. Trophectoderm (TE) and inner cell mass (ICM) cells from blastocytsts were compared for their ability to produce chimeric mice or conceptuses (pluripotency) using nuclear transplantation in which a single cell was transplanted into an enucleated blastomere of a 2-cell embryo. The developmental ability of the embryos reconstituted with TE and ICM cells was dependent on when the recipient 2-cell embryos were recovered after hCG administration. When 2-cell embryos obtained 38-42 and 43-46 h post hCG injection were used, cell division of the reconstituted embryos rarely progressed to the 4-cell stage. However, when embryos recovered 48-51 h post hCG were used, the proportion of cleaved embryos increased significantly, developing to blastocysts with several large blastomeres. When reconstituted embryos were treated with nocodazole after nuclear transplantation, the percentage of cleaved embryos significantly increased, with some developing to blastocysts. Such blastocysts were transferred to recipient females, and no donor nuclei were detected in the conceptuses at midgestation. The in vitro developmental ability of the chimeric 2-cell embryos reconstituted with TE and ICM cells from blastocysts was quite limited and no chimeric conceptuses were obtained.

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Ultrastructural changes in follicular granulosa cells during atresia were examined in pig ovaries. No apoptotic cells were observed in healthy follicles. However, in follicles in the early stage of atresia apoptosis occurred in granulosa cells located on the inner surface of the follicular wall. In these apoptotic granulosa cells, nuclear condensation with large clumps of chromatin and cytoplasmic condensation but with retention of the integrity of organelles were observed by electron microscopy. As atresia progressed, pyknotic granulosa cells and apoptotic bodies increased in number in the follicular wall. In follicles in the late stage of atresia, apoptotic bodies were suspended in the follicular antrum. Concurrently, cell debris without distinct membranes but with condensed chromatin, degenerated apoptotic bodies, occurred in follicles. The apoptotic bodies and cell debris were occasionally surrounded and phagocytosed by neighboring granulosa cells. In follicles in the advanced stage of atresia, apoptotic bodies and cell debris were fed by macrophages.

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The present study examined the role for insulin-like growth factor-I (IGF-I) in preimplantation development in rats. The 8-cell stage rat embryos were cultured with 0 (control), 0.02, 0.2, or 2.0 nM human recombinant (hr) IGF-I for 36 h. Morphological development of embryos to the blastocyst stage was stimulated by hrIGF-I at all examined concentrations. Human recombinant IGF-I at ≥ 0.2 nM increased the number of live cells in the inner cell mass (ICM), the embryonic lineage, of resulting blastocysts by increasing total cell number and decreasing the rate of dead cells in the ICM. Levels of protein synthesis by blastocysts cultured with ≥ 0.2 nM hrIGF-I increased to the same levels as that of in vivo grown counterparts. When blastocysts were transferred to a receptive uterus, the rates of implantation and fetal development to day 18 of pregnancy in the 0.2 and 2.0 nM hrIGF-I groups were greater than those of the control group. In conclusion, hrIGF-I at ≥ 0.2 nM may promote development of 8-cell rat embryos to blastocysts that are fully-potent to postimplantational development.

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Glycosylated components of boar spermatozoa and epididymal plasma from the central caput, distal corpus and distal cauda epididymides were compared by electrophoretic and lectin blotting techniques. The four lectin reagents used in this study were Concanavalin A (Con A), Lens Culinaris Agglutinin (LCA), Ricinus Communis Agglutinin I (RCA I) and Peanut Agglutinin (PNA), which were labeled with peroxidase. The affinity of Con A and of LCA increased for three bands and one smear (A: > 86.8 kDa, B: 80 kDa, C: 65-75 kDa and D: 45 kDa) and for two bands and one smear (A, B and C) of extracts from spermatozoa, respectively, as they passed through the epididymis. RCA I apparently recognized one band and one smear (G: > 86.8 kDa and H: 46-58 kDa) of extracts from the distal cauda spermatozoa, though it hardly or faintly reacted with those from the central caput and distal corpus spermatozoa. PNA showed a similar affinity for band "G" to RCA I. Moreover, bands and smears with similar mobility (A, B, C, D, G and H) were also detected in epididymal plasma by lectin blotting techniques. On the other hand, several sperm components were detected more visibly with Con A (E: 37-41 kDa and F: 32-37 kDa), RCA I (I: 41 kDa) and PNA (I: 41 kDa and J: 47-51 kDa) in sperm extracts from the distal cauda than in those from the central caput, though no corresponding components were observed in epididymal plasma. These epididymal maturation-dependent modifications related to glycosylation in boar spermatozoa are discussed.

