In Japan, low availability of equine oocytes makes it very difficult to improve the in vitro fertilization (IVF) system of horses. In this situation, an assay for sperm penetrability into zona-free hamster oocytes could be useful as a simulator of the IVF system. The aim of this study was to clarify the optimum conditions of the pretreatment of frozen-thawed stallion spermatozoa with caffeine and ionophore A23187 (IA) in the penetration assay. Frozen-thawed stallion spermatozoa were washed and then pretreated with IA in modified BO solution (mBO) supplemented with caffeine. After the pretreatment, the spermatozoa were used for the penetration assay with zona-free hamster oocytes. At first, effect of increasing concentrations of caffeine (0-10 mM) were examined on the penetration of spermatozoa pretreated with 1 μM IA for 60 sec. The highest penetration rate after 10 h co-culture with oocytes was obtained for spermatozoa penetrated in the presence of 1 mM caffeine (49.0%). Next, when spermatozoa were pretreated with 0-1.0 μM IA for 0-180 sec in the presence of 1 mM caffeine, significantly higher penetration rates after 10 h co-culture with oocytes were obtained under the condition of 0.1 μM IA for 120 sec (52.2%) and 1.0 μM IA for 60 sec (40.9%). When oocytes were fixed 2, 4, 6, 8 and 10 h after IVF using spermatozoa treated with 0.1 μM IA for 120 sec in mBO containing 1 mM caffeine, the penetration rates increased significantly from 2 h (10.4%) to 4 h (21.7%) and from 6 h (27.5%) to 8 h (49.0%). These results indicate that pretreatment with 0.1 μM IA for 120 sec in the presence of 1 mM caffeine effectively promotes the penetration of frozen-thawed stallion spermatozoa into zona-free hamster oocytes, and that under this condition, spermatozoa might complete the acrosome reaction during 6 to 8 h of incubation after IA treatment.

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This study was carried out to investigate changes in pituitary response to GnRH on gonadotrophin secretion in prepubertal heifers. A total of 50 prepubertal Holstein-Friesian heifers were treated with 1 μg/kg GnRH intravenously at 1, 2, 4, 6 and 8 months of age, and plasma samples were collected from the jugular vein at 0 to 360 min after GnRH treatment. Plasma concentrations of LH and FSH were measured by RIA. Increase in concentrations of LH and FSH, induced by GnRH, were observed in all heifers from 1 month of age, and the concentrations of LH and FSH at 30 min after GnRH treatment were significantly greater than those before GnRH treatment in heifers of all ages. The time from the GnRH administration to the appearance of the peak of LH and FSH rise was similar at each age and, was prolonged with age. The peak concentration and the amount of release from the pituitary of LH rise increased with age, while those of FSH had a tendency to decrease with age. These results indicate that the pituitary gland already has reactivity to GnRH in heifers by 1 month of age, that the capacity of LH release to GnRH in the pituitary gland develops with age, and that the regulatory mechanism for the secretion of LH and FSH develops differently in prepubertal heifers. This study suggests that the development of the capacity to secrete LH secretion in response to GnRH in the pituitary gland before puberty is one of the factors for deciding the time of the onset of puberty in heifers.

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The present study aimed to determine the relationship among changes in the number of primordial and primary follicles, ovarian weight and concentrations of androstenedione (A), estradiol-17 β (E₂), progesterone (P₄), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the bovine fetus between day-90 and -150 of gestation. Fetal ovaries were obtained from 25 pregnant cows. Histological sections of the ovaries were examined to determine the number of preantral follicles. The hormone concentrations were determined by enzyme immunoassay. Ovarian weight increased in parallel with fetal age. No clear changes in the number of primordial follicles were observed in this period. Primary follicles were observed in ovarian sections at day-100 and the number of primary follicles increased in parallel with fetal age between day-90 and -120, and afterwards it significantly increased (p<0.05) at day-130. Around day-120, the number of primary follicles increased with the serum concentrations of A and E₂. However, no relationships among number of primary follicles and concentrations of FSH and LH were observed in this period. These results indicate that the number of primary follicles markedly increases around day-130 of gestation, the increase in the number of primary follicles is associated with an increase in the concentrations of A and E₂ in the fetal serum, and the growth and differentiation of primordial and primary follicles are independent of FSH and LH.

