The mammalian ovary contains a huge number of small follicles of various sizes, and each follicle encloses a small oocyte. Only a small number of non-growing oocytes (30 μm in the pig and cow) grow to their final size (120 μm), mature, and are ovulated. In vitro growth (IVG) culturing of small oocytes will provide a new source of mature oocytes for livestock production. Using the IVG culture system, non-growing mouse oocytes in primordial follicles grow to their final size and acquire full developmental competence. Among large animals, babies were produced from ovarian oocytes by IVG culture only in the cow. However, the oocytes used were not non-growing ones but at the mid-growth stage (90-99 μm in diameter) in early antral follicles. Xenotransplantation of the follicles at an early stage to immuno-deficient mice is a substitute for an effective long-term IVG culture of much smaller oocytes. IVG and xenotransplantation of small oocytes at a specific size will provide a new understanding of the mechanisms regulating oogenesis and folliculogenesis in the complex mammalian ovary.

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Recently, reproductive management has become more difficult as a result of increased herd size. Problems with missing estrous signs and decrease in conception rate by artificial insemination (AI) performed at wrong timing have caused low AI conception rates. In 1995, ovulation synchronization and fixed-time AI (Ovsynch/TAI) was developed in the USA as a new reproductive technology, which was accepted as an useful reproductive management tool in many countries. However, no information on the use of Ovsynch/TAI was available in Japan. It was, therefore, warranted to show the ovulation rate and conception rate after Ovsynch/TAI using gonadotropin releasing hormone analogue (GnRH-A, fertirelin acetate) and prostaglandin F_2α (PGF_2α)-THAM, both were commercially available in this country. The conception rate after Ovsynch/TAI has been known to vary among different herds and individuals. Investigation and analysis of factors affecting the conception rate was also warranted to improve the conception rate. A series of experiments were carried out to establish Ovsynch/TAI using domestically produced GnRH-A and PGF_2α and to study factors affecting conception rate after Ovsynch protocol. Ovsynch using 100 μg GnRH-A and 25 mg PGF_2α were observed using ultrasonography. As a result, a high synchronization rate of ovulation at 16 to 20 h after the second GnRH injection was confirmed. The conception rate after Ovsynch/TAI was compared in 87 cows with the conception rate after AI at estrus induced by PGF_2α (139 cows). Conception rate after Ovsynch/TAI was higher than the figure after AI at induced estrus (59.1% vs 20.9%, P<0.05). The dose of GnRH-A was also studied and a practical dose of GnRH-A was found to be 50 μg per cow. To clarify some factors affecting the conception rate after Ovsynch/TAI, 1,558 cows were investigated for the state of their ovaries, days after calving, parity, season, ovarian cyclicity postpartum and nutritional state at the day of Ovsynch. The overall conception rate after Ovsynch/TAI was 51.5%. Fifty-six cows (3.6%) showed estrus at 6 to 7 d after the first injection of GnRH-A. The conception rate after Ovsynch/TAI was low in cows that were 40 to 60 d postpartum, those in their 5th lactation or more, those bred in July to August, and those recovering ovarian cyclicity later than 56 d postpartum. The conception rate after Ovsynch/TAI was high in cows in which body condition score (BCS) was 3.75 at dry period and 3.0 at the day of Ovsynch. In conclusion, Ovsynch/TAI is an effective tool for the reproductive management of dairy cows. A steady and sufficient conception rate after Ovsynch/TAI could be expected by taking the factors affecting the conception rate into the consideration.

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We examined morphological nuclear events during the first cell cycle of bovine embryos reconstructed with somatic cells at the M and G1 phases (M-embryos and G1-embryos, respectively) by intracytoplasmic nuclear injection, and the subsequent development of these embryos in vitro and in vivo. Bovine fetal fibroblasts (BFFs) at the M or G1 phase were directly injected into enucleated oocytes, and activated immediately. Only half (48%) of the M-embryos extruded polar body-like cells (PBCs) at 6 h post injection (hpi). At 15 to 19 hpi, 54% of the M-embryos formed a single pronucleus-like nucleus. Nuclear envelope-breakdown, premature chromosome condensation and single nuclear clusters were observed in most of the G1-embryos (88%) within 30 min following the nuclear injection. At 15 to 19 hpi, single pronucleus-like nuclei were formed in most G1-embryos (83%). The potential of G1-embryos to develop to blastocysts was significantly higher than that of M-embryos (31% vs 16%). Three of five recipients following transfer of blastocysts derived from the G1-embryos became pregnant on Day 30, and one recipient delivered a calf. Our results indicate that almost a half of the M-embryos failed to extrude PBCs and that the G1-embryos developed to blastocysts at a higher rate than the M-embryos.