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Inbred BALB/c strain mouse was characterized by a low in vitro fertilization (IVF) rate. However, this strain has been used broadly for various researches, especially for researches in immunology. For improving the fertilization rate in this strain, we used taurine, a sulfur-containing β-amino acid that had been reported to be effective in improving fertilization in several species. Taurine (1 or 10 mM) did not change the proportion of capacitated spermatozoa in vitro. However, 1 mM taurine significantly (P<0.05) improved the fertilizaton rate compared to that of the control. The highest fertilizaton rate (78%) was obtained by addition of 1 mM taurine to TYH medium. The taurine effect on the early embryonic development was also examined in a simple culture medium (KSOM) for 4 days culture. There was no effect of taurine on embryo development. More than 50% of fertilized embryos developed to the blastocyst stage without taurine. The viability of fertilized embryos was proved by embryo transfer to ICR recipient mice. A total of 13 offsprings were born from two recipient mice. The present results indicate that taurine has a supporting effect on the improvement of IVF in inbred BALB/c mouse strain. Fertilized embryos developed to the blastocyst stage in in vitro culture and normal offspring after embryo transfer.

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Changes of glycoconjugates in granulosa cells during follicular atresia were examined by lectin histochemical and biochemical techniques. Twenty-four lectins were used to visualize the different glycoconjugates of granulosa cells in frozen sections of the pig ovary. Four lectins showed stronger staining of the granulosa cells in atretic than in healthy follicles. Very strong and/or strong staining with Sambucus sieboldiana agglutinin (SSA) was seen in scattered granulosa cells of atretic follicles, but those of healthy follicles were not stained with this lectin. In atretic follicles, very strong-moderate diffuse staining was seen with Aleuria aurantia lectin (AAL), Pisum sativum agglutinin (PSA) and Lens culinaris agglutinin (LCA) in granulosa cells, in contrast with very weak staining in those of healthy follicles. Furthermore, lectin blot analysis with three lectins (SSA, AAL, and PSA) indicated several glycoproteins with increased staining in atretic relative to healthy follicles for these three lectins. Especially, two SSA-positive glycoproteins (62 and 64 kD) of granulosa cells in atretic follicles were specific and characteristic which were not detected in those in healthy follicles. These results suggest that changes of some glycoconjugates in granulosa cells may be involved in granulosa cell apoptosis and follicular atresia.