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To investigate whether progesterone plays an essential role in differentiation of uNK cells, we ectopically transplanted tissue grafts from different developmental stages of uteri under various conditions. When female mice were used as recipients, decidualization and differentiation of uNK cells were observed in grafts from mature (8 weeks old) and immature (4 weeks old) donors, but there was no change in grafts from postnatal (10 days after birth) donors. When immature female mice were used as recipients, uNK cell differentiation was observed in grafts from both mature and immature donors, but decidualization and differentiation of uNK cells were not observed in the recipient uterus even in the presence of progesterone. These results suggest that uNK cell differentiation is not associated with the sexual maturation of recipients, and that the effect of progesterone on uNK cell differentiation is not direct, even though progesterone is an essential factor for uNK cell differentiation. When male mice were used as recipients, decidualization and differentiation of uNK cells were not observed in uterine grafts from normal mature mice. On the other hand, in castrated recipients uNK differentiation was induced in the graft, suggesting that uNK cell cannot fully differentiate in the presence of testosterone.

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We prepared an IgM monoclonal (PAG-1) antibody against follicular granulosa cells of porcine ovaries. Immunohistochemical reaction of the antibody was detected only in follicular granulosa cells, not in any other ovarian tissues or organs. Western blotting analysis demonstrated that 38- and 42-kD bands were present in the granulosa cell membrane samples prepared from healthy follicles. In the samples from early atretic and progressed atretic follicles, 38- and 64-kD bands were shown. During follicular atresia, the 64-kD band increased. After glycosidase treatment, the 64-kD band shifted to 42-kD, but no change was seen in the 38- and 42-kD bands. The present results indicate that the 42-kD band membrane protein is glucoconjugated during atresia, resulting in a glycoconjugated 64-kD protein appearing in the granulosa cells of the atretic follicles. We hypothesize that the glycoconjugated cell membrane protein (64-kD glycoprotein) may act as a trigger for phagocytosis in neighboring granulosa cells in atretic follicles.

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Recent observations suggest that vasoactive peptides angiotensin II (Ang II) and endothelin-1 (ET-1) regulate ovarian functions. The aim of this study was to investigate mRNA expression by semi-quantitative RT-PCR for the members of the angiotensin system such as angiotensin converting enzyme (ACE), angiotensin type 1 and 2 receptors (AT1R and AT2R), and also for the members of the endothelin system such as ET-1, endothelin converting enzyme-1 (ECE-1), endothelin A and B receptors (ETR-A and ETR-B) in theca interna (TI) and granulosa cell (GC) compartments of bovine follicles during final follicular growth. A classification of follicles into five groups (<0.5; >0.5-5; >5-20; >20-180; >180 ng/ml) was performed according to the estradiol-17β (E) content of follicular fluid. For the angiotensin system, the mRNA expression of ACE in TI significantly increased with E content in preovulatory follicles. In contrast, the GC of follicles did not express ACE mRNA. The mRNA expression of AT1R and AT2R in TI and GC was consistently detected throughout the different follicular stages. For the endothelin system, mRNA expression of ECE-1 in TI and GC increased during final growth of follicles. The mRNA expression of ET-1 in TI decreased in large follicles, whereas in GC it was completely negative at all stages of the follicle. In TI, mRNA expression of both ETR-A and B was constant, while in GC the expression of ETR-A mRNA increased with follicular E content. In conclusion, it is clear from our results that distinct regulatory changes of mRNA expression for some members of the angiotensin and endothelin systems occur in mature follicles. Particularly, a significant increase in the mRNA expression of both ACE and ECE toward the final maturation of follicles suggests the possible modulatory roles of Ang II and ET-1 in supplying the optimal microenvironment of ovulatory follicles.

