The viability of rat uterine natural killer (NK) cells in vitro was examined. Uterine NK cells were obtained from rats on days 10 and 16 of pregnancy and they were cultured in RPMI 1640 medium supplemented with 10% v/v fetal calf serum in the presence or absence of human recombinant IL-2 or cycloheximide for 24, 48, 72 and 96 h. In both preparations of cells collected on days 10 and 16 of pregnancy, about 60% of the cells died during the first 48 h of culture regardless of the presence of IL-2. Gel electrophoresis of genomic DNA showed ladder fragmentation with 180 to 200-bp fragments, indicating that the cell death was due to apoptosis. Apoptosis of uterine NK cells in vitro was promoted, not prevented, by cycloheximide. The remaining uterine NK cells (40%) were maintained alive under these culture conditions for, at least, 96 h. Therefore, rat uterine NK cells undergo apoptosis and are consist of subpopulations with referring viabilities in vitro.

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The aims of the present study were twofold. The first aim was to examine whether or not early pregnancy-associated thrombocytopenia (EPAT) occurred in cows following nonsurgical embryo transfer by observing the daily patterns of the concentration of peripheral platelets (PLT) during the preimplantation period of pregnancy. The other goal was to assess the use of PLT count as a diagnosis of early pregnancy in cows. In Experiment 1, out of 50 Holstein parous cows, 40 had one embryo nonsurgically transferred to each uterine horn on Day 6 or 7 (Day 0=onset of standing estrus). The remaining 10 cows served as controls having no embryos transferred. Blood was taken daily at the same time, from Day 6 or 7 following estrus (the day when the embryos were transferred in the non-controls) to the day of return to estrus for control cows and non-pregnant cows, or to Day 36 for pregnant cows. Bilateral embryo transfers resulted in 9 twin, 14 single, and 17 non-pregnancies. Some recipient cows showed an increase in PLT following embryo transfer, while others displayed a decrease in PLT. If significant PLT change was characterized either by a decrease or an increase of >20% compared to the value of pre-transfer, the proportions of cows indicating significant change were 78% (7/9) in twin, 64% (9/14) in single, 76% (13/17) in non-pregnancies and 30% (3/10) in controls. The proportions in twin (P<0.05), single (P<0.05) and non-pregnancies (P<0.01) were higher than that in controls. The effect of the particular group on PLT was significant (P<0.0025). In Experiment 2, each of several in vitro-produced blastocysts were nosurgically transferred to 9 Japanese Black cows on Day 7 or 8 following onset of estrus. Seven days after embryo transfer, an attempt to recover the previously transferred embryos by nonsurgical flushing was performed. Embryos could be recovered on Day 14 or 15 from 5 animals, whose blood was taken from the day of embryo transfer to the next day of embryo recovery and during the corresponding days of the following estrous cycle. For PLT, differences were detected between the both phases of 3 out of the 5 cows. The individual PLT variation in response means that EPAT does not necessarily occur in cows following embryo transfer, and that the use of PLT counts is unlikely to be clinically useful in assessing early pregnancy in cows. These results may suggest, however, that maternal platelet activation would occur during early pregnancy in cattle.

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The timing of shedding and disintegration of cytoplasmic droplets from boar and goat spermatozoa was determined. More than 90% of spermatozoa from the cauda epididymidis and ductus deferens had a droplet each at the distal region of the middle piece in both species. In boars, the droplets were still observed in 97% of spermatozoa immediately after ejaculation. After 1 min of ejaculation, however, the percentage of spermatozoa without the droplet greatly increased to 65%, with no significant changes thereafter. Moreover, most of the free droplets shed from spermatozoa had already disappeared after 1 min of ejaculation. In goats, most spermatozoa (90%) had no droplet even immediately after ejaculation, and the rate of free droplets remaining in the semen was 63%. However, within 2 min of ejaculation, the rate of free droplets remaining in the semen dropped to 8%. These results indicate that shedding of cytoplasmic droplets from boar spermatozoa and their disintegration occur within 1 min after ejaculation. They also suggest that droplets are shed from most of goat spermatozoa during their transit through the urethra and/or immediately after ejaculation, and that their disintegration occurs within a few minutes after ejaculation.