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The present study was undertaken to examine the effects of recombinant bovine growth hormone (rbGH) on nuclear and cytoplasmic maturation of bovine oocytes in vitro and subsequent embryonic development after in vitro fertilization. When cumulus-oocyte complexes (COCs) and cumulus-free oocytes were cultured in modified Medium 199 supplemented with 0, 0.1, 1, 10, 100 or 1000 ng/ml rbGH for 24 h, no cumulus expansion was observed in most COCs in the presence of any of the tested concentrations of rbGH. The efficacy of nuclear maturation of oocytes to metaphase II was promoted by rbGH only in COCs showing higher proportions at 10-1000 ng/ml (77-78%) than at 0-1 ng/ml (57-64%). When COCs were cultured in the presence or absence of 10 ng/ml rbGH for 0-9 h, the proportions of oocytes that reached prometaphase I and metaphase I after 6 and 9 h of culture, respectively, were higher in the presence (53% and 51%, respectively) than in the absence (34% and 30%, respectively) of rbGH. After in vitro maturation and fertilization, no differences were observed in the penetration rate (82-91%), pronuclear formation (27-45%) and polyspermy (23-29%) at 8 h post-insemination among cumulus-enclosed oocytes cultured in the presence of different concentrations (0-1000 ng/ml) of rbGH. When starting 8 h after insemination, the oocytes were cultured for 136 h in a chemically defined medium, 21-25% of oocytes developed to the morula stage or beyond without difference among different concentrations of rbGH during oocyte maturation. However, at 192 h post-insemination higher proportions of oocytes developed to the blastocyst stage when COCs were matured in the presence (13-15%) than in the absence (6%) of 10-1000 ng/ml rbGH. These results indicate that a stimulatory effect of rbGH on bovine oocyte maturation is dependent on the cumulus cells. Presence of rbGH in the media improved maturation rate and subsequent embryonic development to the blastocyst stage.

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This study was conducted to examine whether or not the post-luteinizing hormone (LH) surge termination of estrus could be delayed by prolonged exposure to estradiol. Subcutaneous tubings containing estradiol were implanted in eight ovariectomized goats to maintain blood estradiol at the follicular phase level for 56-80 h. Behavior observation and blood sampling were carried out every 2 h for 114 h from 6 h before through 108 h after the initiation of estradiol treatment. As a result, the duration of proceptivity (defined as an active approach to the male) and receptivity (defined as an acceptance of the male) were extended in the manner reflecting the length of estradiol administration. The proceptivity and receptivity disappeared at 14.5 ± 5.0 h and 8.0 ± 3.6 h (mean ± SEM, n=8) after the removal of estradiol tubings, respectively. These results suggest that the termination of estrus in female goat is due to a decline of circulating estradiol level following the LH surge, and that the central nervous system regulating behavioral estrus maintains its responsiveness to estradiol even at the post-LH surge period.

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The required concentration and time of vinblastine sulfate (a mitotic inhibitor) treatment were determined for successful cytogenetic diagnoses, such as normality and sex in bovine blastocysts, derived from in vitro fertilization. Chromosome preparations were made from blastocysts treated for 17 h, in media containing vinblastine sulfate, with various concentrations of 0, 30, 100, 200 and 300 ng/ml. Vinblastine sulfate was effective at a concentration of 100-300 ng/ml, with respect to the average numbers of metaphase plates and the mitotic indexes (% of mitoses) in the embryos. To determine the sufficient treatment time, embryos were placed in a medium containing 100 ng/ml vinblastine sulfate for 10, 17 and 24 h. The required time of vinblastine sulfate treatment was 10 h. The average length of the no.1 chromosome shortened with the prolongation of treatment time but not with significant difference. Most of the embryos (approximately 98%) treated with vinblastine sulfate had metaphase plates, and approximately 75% of them were numerically analyzable. A high incidence of polyploidy and a sex ratio showing male advantage were found in the present study.

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The information concerning the transcription of growth factor receptor mRNA in porcine cumulus-oocytes complexes (COCs) during oocyte maturation is limited. The expression of mRNA was analyzed for growth factor receptors with receptor tyrosine kinases [epidermal growth factor receptor (EGF-R), ErbB3 (a member of EGF-R subfamily), insulin-like growth factor-I receptor (IGF-I-R), basic fibroblast growth factor receptor (bFGF-R) and platelet-derived growth factor receptor α (PDGF-Rα)] by reverse transcription-polymerase chain reaction in porcine COCs during oocyte maturation in vitro. Porcine oocytes or their surrounding cumulus cells were analyzed immediately after collection (0 h), at 12, 24, 36 and 42 h of in vitro maturation, respectively. Whereas EGF-R transcript was not observed in both oocytes and cumulus cells throughout in vitro maturation, ErbB3, IGF-I-R, bFGF-R and PDGF-Rα mRNAs were detectable in both oocytes and cumulus cells at all stages of in vitro maturation. The intensity of bands for ErbB3, IGF-I-R, and bFGF-R transcripts in oocytes was higher than that for PDGF-Rα transcript in oocytes at all stages of in vitro maturation. These results provide evidence that several growth factor receptor mRNAs are present in both oocytes and cumulus cells during in vitro maturation, and support the concept that those factors are involved in the regulation of porcine oocyte maturation.