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It is well known that prostaglandin (PG) F_{2 α} and PGE₂ are actively produced by bovine early corpus luteum (CL) at higher levels than in the CL of later stages. However, there is no in vivo information about the local secretion of PGs within bovine developing CL so far. Thus, the objective of the present study was to determine in detail the real-time changes in PGF_2α and PGE₂ secretion within developing CL. A microdialysis system (MDS) was surgically implanted into the newly formed CL of 6 cows on Day 3 after GnRH administration (about 1.5 days after ovulation) to induce superovulation following FSH treatment. Both PGF_2α and PGE₂ release were high during the first 24 h within developing CL but decreased thereafter. These profiles were well reflected in the PG concentrations in ovarian venous plasma. Furthermore, reverse transcription polymerase chain reaction analysis using CL from other animals independent from the MDS study revealed that the level of cyclo-oxyganase-2 mRNA expression in early CL was higher than that of later luteal phases. The present results provide the first direct in vivo evidence that active PGs production in CL occurs up to Day 4 after estrus in the cow.

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In this study, the toxicity of bis (tri-n-butyltin) oxide (TBTO), a marine pollutant, in premature mouse testes and the correlation of a total tin concentration with toxicity were examined. Mice were treated by oral administration twice a week for four weeks from five weeks of age at doses of 0 mg/kg (control), 0.4 mg/kg, 2.0 mg/kg, or 10 mg/kg, and sacrificed on the day following the final administration. The control mice were administered with distilled water containing 0.2% ethanol as vehicle. Removed testes were used for determination of the testicular sperm head counts and for histological study or determination of the total tin concentration. The sperm head count was significantly decreased in the 2.0 mg/kg and 10 mg/kg administration groups, although the testicular weights of both groups were unchanged compared to that of the control. The histological study revealed that TBTO caused vacuolization of Sertoli cells in several seminiferous tubules that failed to organize, however, the frequencies of occurrence were low. The total tin concentration in the testis increased in a dose-dependent manner in inverse proportion to the reduction of sperm head counts. These results suggest that TBTO is potentially an anti-testicular compound in mice.

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To explore a strategy for superovulation in heifers, effects of immunization and re-immunization of Japanese beef heifers against inhibin on ovarian response and hormonal profiles were investigated. On day 9 of the estrous cycle (day 0=day of estrus), ten heifers were primary injected intramuscularly (i.m.) either with inhibin vaccine (recombinant ovine inhibin α-subunit in oil emulsion, 125 μg/ml) or a placebo (Montanide:Marcol adjutant alone) followed by two booster injections at one estrous cycle intervals (immunized group). One year later, 3 injections of inhibin vaccine were given the same as in the first year (re-immunized group). Inhibin antibody titers in plasma of immunized heifers increased after the second injection of inhibin vaccine and in 3 out of 5 immunized heifers and multiple-ovulation (2.4 ± 0.6, n=5) was induced after the third injection. On the other hand, in the re-immunized heifers, the plasma inhibin antibody titers increased sharply after the first injection of inhibin vaccine and in 4 out of 5 re-immunized heifers multiple ovulation (4.4 ± 1.1, n=5) was induced after the first injection of inhibin vaccine. In all re-immunized heifers multiple-ovulation was induced after the third injection of inhibin vaccine (5.0 ± 1.1, n=5). The results also showed that the ovulation rates were positively correlated to circulating inhibin antibody titers and plasma levels of FSH. These results confirmed that active immunization against inhibin α-subunit induced multiple ovulation mainly by attenuation of the suppressive effect of inhibin on FSH secretion in heifers and indicated that re-immunization against inhibin was a good way to induce multiple-ovulation in cattle.

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Effects of intracervical prostaglandin E₂ (PGE ₂)-gel treatment on ripening of the cervix at term pregnancy were investigated. Concentrations of relaxin, LH estradiol-17β and progesterone were measured by radioimmunoassays in the maternal peripheral plasma during 48 hours after treatment with PGE₂-gel. Treatment with PGE₂-gel induced cervical ripening without induction of labor at term. Plasma levels of estradiol-17β increased after administration of PGE₂-gel, whereas plasma levels of relaxin, LH and progesterone were unchanged during the period studied. These results suggest that increased levels of estradiol-17 β may be involved in ripening of the cervix without increasing circulating relaxin during the period of PGE₂-gel treatment in the Japanese monkey.

