RNA synthesis in bovine embryos produced in vitro was investigated to clarify changes in transcriptional activity. Overall 1003 follicular oocytes were matured and fertilized in vitro and subsequently cultured with a cumulus cell monolayer in TCM 199 with 5% fetal bovine serum (FBS). Embryos were transferred to TCM199 supplemented with 100 μCi/ml of [5-³H]uridine at 26, 35, 43, 50, 68 and 116 h post insemination (hpi) and cultured for 8 h at 39 C. Treated embryos were washed in PBS and the zona pellucida weakened with 5% proteinase K followed by fixation in acetic-alcohol. Preparations were air-dried, dipped in K5 emulsion, and exposed at 4 C. After 4.5 days exposure, the preparations were developed in D19 and the density of ³H-uridine incorporation was analyzed by image analysis (NIH-157 analysis system). ³H-Uridine incorporation into nuclei was observed in 43% of 2-cell embryos at 26 hpi although the level was low. The change of ³H-uridine incorporation level was not consistent until 4-cell stage and thereafter the level increased in a time-dependent manner but not dependent on cell number. Incorporation of ³H-uridine into nucleoli was observed from 50 hpi. Twenty-two embryos at 5-cell to morula were incubated with labelled and unlabelled uridine as negative control showed no incorporation of ³H-uridine. These results show that the transcription of early bovine embryos fertilized in vitro is already active in 2-cell embryos at 26 hpi, the level increases in a time-dependent manner after 5-cell stage and is not dependent on the cell number.

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Present study was conducted to investigate roles of the basal level of FSH and LH in the follicular maturation and maintenance during pseudopregnancy of the rat. Antiserum against luteinizing hormone releasing hormone (LHRH-AS) or charcoal-treated porcine follicular fluid (PFF; a crude inhibin preparation) was given to individual animals on day 5 of pseudopregnancy. Plasma LH and inhibin decreased after treatment with LHRH-AS. Contrarily, plasma FSH increased after LHRH-AS injection. The number of follicles capable of ovulating in response to human chorionic gonadotropin (hCG) in animals given LHRH-AS declined gradually, reached a nedir at 24 h after the treatment, and then returned to control levels during the subsequent 24 h. On the other hand, plasma FSH levels in rats given three injections of 0.5 ml PFF at 6-h intervals, compared to those in control animals, remained significantly lower at nearly all time points during the first 24 h after the initial PFF injection, and thereafter the hormone levels temporarily increased, being significantly elevated above the control value at 30 and 42 h after the initial PFF injection. Only in the rats given intraperitoneal injections of 0.5 ml PFF, compared to the control animals, plasma LH concentrations remained significantly lower at 9 h after the initial PFF injection and those values were significantly higher at 36 h after the initial treatment. The number of follicles capable of ovulating in response to hCG decreased in PFF-treated animals in a dose-dependent-manner. The follicular development in animals given injection of 0.5 ml PFF were virtually or completely inhibited from 12 to 36 h after the initial injection of PFF. Present results clearly indicate that the basal secretion of both LH and FSH are essential for follicular development and maintenance during pseudopregnancy of the rat. These results also suggest that inhibin is a primary factor to suppress FSH secretion in the pseudopregnant rat.

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Mouse oocytes were pulsed in 300 mM mannitol solutions containing (MS⁺) or not containing (MS^-) Ca²⁺ and stored at different room temperatures in M2 with (M2⁺) or without (M2^-) Ca²⁺ for different periods of time before culture in Whitten medium. The role of extracellular Ca²⁺, the formation and duration of pores on the oolemma, and the effect of low temperature during oocyte activation were studied by measuring the rate of oocyte activation. The results were as follows: 1. At 20 C, when oocytes were pulsed in MS⁺ and transferred to and stored in M2⁺ for 10 min, the activation rate was 61.9%, significantly (P<0.01) higher than that obtained when oocytes were pulsed in MS^- and stored in M2^- for 10 min (1.8%), indicating that extracellular Ca²⁺ was absolutely necessary for oocyte activation. 2. When oocytes were pulsed in MS^- and stored in M2^- for 10 min, 5 min and 30 sec before transfer to M2⁺, the activation rates were very low, being 1.8, 6.7 and 3.8%, respectively, significantly (P<0.05) lower than that (16.4%) when oocytes were transferred to M2⁺ immediately (within 15 sec) after pulsing in MS^-, suggesting that pores were formed on the oolemma during pulsing and they began to close within 30 sec after stimulation. 3. When oocytes were pulsed in MS⁺ at 4 C and stored in M2⁺ for 5 min at 4 C, the activation rate was only 22.5%, significantly (P<0.05) lower than that (61.9%) of the oocytes treated under the same conditions but at 20 C. When oocytes pulsed in MS⁺ at 4 C were stored in M2^- for 5 min and 10 min at the same temperature, the activation rates were 25.8 and 10.8%, respectively, and the difference between them was significant (P<0.05). Although there might be other reasons for the low activation rate in 4 C experiments, these two results might indicate that the reduction in fluidity of the oolemma at low temperature made the pore formation more difficult, but the pores persist longer than at normal temperature.

