8 the circumference in the zona. (c) Partial zona dissection-large (PZD-large): Enlarge the incision about 1/4 the circumference. (d) Zona drilling (ZD): Spray acid solution to the zona, which dissolves the zona to create a hole about 30 μm in diameter. (e) Zona thinning-partial (ZT-partial): Thin down a part of the outer area of the zona to about half the thickness by the same procedure as in ZD. (f) Zona thinning-total (ZT-total): Thin down the total outer area of the zona. [Results] When these AHA were each administered to 4-cell-stage embryos, the effects of PZD-large, ZD, ZT-partial and ZT-total on the hatching were observed, but the effects of PIC and PZD-small were not recognized. Furthermore the effects of AHA on 8-cell-stage embryo, morula and frozen-thawed embryo were recognized. With PZD-partial in which the opening of incision is small, the embryo in the halfway through hatching was trapped in the thick zona pellucida. With PZD-large or ZD in which an incision or a hole is large, hatching started at the earlier stage than normal (premature hatching). [Conclusion] With the role of the zona and the time to start hatching taken into consideration, zona thinning (ZT-partial or ZT-total) appears to become the most reasonable AHA.

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Distributional changes in lysosomes in hamster eggs and embryos during the folliculogenesis and early development were observed using the acridine orange (AO) staining method. AO-stained lysosomes were densely observed all over the cytoplasm of eggs in secondary follicles and of eggs in antral follicles until 13 hrs before ovulation, but were sparsely observed in the cytoplasm of eggs in primordial follicles. The lyso-somes were distributed throughout the cytoplasm in about a half of eggs in antral follicles at 12 and 10 hrs before ovulation, while they gathered around nuclei in the remaining half of the eggs. The lysosomes were distributed all over the cytoplasmic region of all eggs from 8 to 3 hrs before ovulation and of unfertilized eggs 2 hrs after ovulation. Such a distributional pattern remained unchanged until uncompacted 8-cell embryos. In compacted 8-cell embryos and blastocysts, the lysosomes were densely observed all over the cytoplasm in round blastomeres or inner-cell-mass cells, whereas they were sparsely observed in flattened blastomeres or trophoblast cells, mainly around nuclei. From the results on 8-cell embryos and blastocysts, it was suggested that there is the close relation between distributional changes of lysosomes and differentiation of blastomeres.

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Chromosome preparations of mouse morulae and blastocysts derived from in-vitro fertilization were made by a new air-drying method, and then subjected to various methods of G-band staining. Excellent G-band figures of embryo chromosomes were obtained by a modified method of urea treatment. The G-band method is shown in the present paper.

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To obtain identical twins with predicted sex, an efficient and reliable method for manipulation and analysis of bovine embryos has been developed using in vitro produced embryos and PCR. Expanded blastocysts graded as excellent were bisected into halves following biopsy to remove approximately 10% of trophoectoderm. Viability and cell number of these biopsied demi-embryos at 48 h in culture were compared with demi-embryos which have been only bisected. No significant difference was seen in survival rates between the biopsied demi-embryos (80.7%) and demi-embryos (87.0%). The mean cell number of the biopsied demi-embryos (70.00 ± 18.84) was slightly lower than that in the demi-embryos (77.50 ± 20.82). The sex of small sample and one biopsied demi-embryo was determined by PCR. The sex of 48 (96.0%) of the biopsied demi-embryos survived was consistent with the sample-side with 27 males and 21 females. These findings indicate that the sex of biopsied demi-embryos can be determined with a high accuracy, and their in vitro viability is comparable to that of demi-embryos.

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Culture conditions of in vitro fertilized bovine eggs denuded or surrounded with cumulus cells were examined in order to effectively use the simply defined medium, HECM. In experiment 1, the rate of development to the blastocyst stage of denuded eggs was lower than that of eggs co-cultured with cumulus cells. The rate of development to the blastocyst stage of denuded eggs in the HECM medium was higher than that in the medium TCM199. In experiment 2, the effects of oxygen concentrations on the development of denuded and co-cultured eggs were examined in the HECM medium. The rate of development to the blastocyst stage in denuded ova was significantly higher in the 7% O₂ condition (21%) than in the 20% O₂ condition (0%). In experiment 3, the effect of calf serum (CS) and insulin like growth factor 1 (IGF-1) on the development of denuded eggs cultured in HECM with 7% O₂ was examined. The rate of development of CS and IGF-1 improved significantly (13-27%) compared with the control (PVA) (8%). This suggested that the bovine embryo could be effectively cultured in the simply defined medium in vitro when the culture conditions were sufficiently examined, including the elimination of inhibing factors from the culture medium and gas (O₂, CO₂) pressure.

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Chimeric hamsters were produced by the aggregation of eight-cell embryos obtained from two stains (golden and albino) of Syrian hamster. Eight-cell embryos were collected by flushing of oviduct and uterus using HEPES-HECM-3 medium. The aggregated embryos by PHA were cultured in HECM-3 medium for 20 h under 10%O₂-10%CO₂-80%N₂ at 37.5°C. Of 354 aggregated embryos, 87 (25%) and 234 (66%) had developed into morulae and blastocysts, respectively. Total 90 aggregates (morulae and blastocysts) were transferred to 3 recipients at Day 3 of pseudopregnancy. Of 3 recipients, 2 became pregnant. Seven pups were born from the two recipients. Of 7 pups born, 6 were determined as chimeric hamsters by coat color (agouti). Of 6 chimeras, 5 were phenotypic males, and 1 was a phenotypic female. All chimeric hamsters had normal external genitalia.

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Bovine embryos derived from IVM, IVF, IVC were subjected to analyze of programmed SDS-electrophoresis with silver staining, using Phast system (Pharmacia Co. Ltd.). Embryos in early stage of development with immatured oocytes, matured oocytes, 1 cells (zygotes), 2 cells, 4 cells, 8 cells, morulae and blastocysts were aspirated into 1 μl capillary tube and kept at -60°C until used for electrophoresis. The embryos of each developmental stage were dissolved in saline solution containing SDS and 2-mercaptoethanol, incubate containing for 1 hour at 60°C, and applied to the electrophoresis. One or three embryos were standarized to analyze their protein pattern in each stage. The pattern of protein bands were analyzed with a densitometer (CS-9300-PC, Shimadzu). The band of 59-62 KD in immatured oocytes become high molecular weights eggs developed. The band of 84-91 KD and 101-108 KD were identified in eggs at each stage of the early development except for 101-108 KD in immature oocytes. In addition the bovine embryos during early development had the protein bands that were the same bands recognized as 101-103 KD in granulosa cells, 63-65 KD in FCS and follicular fluid, respectively.

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