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The development of preimplantation mammalian embryos in vitro is less than optimal. Follicular fluid and cumulus cells have both been used independently, to improve preimplantation embryo quality in culture. This study was undertaken to evaluate the influence of a cumulus cell monolayer in human follicular fluid on mouse early embryo development in vitro. One-cell embryos were obtained from NMRI mice after superovulation with eCG and hCG. Cumulus cells were prepared from mouse egg-cumulus mass. These cells were separated from red blood cells using a Percoll gradient. Follicular fluid was collected from patients undergoing an IVF program during oocyte pick-up. The cumulus cell monolayer was prepared in follicular fluid (FC) and Ham's F10 (HC). Mouse one-cell embryos were cultured in FC, HC, Ham's F10 (HF) and follicular fluid (FF) for 120 h. Only 10.5% of embryos passed the two-cell block in HF. However, the proportions of embryos passing the two-cell block were 23.1%, 21.4% and 68.5% in FF, HC, and FC treatments, respectively; which were significantly different from HF (p<0.05). The differences between FC and the two other treatments were also significant (p<0.001). In FC, 33.7% of one-cell embryos continued to grow to the blastocyst stage whereas only 2.1% and 1.9% of one-cell embryos in FF and HC reached this stage and no embryos developed to blastocyst in HF. The proportion of blastocysts in FC was significantly higher than all other treatments (p<0.001). It can be concluded that follicular fluid and cumulus cells in monolayer form synergistically improve the early embryo culture condition.

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Many environmental chemicals including pesticides have been reported to possess hormonal activities, and thus are classified as endocrine disruptors. Permethrin, a synthetic pyrethroid insecticide, is used worldwide, which provides potential environmental exposure. However, relatively few studies have reported on hormonal activities, particularly estrogenic and androgenic activities of permethrin, and the results of these studies are in some respects contradictory. Therefore, this study investigated the potential estrogenic and androgenic activities of permethrin in vitro and in vivo. We conducted an uterine Calbindin-D_9k (CaBP-9k) gene expression assay and an uterotrophic assay for estrogenic activity, and a Hershberger assay for androgenic activity. The CaBP-9k gene, one of the intracellular calcium binding proteins, is estrogen-responsive in the uterus. The rat uterotrophic and Hershberger assays are generally used as in vivo short-term screening assays for detecting the estrogenic and androgenic activities of chemicals, although these assays are still being validated by the Organization for Economic Cooperation and Development (OECD). Northern blot analysis showed the induction of uterine CaBP-9k mRNA level in response to permethrin as well as co-administration of permethrin with E2. In the uterotrophic assay using 18-day-old female rats, subcutaneous treatments with permethrin (10 to 800 mg/kg) for three days increased relative uterine wet weights, and E2-induced uterine weights. These effects were statistically significant at 800 and 200 mg/kg, respectively. Moreover, permethrin-induced uterine weights were inhibited by the co-administration of ICI 182,780, an antiestrogen. In the Hershberger assay, the administration of permethrin orally to testosterone propionate-treated castrated male rats led to statistically significant reductions in androgen-dependent sex accessory tissue (ventral prostate, seminal vesicles, levator ani and bulbocavernosus muscles, Cowper's gland and glans penis) weights at all doses tested (10, 50 and 100 mg/kg). These results suggest that permethrin might have estrogen-like effects on female rats, but antiandrogen-like effects on males.

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Gene expression of bovine trophoblast cell line (BT-1) was analyzed with a custom utero-placenta complementary DNA microarray. Expression comparison with in vivo tissues of trophoblast derivation was performed to investigate characteristics of the expression in BT-1. BT-1 is a cell line established without feeder cells using trophoblast cells that are separated from bovine blastocysts. The bovine in vivo tissues of the trophoblast derivation were collected on day 17 through 56 of gestation. Of 1,773 genes, 933 exhibited an expression difference exceeding two-fold between BT-1 and in vivo tissues. These genes were analyzed by the k-means clustering method and were distributed into six clusters. Some genes, such as placental lactogen, interferon-τ and pregnancy-associated glycoproteins, all known as trophoblast-specific genes, were detected in BT-1 as well as in vivo tissues throughout the experiment period. These trophoblast-specific genes and octamer-binding transcription factor-4, known as a marker for undifferentiation of cells in mice, were detected by RT-PCR in both BT-1 and in vivo trophoblast tissues. The overall gene expression profile in BT-1 suggests that this cell line contains trophoblast-specific characteristics and is similar to trophoblast cells around the implantation period.