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In cultured mouse and rat blastocysts, hatching was found to begin with protrusion of trophectoderm cells from zonae pellucidae at the expanded stage. Protrusion of the cells occurred at the mural trophectoderm and at the polar trophectoderm. After protrusion, a slit was formed in the zona pellucida in all mouse and rat blastocysts. It was also confirmed that hatching was completed in the form of either expansion or contraction in both mouse and rat blastocysts. From these results, the process of blastocyst hatching could be classified into 6 types in the mouse. Since 5 of 6 types of hatching process, classified in mouse blastocysts, were observed in rat blastocysts, it is inferred that the process of hatching differs among blastocysts, but not remarkably between the mouse and rat. The mean duration of hatching from its start to completion was 22.4 h in mouse blastocysts and 40.9 h in rat ones. When the activity of trypsin-like proteinase was histochemically examined in mouse and rat embryos, this enzyme activity was detected in those from the compacted 16-cell stage to the expanded blastocyst stage. This enzyme activity was present in both mural and polar trophectoderm cells of blastocysts, suggesting the reason for no polarity of protrusion of trophectoderm cells in mouse and rat blastocysts.

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The effect of the protein transport inhibitor brefeldin A (BFA) on compaction of early mouse embryos was investigated morphologically and immunocytochemically. After placing 4- and 8-cell mouse embryos in a medium with BFA for 3 or 6 h, the features of compaction were investigated. It was found that the effects of BFA on compaction were dependent upon the cell stage when this drug was applied. Compaction was delayed when embryos were placed in BFA during the mid or late 8-cell stage (3-6 and 6-9 h post division to the 8-cell stage). In embryos treated with BFA, Roh-ConA binding was weakened and detected at the cell contact regions. The distribution of microvilli seemed to be altered. It was concluded that protein transport for compaction was initiated at the early 8-cell stage just before the development of the compacted state.

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To investigate seminal plasma factors which damage the surface of the sperm head during freezing treatment, proteins were recovered from the surface of ejaculated spermatozoa of miniature swine. Semen of miniature swine was washed with phosphate buffered saline and treated with hypertonic saline solutions. The fraction showed significant haemagglutination activity. Proteins in the fraction were separated by SDS-PAGE. Five major proteins were purified by excision from the gels followed by electro-elution, and then antisera against the proteins were produced using mice. Haemagglutination inhibitory assay demonstrated that only anti-13K serum inhibited haemagglutination of the fraction. Immunocytochemistry clearly showed that anti-13K serum reacted to the whole surface of ejaculated sperm. Furthermore the antiserum showed a slightly stronger reaction to the swollen surface of one-hour frozen spermatozoa than untreated spermatozoa. The materials seen in the frozen sperm specimen, considered as aggregates of the released sperm surface complex, also reacted with the anti-13K serum intensively. In addition, the 13K-protein was constantly detected in seminal plasma precipitates during the entire freezing treatment. The present data indicated that one of the sperm surface proteins isolated with a hypertonic saline solution is a haemagglutinin, which might be involved in the aggregation of seminal plasma during freezing.

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To analyze expression of developmental genes and their regulatory mechanisms in mouse preimplantation embryos, pronuclei of in vitro-fertilized eggs were microinjected 8-10 h after insemination (hpi) with a fusion gene composed of the mouse metallothionein-I promoter (MT-I) and the Escherichia coli β-galactosidase gene (lacZ). The eggs were cultured up to the blastocyst stage in Whitten's medium supplemented with 100 μM EDTA. Expression of the lacZ gene was examined by staining the embryos with X-gal as a substrate. Expression was first detected at the 2-cell stage, 27 hpi; the proportion of embryos expressing the lacZ gene reached a maximum of 80% at 33-48 hpi. The proportion declined from the 4-cell stage onward, but some embryos still showed positive staining at the blastocyst stage. α-Amanitin blocked the expression of lacZ gene only when added before 12 hpi, suggesting that transcription had started during the pronuclear stage rather than in cleavage stages. The presence of EDTA in the culture medium reduced the expression markedly from the morula stage on, but 25 μM ZnCl₂ enhanced expression after the 4-cell stage in the presence of EDTA. These results suggest that transcription is active at the pronuclear stage and that the MT-I promoter becomes inducible by ZnCl₂ after activation of the embryonic genome.