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This study was undertaken to determine the mechanism of early pregnancy factor (EPF) secretion from ova. EPF released into the culture medium from in vitro matured bovine oocytes that were sperm-injected and plasmamembrane-perforated was detected. Bovine follicular oocytes were matured in vitro for 19 to 22 h in 5% CO₂ in air at 39 C. A capacitated spermatozoa was injected into each of the three parts of the matured oocyte, namely, the zona pellucida, the perivitelline space and the ooplasm. The plasmamembrane of matured oocytes were also perforated with a glass micropipette into the ooplasm. After sperm-injection or perforation, these ova were cultured separately in TCM199 containing 2% fetal calf serum (FCS) for 24 h in 5% CO₂ in air at 39 C. Then EPF activity in these culture media were assayed by rosette inhibition test. A rosette inhibition titer (RIT) above 4 was considered as a positive expression of EPF activity. Of the medium samples from the sperm-injected oocytes, 4 out of 5 of those injected in the perivitelline space and 4 out of 5 of those injected in the ooplasm showed positive EPF activity. Further, all 7 medium samples of oocytes perforated in the plasmamembrane with a glass micropipette also showed positive EPF activity. However, all 4 medium samples containing ova injected with sperm into the zona pellucida showed negative EPF activity. Consideration of these data led to a remarkable discription of EPF release from oocytes micromanipulated with sperm and inorganic matter, and provided the evidence to show that EPF release is triggered by sperm penetrating through the zona pellucida.

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The antral follicles, 4-6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, high activity of neutral Ca²⁺/Mg²⁺-dependent endonuclease by which the chromatin DNA is degraded at internucleosomal sites in the cells undergoing apoptosis was noted only in granulosa cells but not in cumulus cells. Low activity of the Ca²⁺/Mg²⁺-dependent endonuclease was observed in both types of cells obtained from healthy follicles. Low activities of neutral Ca²⁺-dependent endonuclease, neutral Mg²⁺-dependent endonuclease and acidic cation-independent endonuclease were demonstrated in all cases. A good correlation (r=0.892) between the Ca²⁺/Mg²⁺-dependent endonuclease activity of granulosa cells and the progesterone/estradiol-17β ratio of follicular fluid in each follicle, which provides an index of follicular atresia in pig ovaries, was found. These results suggest that the Ca²⁺/Mg²⁺-dependent endonuclease, and not the Ca²⁺-dependent endonuclease, the Mg²⁺-dependent endonuclease or the cation-independent endonuclease, is involved in granulosa cell apoptosis of the atretic antral follicles in pigs.

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In mammals, the SRY (the sex determining region on the Y; Sry in the mouse) gene is responsible for the function of TDF (testis determining factor; Tdy in the mouse) locus, and plays a key role in the sex determination. In the mouse, the Sry open reading frame (ORF) is flanked at either end by a large inverted repeat, causing the formation of untranslatable circular transcripts; the linear transcripts have so far been known to be expressed only in the genital ridge between 10.5 and 12.5 d.p.c. In past attempts to examine the effects of expression of Sry exogenously introduced into cultured cells, the transfected Sry generated only the circular transcripts. In our experiments, cells from a cultured mouse Sertoli cell line, TM-4, which do not express endogenous Sry, were transfected with either human SRY or mouse Sry DNA where the inverted repeats responsible for the formation of circular transcripts were deleted. The expression of linear Sry transcripts was confirmed in the Sry-transfected TM-4 cell lines (TMHm series). Curiously, in TMHm cells, the gene coding for P450 aromatase (P450arom) was expressed, while P450arom was not expressed in the non-transfected TM-4 cells. No expression of P450arom was induced in the TM-4 cells transfected with the human SRY (hSRY) DNA. Although WT-1 promoter has been shown to possess a SRY binding motif, no evidence for the Sry- or hSRY-induced WT-1 expression was obtained in the transfected cell lines of either TMHm or TMHh series. MIS, another putative target gene of Sry, is expressed in non-transfected TM-4 cells, confirming the previous reports indicating that Sry expression is not a prerequisite for the MIS expression.