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Thyroid hormones permit the annual reproductive transition of seasonal breeders. Although, precise function of thyroid hormones in seasonal breeding is not well understood. In the present study, we examined effects of hypothyroidism on the hypothalamus-pituitary-gonadal axis in adult male golden hamsters after transition of the short-day photoperiod (SD; 8 h light: 16 h dark) condition. We confirmed that hypothyroid, which had been induced by administration of thiouracil in drinking water for 4 weeks, did not have direct effects on testes in male hamsters under the long-day photoperiod. Plasma concentrations of free T3 and T4 decreased 15 weeks after transition of SD condition. Plasma concentrations of testosterone in the hypothyroid group decreased earlier than in the control group after the transition from LD to SD. In animals treated with testosterone after castration, plasma concentrations of LH in the hypothyroid group decreased earlier than in the control group after the transition of SD. On the other hand, pituitary response to GnRH for LH release did not change in castrated hamsters as a result of hypothyroidism. These results suggest that thyroid hormones act the hypothalamus and might be required to maintain GnRH secretion in male golden hamsters.

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N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101), which catalyzes the first step in the conversion of oligomannose to complex or hybrid N-glycans of glycoproteins, was found in media cultured with bovine oviduct epithelial cells (BOEC) obtained from non-pregnant cows during the follicular phase. Combined treatment with specific hormones increased GnT I release from BOEC. Luteinizing hormone (LH; 10 ng/ml) alone slightly, but together with 17β-estradiol (E₂; 1 ng/ml), synergistically increased GnT I activity. Vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF) α, which have been shown to have their highest activities in the bovine oviduct during the periovulatory period, also increased in GnT I activity. This study provides the first evidence of an increase of GnT I release from BOEC in vitro, and shows that endocrine as well as local factors such as LH, VEGF and TNFα increase this activity. The results suggest that GnT I activity in the bovine oviduct may contribute to the induction of glycosylation and thereby contributing to the provision of the optimal microenvironment for fertilization and early development of the embryos.

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To assess the permeability of mouse oocytes and embryos, matured oocytes and embryos at various stages of development were placed in five cryoprotectant solutions at 25 C for 25 min. From the cross-sectional areas of the oocytes/embryos, the relative change in volume was analyzed. In oocytes, shrinkage was least extensive and recovery was quickest in the propylene glycol solution, showing that propylene glycol permeates the oocytes most rapidly. Dimethyl sulfoxide, acetamide, and ethylene glycol permeated the oocytes slightly more slowly than propylene glycol. The oocytes in glycerol shrunk extensively and then expanded marginally, indicating slow permeation. The volume changes of 1-cell and 2-cell embryos were similar to those of oocytes, showing little change in permeability. In 8-cell embryos, the volume recovered much faster than in the earlier stages especially in glycerol and acetamide. In morulae, the volume recovery was much faster in glycerol and in ethylene glycol; in ethylene glycol, the extent of shrinkage was small and the recovery was fast, indicating an extremely rapid permeation. Although the permeability of oocytes/embryos generally increased as embryo development proceeded, the degree of increase varied greatly among the cryoprotectants. Interestingly, the volume change in propylene glycol was virtually unaffected by the stage of development. Such information will be valuable for determining a suitable protocol for the cryopreservation of oocytes/embryos at different stages of development.

