The in vitro capacitation and fertilizing ability of epididymal spermatozoa of BALB/c and ICR mice were compared. The capacitation rate of spermatozoa from BALB/c was significantly (P<0.05) lower than that of those from ICR mice when examined by chlortetracycline (CTC) fluorescence assay after 1 and 2 h of preincubation. An in vitro fertilization technique has revealed that the time taken for sperm penetration into intact and zona-free eggs was longer for BALB/c than for ICR spermatozoa (P<0.01). Fertilization rates for spermatozoa from BALB/c mice were significantly (P<0.01) lower than for spermatozoa from ICR regardless of the type of eggs (BALB/c, ICR). These results suggest that the low fertilizing ability of epididymal spermatozoa from BALB/c mice is associated with a lower efficiency of capacitation in vitro.

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It is necessary to use a suitable marker gene to select embryos expressing transgene prior to transfer into recipient females especially in an efficient production of transgenic farm animals. Recently a green fluorescent protein (GFP) gene has been used as a marker gene for this purpose, because it is a self-fluorescent substance and does not require a substrate such as X-Gal in a lacZ gene. Using these characteristics of GFP, we transfected the GFP gene with the neomycin-resistance gene under the control of the phosphoglycerase kinase promoter into mouse embryonic stem (ES) cells, and attempted to produce a transgenic mouse derived from aggregates of GFP-positive ES cells after the selection with G418 for 8 days with diploid or tetraploid embryos. There were some ES colonies expressing GFP at different levels after the selection with G418. These GFP-positive ES cells were used as a complete tight adhered clump for aggregation with diploid or tetraploid embryos and resulted in GFP expression in the inner cell mass (ICM) cells of aggregates, but no embryos expressing GFP in trophoblasts. At 9.5 days p.c., 6 out of 10 live fetuses (60%) for diploid⇔ES chimera, and all of 14 live fetuses (100%) for tetraploid⇔ES chimera, in spite of head deformity, were transgenic fetuses. At 14.5 days p.c., among 7 normal embryos obtained from 21 embryos transferred to recipients, one fetus showed GFP expression in five organs (heart, kidney, liver, gonad and intestine), and 4 fetuses strongly expressed GFP in more than one organ as seen under the fluorescent dissecting microscope. These results indicate that enough transfected ES cells selected in a short time with G418 were able to be used for the selection of transgenic embryos prior to transfer to the recipient. Moreover, by using a complete tight adhered clump of ES cells for aggregation, the clump is able to completely contribute to a part of the ICM. The GFP gene may be useful and efficient as a marker especially for farm animals which have a long term pregnancy and a small litter size.

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We attempted to determine the sex by polymerase chain reaction (PCR) in single bovine blastomeres at the 8-cell stage. PCR was performed on male-specific primers attached to a bovine embryonic sex determination kit, an XY Selector. Embryos at the 8-cell stage were isolated by the EDTA method, and one (1/8 embryo), two (2/8 embryo), and four (4/8 embryo) blastomeres were subjected to PCR. The detection rates for male-specific PCR product were 25.8, 25.8 and 45.2% for 1/8, 2/8 and 4/8 embryos, respectively. In some embryos, despite detection of the male-specific product in 4/8 embryos, the male-specific product was not detected in 1/8, 2/8 or both 1/8 and 2/8 embryos derived from the same embryo (3.2, 3.2 and 16.1%, respectively). Collecting single blastomeres by the extrusion method affected neither the rate of development to blastocysts nor the number of cells in blastocysts. PCR was performed in 1/8 embryos collected by the extrusion method, and the male-specific PCR product was detected. Nevertheless, in 27.5% of embryos, despite detection of male-specific PCR product in 7/8 embryos, the male-specific product was not detected in the 1/8 embryo from the same embryo. These findings indicated that collecting single blastomeres at the 8-cell stage allows the selection of the male embryo by PCR, and also the extrusion method is useful for biopsy of embryo at the 8-cell stage.

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Development and enzyme activities of embryos during the preimplantation period were examined to reveal causes of the age-related embryonic loss in the golden hamster. Embryos were obtained from young (9-12-week old, n=40) and aged hamsters (43-53-week old, n=40) on Days 2, 2.5, 3 and 3.5 of pregnancy and the number and developmental stages were recorded. In order to compare the viability of the embryos, the metabolic activities of mitochondria, microsomes, plasma membrane and lysosomes were examined histochemically. Early embryos from aged hamsters showed delays in their development and transport through the oviduct compared to those from young females. The embryos exhibiting strong activities of succinate dehydrogenase, isocitrate dehydrogenase and Δ⁵-3β-hydroxysteroid dehydrogenase were significantly diminished in the aged females. The present results suggest that the reduced metabolic activity may be one possible reason for the retarded development of embryos and that the asynchronous development and delayed migration of the embryos into the uterus may partly account for the embryo loss in the aged hamsters.

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This study was carried out to examine the underlying mechanism for the high susceptibility of bovine aged oocytes to activation stimulus. The oocytes were matured for 21, 27, 33, and 39 h and were then parthenogenetically activated with different concentrations of cycloheximide (CHX); their activation rates were then assessed. Additionally, the p34^cdc2 kinase activities in oocytes matured for different time periods were determined. The activation rates of oocytes treated with CHX increased with oocyte aging. In oocytes cultured for as long as 39 h in maturation medium, the treatment led to maximal activation rate of oocytes even in those with the lowest concentration of CHX. The p34^cdc2 kinase activity in oocytes decreased progressively with prolonged incubation time and the activity of the oocytes cultured for 39 h was approximately 40% of that of oocytes cultured for 21 h. In conclusion, the sensitivity of bovine oocytes to activation stimulus increases during in vitro aging; also, the time dependent decline in the capacity of oocytes to synthesize the regulatory protein required for maintaining MII stage may contribute to a high susceptibility of aged oocytes to protein synthesis inhibitor (activation stimulus).