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The present study was carried out to examine whether the sex of giant anteaters (Myrmecophaga tridactyla), which are not easy to sex from their external genitalia, could be identified using hair root by polymerase chain reaction (PCR) amplification. DNA was extracted from the hair root(s) and amplified by PCR with a pair of primer designed from human sex-determining region Y (SRY) gene. When PCR amplification was applied to the DNA samples extracted from 5 hair roots of each 5 individual of known sex, a signal was only detected in the male, but no signal was detected in the female. In addition, a clear male-specific signal could be detected even when DNA was extracted from a single hair root of the male. Moreover, the nucleotide sequence of this segment in giant anteater exhibited the high similarity with human SRY gene. When PCR amplification was applied to the DNA samples extracted from hair roots of each 10 individual of unknown sex, a male specific signal was detected in 8 individuals including 2 pairs kept in 2 places, but no signal was detected in 2 individuals, suggesting that some institutions had kept 2 giant anteaters as the same sex, not as a couple. The results of this study indicate that PCR amplification with SRY primer pair using DNA from hair root made possible the sensitive, reliable and rapid molecular identification of the sex in giant anteater.

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The intracellular mechanism that mediates the stimulatory effect of prostaglandin F_2α (PGF_2α) on progesterone secretion by bovine luteal cells is not well understood. In the present study, we investigated to test the hypothesis that PGF_2α stimulates progesterone secretion through activation of protein kinase C (PKC) by using a cell culture system. Bovine mid-luteal cells (Days 8-12 of the estrous cycle) were cultured for 24 h and then exposed to varying concentrations of PGF_2α (10^-8, 10^-7, 10^-6 M) or luteinizing hormone (LH; 100 ng/ml) for 4, 6, or 16 h. Treatment of the cells with the highest dose of PGF_2α (10^-6 M) or LH resulted in increases in progesterone secretion for all three exposure times. Following down-regulation of PKC with a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA; 10^-6 M), the stimulatory effect of PGF_2α was no longer evident, whereas the response to LH was unaffected. These results support the hypothesis that the luteotropic action of PGF_2α is mediated by the activation of intracellular PKC.

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A simple immunomagnetic cell sorting method is described for the isolation and the purification of mouse embryonic stem (ES) cells from a heterogeneous cell mixture containing both ES cells and mouse embryonic fibroblast cells (MEF). The cells (1.5 × 10⁶ cells per experiment), which labelled with the monoclonal SSEA-1 (stage specific embryonic antigen 1) antibody followed by the microbead coated with anti mouse IgM antibody, were retained in the separation column held in a magnetic field. Over 95% of the cells collected were viable after immunomagnetic cell sorting. Recoveries of 68, 82 and 90% and purity 83, 82 and 73% of the ES cells were obtained using 10, 20 and 50 μg of SSEA-1 antibody per experiment, respectively. Procedure for isolation did not affect the proliferation of the isolated cells on MEF in culture. These results suggest that the immunomagnetic cell sorting using undifferentiated cell marker antibody could be highly effective for isolating the undifferentiated cells such as ES cells.