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Our previous studies demonstrated that the gene coding for whey acidic protein (WAP) is expressed in a variety of tissues other than mammary glands in lactating mice (Wen et al., 1995). It was suspected, therefore, that WAP plays important physiological roles in tissues of rodents. To clarify biological functions of mouse WAP (mWAP), we produced transgenic mice introducing a 6.4 kb metallothionein-I (mMT-I)/mWAP construct where 1.8 kb mouse MT-I promoter was linked to 4.6 kb WAP structural gene. Three male mice were confirmed to be transgenic by Southern blot and PCR analyses, and served as the founders transmitting the genes to their offspring. The founders, their offspring and normal mice (control) were given drinking-water containing 25 mM ZnSO₄ for 4 weeks. The expression levels of WAP genes induced by Zn²⁺ were about 3 times higher in the liver of transgenic mice than those of normal mice. Unexpectedly, it turned out that the expression of the endogenous WAP was also enhanced by the prolonged exposure to Zn²⁺. Despite the enhanced expression of WAP, no alteration in phenotypes, e.g., morphological characteristics, growth rates, reproductive performance and/or behavior have so far been detected in all the transgenic mice examined.

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The AKR/N mouse eggs fertilized in vitro with ICR mouse epididymal spermatozoa were cultured in a phosphate-free medium. At 96 h after insemination, 48% of the fertilized 1-cell embryos developed to the ≥morula stage. When a total of 96 blastocysts obtained 96-100 h after insemination was transferred to 6 ICR mice, 52 (54%) implantation sites and 27 (28%) fetuses without detectable abnormality were observed in the uterine horns on Day 17.

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Clinical ultrasonographic observations of the ovaries of 5 cross-bred sows were carried out during the estrous cycle and during treatment for ovarian disease (1 case of normal estrous cycle, 2 cases of ovarian quiescence, 1 case of large multiple follicular cysts, and 1 case of small multiple follicular cysts). Ultrasonography allowed clear observations of follicle growth, ovulation, luteinization and luteal regression during the porcine ovary estrous cycle. Observation of differences in the response of the ovarian structure between effective and ineffective pregnant mare serum gonadotropin (PMSG) treatment in cases of sows with ovarian quiescence was also possible by ultrasonography. Ultrasonography additionally revealed that the administration of bovine anterior pituitary gonadotropin (bAPG) for treatment of sows with ovarian follicular cysts lead to temporary enlargement of the cysts. The results of this study have shown that ultrasonography is a superior method for detailed observation the porcine ovarian structure, and has promising possibilities for clinical applications.

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The brain of salmonid fishes contains two types of gonadotropin-releasing hormone (GnRH) peptide, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). Although cDNA encoding the mammalian GnRH (mGnRH) receptor has been cloned and characterized in several mammalian species, structures of cDNAs of other GnRH receptors in non-mammalian vertebrates have not been determined yet. As a step to isolate cDNA clones for the sGnRH and the cGnRH-II receptors from masu salmon (Oncorhynchus masou) brains, we examined functional expression of these receptors electrophysiologically using Xenopus oocyte expression system. Oocytes injected with poly(A)⁺ RNA of the brain or the pituitary of the masu salmon responded to both sGnRH and cGnRH-II. Oocytes injected with poly(A) ⁺ RNA of pituitaries of the masu salmon responded to only sGnRH. The poly(A) ⁺ RNA of the brain was further fractionated according to the size. Injection of pooled fractions of poly(A) ⁺ RNA of a size range between 5.5 and 6.0 kb induced the oocyte response to sGnRH, but the response to cGnRH-II was not clearly induced. Injection of poly(A) ⁺ RNA of about 5.0-5.5 kb in size induced the response to cGnRH-II, but the response to sGnRH was not clearly detected. These results suggest that the sGnRH and cGnRH-II receptors are encoded by different RNA, and that the preference of the agonists of these two receptors are clearly different each other.

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