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In this study, we performed immunohistochemistry of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), cytochrome 17α-hydroxylase P450 (P450c17), and cytochrome P450 aromatase (P450arom) in the corpus luteum and placenta of Shiba goats. The aim was to clarify the steroidogenic capability of the corpus luteum and placenta of Shiba goats. Ovaries containing corpora lutea were obtained from four adult Shiba goats during the luteal phase (day10; n=2) and pregnancy (90 and 120 days of gestation). Placenta was obtained from one Shiba goat on day 120 of gestation. The sections of the ovaries and placentae were immunostained using the avidin-biotin-peroxidase complex method (ABC) with polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. All luteal cells expressed P450scc, 3βHSD, P450c17 and P450arom. The distribution of P450scc, 3βHSD, P450c17 and P450arom were not different during the luteal phase and pregnancy. P450arom showed a weak positive staining in late pregnancy (120 days). In addition, immunoreactions for P450c17 and P450arom were observed in syncytiotrophoblast of the placenta of one Shiba goat. These results indicate that, in Shiba goats, corpus luteum is not only an important source of progesterone but also has the ability to synthesize androgen and estrogen during the luteal phase and pregnancy. Also the placenta has the ability to synthesize androgen and estrogen in late pregnancy.

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Phthalates are suspected to disrupt the endocrine system, especially through estrogenic effects. In the present study, we investigated the effects of various phthalates and compared them with those of estrogenic compounds that disrupt the female reproductive system. To assess the effects of these phthalates, alteration of the Calbindin-D_9k (CaBP-9k) gene was measured as a biomarker because rat CaBP-9k gene carries an estrogen response element (ERE) which is involved in estrogen responsiveness of the gene during the estrous cycle. In this study, phthalates were tested for estrogenic properties in in vitro and in vivo models. First, the E-Screen assay was used to measure the proliferation of MCF-7 cells, a human breast cancer cell line. Treatments with 17β-estradiol (E2; 9-fold) and 17α-estradiol (EE; 9-fold) induced MCF-7 cell proliferation at concentrations of 10^-9 M. Phthalates induced an increase in MCF-7 proliferation at concentration of 10^-6 M up to 10^-4 M. Nbutyl benzyl phthalate (BBP; 6-fold vs. vehicle), dicyclohexyl phthalate (DCHP; 8-fold), 2-ethylhexyl phthalate (DEHP; 6-fold) and di-n-butyl phthalate (DBP; 7-fold) at the concentration of 10^-4 M induced in an increase in MCF-7 proliferation after 6 d of treatment compared to vehicle. However, significant increase in MCF-7 proliferation was induced by diethyl phthalate (DEP). Second, we investigated the expression of CaBP-9k in the uterus of immature rats after oral treatment with BBP, DCHP, DEHP, DBP or DBP (600 mg/kg per day) in this in vivo model, because the immature rat model is highly sensitive to exposure to estrogenic chemicals. None of the phthalates induced the expression of CaBP-9k mRNA and its protein in the neonatal uterus as analysed by Northern and Western blot analyses, respectively. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce CaBP-9k expression in the in vivo system, suggesting that the assays of estrogenic effects of various phthalates conducted in vitro and in vivo expression of CaBP-9k may produce conflicting results.

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20α-Hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form 20α-dihydroprogesterone (20α-OHP). 20α-HSD is expressed in the murine placenta, suggesting a role, yet unidentified, played by this enzyme during the course of pregnancy. To elucidate the possible roles of 20α-HSD during pregnancy, 20α-HSD gene expression in the placenta was examined by Northern blot analysis, and progestin (progesterone and 20α-OHP) concentrations in the maternal and fetal sera and the amniotic fluid were measured by radioimmunoassay in pregnant Shiba goats. The expression of 20α-HSD mRNA was observed in the placenta and the intercaruncular part of the uterus during mid to late pregnancy. Analysis by in situ hybridization revealed that 20α-HSD mRNA was mainly localized in the endometrial epithelium of the caruncle side of the placenta. Considerable enzyme activity of 20α-HSD was also detected in the cytosolic fraction of the placenta and intercaruncular part of the uterus. Although concentrations of progesterone and 20α-OHP in the maternal serum showed similar profiles, progesterone levels in the fetal serum stayed extremely low throughout the pregnancy. The 20α-OHP concentration in the fetal serum was always higher than that in the maternal serum. In the amniotic fluid, the concentrations of both progesterone and 20α-OHP remained at very low levels throughout the pregnancy. These results support the notion that 20α-HSD protects the fetus from the cytotoxic effects of progesterone, and thereby maintains the normal development of the fetus.