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The aim of this study was to examine whether or not uterine infusion with a medium showing positive early pregnancy factor (EPF) activity influences maternal physiology in cattle. Trophoblastic cells derived from 12-day-old bovine embryos were cultured in Dulbecco's PBS (+) at 39 C in air for 24 h. EPF activity was assayed using the rosette inhibition test. A rosette inhibition titer (RIT) above 4 was regarded as indicating positive EPF activity. The trophoblastic cell culture medium (Tro-CM) showed positive EPF activity. On days 7 to 12 after estrus, five cows each received a nonsurgical infusion of Tro-CM (0.25 ml) in the lumen of the uterine horn. Blood samples were collected daily by the tail artery or vein puncture from day 0 (the day of infusion) to day 5. All five cows showed positive EPF activity in their sera on days 2 to 4, and three cows out of the five kept showing positive EPF activity for 2 to 3 days. The result demonstrated that sera of cows infused with a small amount of EPF-positive medium (Tro-CM) into the uterus showed temporary positive EPF activity in maternal serum, further suggesting that EPF released from Tro-CM would induce a release of maternal EPF, but not by directly entering into the maternal blood flow.

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The morphology, motility and viability of frozen-thawed southern minke whale (Balaenoptera acutorostrata) spermatozoa was evaluated. The whales used in this study were captured during the period from December 1994 to February 1995 for the Japanese Whale Research Programme under Special Permit in the Antarctic. Each sperm sample recovered from the vasa deferentia of eleven whales was diluted to a 1:9 ratio with Tris (300 mM)-citrate (94.75 mM)-glucose (27.75 mM)-egg yolk (15%; v/v)-glycerol (5%; v/v) diluents. The diluted samples were cooled gradually at 5 C for 2 h, frozen at -80 C and kept in liquid nitrogen. After thawing, sperm motility was evaluated subjectively. Sperm viability was assessed by eosin-nigrosin staining. Sperm abnormality and head morphology were assessed by carbol-fuchsin staining using phase-contrast microscopy. Sperm dimensions were measured using a calibrated projection microscope. Sperm motility and viability respectively varied from 1.0 to 20% (7 out of 11 samples), and 1.0 to 11.6%. Sperm concentration ranged from 13 to 585.3 × 10⁶ spermatozoa/ml. The mean proportion of morphologically abnormal spermatozoa was 84.0 ± 6.7%. Seven morphotypes of sperm heads were observed, and the most common types were conical and elliptic in shape. The mean total length of spermatozoa was 56.7 ± 0.5 μm, with their sperm heads measuring 5.2 ± 0.1 μm for length and 3.0 ± 0.0 μm for width; the mean length of sperm tail was 51.7 ± 0.5 μm.

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To determine a site producing a low molecular weight haemagglutinin isolated from the miniature swine sperm surface, Western blot and immunohistochemistry with boar tissue extracts and paraffin sections were employed. Using anti-13K and anti-16K sera raised against 13K and 16K-proteins purified by electroelution, immunoblotting clearly showed that out of six different tissue extracts and two secretory fluids, only seminal vesicular extract and seminal vesicular fluid were positive to these antibodies. Immunohistochemistry also demonstrated that the cytoplasm of epithelial cells of seminal vesicle and secretory materials seen above the epithelial cells were strongly stained with these antisera. The present data indicated that 13K and 16K-proteins were synthesized in the seminal vesicle and then bound to sperm surface in male reproductive tracts.

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Significant numbers of cells known by the name granulated metrial gland (GMG) cells, or uterine natural killer (NK) cells, are present in the murine uterus only during pregnancy. The functions of GMG cells remain incompletely defined, largely due to technical difficulties in isolating and culturing these cells. New methods for purifying large numbers of GMG cells are required. This study examined two methods for isolating GMG cells from the murine pregnant uterus based upon magnetic immunobeads and immunoparticles. By either approach, the yield of GMG cells was 1 × 10⁴ cells/metrial gland. Isolations using immunobeads gave cell suspensions with 82% in purity and 70% in viability. Isolations using immunoparticles gave cell suspensions with 62% in purity and 67% in viability. GMG cells isolated by the magnetic method retained an ultrastructure similar to in vivo GMG cells. These results establish that immunomagnetic approaches are useful for isolation of GMG cells from the pregnant murine uterus.

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