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Although sika deer is valuable for deer farming in Japan, little is understood about the reproductive endocrinology of the male. Especially, there is no information on the blood LH concentration in male sika deer. The present study aimed to determine the annual changes of serum LH and testosterone (T) secretion in male sika deer. Blood samples were collected monthly from three stags for 3 years (1998-2000). Serum LH and T concentrations were determined using a second-antibody enzyme immunoassay and a time-resolved fluorescent immunoassay, respectively. The serum LH and T concentrations fluctuated obviously in an annual fashion. The LH concentrations peaked in May, June and July, while the T concentrations peaked in September and October. These results suggest that LH and T secretion in adult male sika deer are mainly controlled by annual rhythm in the same manner as in other temperate cervids.

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This study investigated bovine somatic nucleus transplantation (NT) using an intracytoplasmic injection system in which cumulus cell nuclei were injected into enucleated oocytes by a piezo micromanipulator. Activation of NT embryos was carried out by (1) simultaneous injection of 2-4 pl of inositol 1,4,5 tri-phosphate (IP3) and a donor nucleus into a recipient cytoplasm (IP method), (2) IP method followed by 1.9 mM 6-dimethylaminopurine (DM) treatment for 3.5 h (IP+DM method), or (3) activated after NT by an ionomycin treatment (IA) followed by DM treatment (IA+DM method). We found the frequency of remodeling nuclei and development into blastocyst stage of NT embryos to be: IP, 44% and 9%; IP+DM, 58% and 29%; and IA+DM, 61% and 27%. These results indicate that IP3 injection into the intracytoplasm followed by DM treatment produces a similar bovine oocyte parthenogenetic activation as IA+DM activation treatment, and that intracytoplasmic nucleus injection is an effective method for bovine somatic NT.

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mRNA expression of receptors, retinoic acid inducible gene 2 (GPRC5B/Raig2), estrogen receptor α (ER α), retinoid X receptor α (RXR α), pregnane X receptor (PXR) and germ cell nuclear factor (GCNF), in murine placentae at 14.5, 16.5 and 18.5 day-post coitum (dpc) was examined by reverse transcription-polymerase chain reaction technique. Strong expression of mRNA of GPRC5B/Raig2, which is a novel member of orphan receptor/G-protein coupled receptor-C and has great effects on cell growth, differentiation and embryogenesis, was first detected in placentae of mice. Moreover, mRNAs of nuclear receptors, ERα, RXRα, PXR and GCNF, were also detected in murine placentae. Wide individual differences in these mRNA expression levels were noted. These data indicated that GPRC5B/Raig2 and other nuclear receptors examined in the present study play important roles in placental function of mice. The findings will contribute to the assessment of the toxic effects of endocrine disruptors on placental function.

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The Japanese wolf, Canis lupus hodophilax Temminck, which putatively became extinct decades ago, remains a taxonomical or phylogenetic enigma. In Japan, 3 mounted taxidermic pelt specimens believed to represent C. lupus hodophilax have been stocked at the University of Tokyo, the National Science Museum and Wakayama University, respectively. Using an improved method developed by our group for the extraction and the PCR amplification of ancient DNA from preserved mammalian pelts, a 351-nucleotide partial genomic DNA stretch of the X-linked gene coding for amelogenin (AMELX) was successfully cloned from a skin specimen derived from the mounted female taxidermic pelt specimen of the University of Tokyo, and its nucleotide sequence was determined. Two allelic lupine AMELX sequences, i. e., the J-type (accession no. AB080688, submitted to DDBJ on 1/Mar./2002) and the R-type (accession no. AB080689, submitted to DDBJ on 1/Mar./2002) were identified. Both the J-type and the R-type alleles differed considerably at the nucleotide as well as the amino acid level from the corresponding AMELX region shared by the dog and the Mongolian wolf. Although the presently discovered lupine variants of the AMELX partial sequence are of great interest from the point of view of taxonomical identity of the Japanese wolf, our preliminary results (unpublished) strongly indicate that polymorphism in the nucleotide as well as the amino acid sequence is likely to exist in this particular region of AMELX among different breeds of domestic dogs, Canis familiaris. Further molecular analysis of the intraspecific as well as the interspecific variations in the AMELX DNA will be needed to gain clear insight into the taxonomical and phylogenetic positions of the Japanese wolf.

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