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Increase in the blood supply to individual follicles appears to be associated with follicular growth rates and the ability to become the dominant follicle, while reduced thecal vascularity appears to be closely associated with follicular atresia. Therefore, this study aimed to determine the real-time changes in the vascularity of the follicle wall during the first follicular wave in cycling Holstein cows. Normally cycling and lactating cows (n=5) were examined by transrectal color Doppler ultrasonography (the sensitivity for velocity: > 2 mm/sec) to determine the changes in the vasculature of the follicle wall (presence or absence of blood flow) and the diameter of follicles. A new follicular wave and ovulation were induced by GnRH injection at 48 h after an injection of PGF_2α analogue. The ovaries were scanned daily for 7 days after GnRH injection. Follicles >2.5 mm were classified into 3 groups by the changes in diameter as follows: 1) largest follicle, 2) second largest follicle, and 3) small follicles, which included all other follicles >2.5 mm. Before the follicle selection, there was no significant difference in the percentage of follicles with detectable blood flow between the subsequently determined largest and second largest follicles. After the follicle selection, the percentage of follicles with detectable blood flow significantly decreased among the second largest follicles. In addition, small follicles with detectable blood flow kept larger diameters than those without detectable blood flow from one day before the occurrence of follicle selection. It is likely that maintenance of follicle vasculature and appropriate blood supply to the larger follicles is essential for follicle dominance. In small follicles, the presence of blood flow within the wall also appears to be required for recruitment. Consequently, the data suggest that the change of the blood supply to an individual follicle closely relates to the dynamics of follicular growth in the first follicular wave in the cow.

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This study examined the association between redox status in the oviduct and early embryonic death in heat-stressed mice. In Experiment 1, non-pregnant mice were heat-stressed at 35 C with 60% relative humidity for 12, 24, or 36 h, and the maternal redox status was verified by measuring the levels of reactive oxygen species (ROS) and free radical scavenging activity (FRSA) in the oviduct, and thiobarbituric acid reactive substances (TBARS) and glutathione peroxidase (GSH-Px) activity in the liver. In Experiment 2, zygotes were collected from mice heat-stressed for 12 h on the day of pregnancy, and their developmental abilities were assessed in vitro, along with the intensity of DNA damage at the 2-cell stage. The TBARS value and GSH-Px activity in the liver, and ROS level in the oviduct were significantly higher in heat-stressed mice, and this increase appeared to depend on the duration of the heat stress. Maternal heat stress significantly reduced the percentage of zygotes that developed to the morula and blastocyst and the total cell number in the blastocyst. In addition, DNA damage at the 2-cell stage was significantly higher in maternally heat-stressed embryos. These results suggest that heat stress induces systemic changes in redox status in the maternal body, and the resultant increase in oxidative stress in the oviduct is possibly involved in heat stress-induced early embryonic death .

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The storage of unfertilized oocytes, either immature, maturing or mature, is still unsatisfactory. Here we describe an approach in which germinal vesicles isolated as karyoplasts from immature oocytes are vitrified by open the pulled straws (OPS) method in evacuated porcine zonae pellucidae. After thawing, their survival was almost absolute. Moreover, when thawed GV-karyoplasts were fused to immature oocyte cytoplasts the maturation of reconstructed cells resulted in the production of secondary oocytes - metaphase II.

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The atomic force microscope (AFM) provides nanometer resolution, topographic data of the natural surface structure of materials. We studied the topology of the surface structure of bovine sperm heads during the acrosome reaction by AFM. In addition, we numerically analyzed the areas of the median sagittal plane of the sperm heads. Bovine frozen-thawed spermatozoa were washed, capacitated by heparin, and incubated with lysophosphatidylcholine (LPC) to induce the acrosome reaction, smeared on a cover glass, air-dried, and observed with AFM using the dynamic force (tapping) mode. AFM analysis of spermatozoa showed the clear surface structure of acrosomes, equatorial segments, postacrosomal regions and necks. Although AFM images of spermatozoa capacitated by heparin had complete acrosomes, most spermatozoa treated with LPC had no acrosomal caps as shown by AFM. These observations coincided with those obtained by light microscopy after staining with naphthol yellow S and erythrosin B. Furthermore, numerical analysis of AFM images indicated that areas of the median sagittal plane of the anterior portions of acrosome-reacted sperm heads (2679 ± 616 pixels) were approximately 40% less than those of intact heads (4535 ± 174 pixels, P<0.05). These results indicate that AFM can usefully observe and numerically analyze the fine surface structures of bovine spermatozoa.

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