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A new medium which contains amino acids, electrolytes, glucose, lactate and pyruvate at concentrations adjusted to those in human follicular fluid was prepared and named human follicular fluid (HFF) medium. We compared the developmental ability of mouse embryos cultured in HFF and in three commercially available media, i.e., human tubal fluid (HTF), α-modified minimal essential medium (αMEM) and Ham's F-10. When mouse maturing oocytes obtained from ovarian follicles were cultured in HFF or αMEM/10% fetal calf serum (FCS), extrusion of the first polar body was observed in 71 and 72% of the oocytes, respectively. The oocytes matured in HFF/10% FCS showed good developmental competence to the blastocyst stage compared with those matured in HTF or Ham's F-10/10% FCS after in vitro fertilization. The supplementation of amino acids in follicular fluid to HTF medium (HTF(FF)) did not improve the development to the blastocyst stage after IVF of maturing oocytes. The HFF medium was applied to in vitro culture of blastocysts using mouse embryos obtained from oviducts. After 80 hours of incubation in each medium containing 0.5% bovine serum albumin (BSA), the percentages of expanded blastocysts in HFF and HTF(FF)/0.5% BSA were significantly higher than those in HTF and αMEM (89% and 78% vs. 39% and 34%, respectively). Blastocysts and morulae were transferred to the medium CMRL 1066/20% FCS, and their developmental competence was assessed. Embryos derived from blastocysts and morulae cultured in HFF and HTF/0.5% BSA appeared to have good developmental competence, with the formation of an inner cell mass and outgrowing trophoblast, but those cultured in HTF(FF) and αMEM/0.5% BSA did not. Amino acids at high concentrations supplemented in αMEM produced a large amount of ammonium which is harmful to embryo development in extended cultures. These results indicate that the HFF medium was beneficial for both in vitro oocyte maturation and embryo development in the extended culture.

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A comparison was made of good quality blastocysts obtained after either conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). After IVF or ICSI, fertilized oocytes were kept in culture for a further 5 to 7 days before embryo transfer (ET) or embryo freezing. No differences were found in the number of oocytes showing two-pronuclei between conventional IVF (76.9%) and ICSI groups (85.1%). A cohort of 60 and 74 oocytes in the pronuclear stage were cultured after IVF and ICSI, respectively. The number of fertilized oocytes reaching the blastocyst stage was significantly higher (P<0.05) in the IVF group (73.3%) than in the ICSI group (56.8%). A total of 86 blastocysts were categorized into three grades depending on their morphology. The number of blastocyst embryos achieving blastocyst grade 1 (BG1) was significantly higher (P<0.05) in the IVF group than in the ICSI group, 63.3% and 13.5%, respectively. In the IVF group, 10.0% and 0% of 2PN oocytes developed to BG2 and BG3, and 2.7% and 4.1% in the ICSI group, respectively, with no significant differences between the two groups. Pregnancy rate was higher in the IVF group than in the ICSI group, 50% and 20%, respectively. It was concluded that fertilized oocytes resulting from ICSI cannnot be successfully cultured for 5-7 days for blastulation and blastocyst-ET.

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There is abundant evidence that cAMP plays an important role in maintaining meiotic arrest in mammalian oocytes. In mouse oocytes, cAMP and cAMP-dependent protein kinase (PKA) form part of a negative pathway which participates in the maintenance of meiotic arrest. In the present experiment, involvement of PKA on the meiotic arrest of pig oocytes was examined using PKA specific inhibitor H89. Cumulus-enclosed oocytes with or without parietal granulosa tissue were incubated in H89-supplemented medium for various durations, and subsequently cultured in H89-free medium for a total of 24 hr. Over 95% of both types of oocytes were arrested at the germinal vesicle stage after 24 hr in H89-free medium. On the other hand, after H89 treatment, about 40% (75 μM H89 for 6 hr, and 100 μM for 4 hr) of cumulus-enclosed oocytes with parietal granulosa tissue and about 70% (50 μM for 6 hr, and 75 μM for 4 hr) of cumulus-enclosed oocytes without granulosa tissue underwent germinal vesicle breakdown (GVBD), respectively. Pig denuded oocytes released from granulosa cells resumed meiosis spontaneously (75% GVBD), but the spontaneous resumption was inhibited by a component of pig follicular fluid, hypoxanthine (12% GVBD). Under the inhibition of hypoxanthine, 42% of denuded oocytes underwent GVBD after treatment with 100 μM H89 for 30 min. These results suggest PKA involvement in the meiotic arrest of pig oocytes.

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Cell surface marker analyses revealed that sodium alginate-induced reddish purple-stained cells (ARPC) expressed high levels of CD45 (common leukocyte antigen), F4/80, MHC class II antigen and c-kit but little Gr-1 and FceR. This expression pattern is the same as those of resident peritoneal macrophages and thioglycollate-induced macrophages (TG-macrophages), indicating that ARPC are macrophages. Mac-1 and Mac-2 expression patterns of ARPC were similar to TG-macrophages, but F4/80 expression was higher than that of TG-macrophages. In addition, the average size of ARPC is about 1.3 times as large as that of TG-macrophages. These results indicate that ARPC may be a distinct macrophage subset.

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