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Bovine early pregnancy factor (EPF) has been detected in the recipient serum after embryo transfer which was performed on day 7 to 8 after estrus. However, there have been no reports of the detection of EPF in the recipient serum with embryo transfer on the day more than 3 days different from the estrous cycle of the donor. The aim of this study was to determine if EPF is detectable from serum of Holstein cow recipients with embryo transfer on day 12 to 16 after estrus which was 5 to 9 days earlier than that of the donor. Embryos were collected nonsurgically from superovulated Holstein cows on day 7 to 8 after artificial insemination (AI). The embryos were transferred nonsurgically to seven recipients on day 12 to 16 after estrus. Blood samples of the recipients were collected on day 0 (the day of embryo transfer) to 15. EPF activity was assayed by the rosette inhibition test. A rosette inhibition titer (RIT) above 4 was regarded as indicating positive EPF activity. EPF activity was positive in the serum of seven recipients on day 2 to 3 after embryo transfer. Subsequently, six recipients out of seven remained positive for 2 to 3 days. As for the other recipient, positive EPF activity was maintained until day 15 and then a female calf was delivered on day 268 after embryo transfer. These results provided evidence that EPF was detected in the serum of recipients of transferred embryos and was indicative of embryonic life (or existence of the embryo) on even day 12 to 16 after estrus, which was 5 to 9 days earlier than that of the donor

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The 5' flanking region (1.2 kb) and the first exon and intron (1.5 kb) of the human αS1-casein (CSN1) gene, and the 5' flanking sequence (2.0 kb) of the human β-casein (CSN2) gene were cloned and sequenced. Strong sequence homology was observed between the 5' flanking regions of the casein genes of humans and other mammals. Several transcriptional regulatory elements (GRE, C/EBP, YY1, STAT5) identified in the rat β-casein gene were conserved in both human casein genes.

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In Exp. 1 oocytes were collected from follicles (4-6 mm) in ovaries from prepubertal gilts by aspiration of the follicles with a syringe or dissection from healthy follicles. After aspiration some oocytes with compact cumulus and granulosa cells were selected. In Exp.2 oocytes were collected from follicles (4 mm, 5-6 mm and 7-8 mm) of gilts on the 18th-19th day of the cycle. Germinal vesicle (GV) stages of oocytes were examined and categorized into GV1, GV2, GV3 and GV4. In Exp. 1 when collected by aspiration, a significantly lower percentage of oocytes (45%) was arrested at the GV1 stage than oocytes from dissected follicles (78%). However, after morphological selection of oocytes, the percentage (58%) significantly increased. In Exp 2, significantly higher percentages (86-90%) of oocytes from follicles of 5-8 mm in diameter were at the GV1 stage than oocytes from follicles in diameter of 4 mm (24%) and atretic follicles (57%). These results indicate that by aspiration some oocytes are collected from follicles that were inadequate to maintain arrest at GV1, but significantly higher percentages of oocytes at GV1 can be obtained after morphological selection of oocytes or isolation from large follicles of gilts on the 18th-19th day of the cycle.

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An attempt was made to measure the qualitative fecal gestagen concentration in the sow using a commercial bovine milk progesterone qualitative test EIA kit (qualitative kit), which can measure the gestagen concentration in approximately 10 min. Additionally, the possibility of applying this method of gestagen concentration measurement to early pregnancy diagnosis in the sow was investigated. Feces were collected from the rectum of the sow, and 0.5 g of feces was placed in 20 ml of distilled water and stirred. The supernatant was used as the fecal solution, which was subjected to measurement by the qualitative kit. The accuracy of pregnancy diagnosis at 21-25 days after last mating for 111 sows was 97.6% (80/82) for positive cases and 100% (17/17) for negative cases. The overall pregnancy diagnosis accuracy, including 12 indeterminable cases, was 87.4% (97/111). A comparison of the diagnoses based on gestagen concentrations that were measured using the qualitative kit, and the fecal gestagen concentrations of identical samples that were measured using the quantitative kit showed close agreement: 17 cases diagnosed as negative pregnancy by the qualitative kit all had a gestagen concentration of less than 200 ng/g, while 79 of 82 cases diagnosed as positive pregnancy by the qualitative kit showed a gestagen concentration of over 200 ng/g. The results of this study showed that qualitative measurement of the fecal gestagen concentration in the sow using a bovine milk progesterone qualitative test EIA kit is a practical method for early pregnancy diagnosis.

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