In cattle, the mechanisms underlying implantation and placental development are still unclear. Synepitheliochorial placentation in cattle is noninvasive, and thus generates limited interest in terms of degradation and remodeling of endometrial tissues. The overall purpose of this study was three-fold: (1) to examine the gene circuitry around the implantation window, (2) to understand development of the placenta during the peri-implantation period by using a uteroplacental cDNA microarray, and (3) to study the roles of molecules involved in endometrial remodeling. Bovine trophoblastic binucleate cell-specific molecules, such as pregnancy-associated glycoproteins (PAGs), placental lactogen (PL), and prolactin-related proteins (PRPs), were markedly expressed in binucleate cells (BNCs) around implantation. The expression of PRP-1 was specific to the caruncular (CAR) area of the gravid uterine horn. Gelatinases (MMP-2 and -9) in association with heparanase may be central to endometrial remodeling. In situ hybridization analyses of PAGs, PRPs, PL, and heparanase suggested that BNCs expressed these molecules simultaneously. Future studies will further investigate the specific roles of these molecules in placentogenesis. The uteroplacental cDNA microarray presented cascades of molecular signatures not only for the endometrium but also for the intricate dialogue at the level of the feto-maternal interface in cattle. Placentome morphogenesis potentially parallels the dynamic multigenic circuitry and regulates the cell cycle in the endometrium. The roles of BNCs and their secreted molecules remain an enigma, particularly with regard to the adhesion process and endometrial remodeling, which is the focus of this study.

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Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

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Flow cytometrically-sorted sperm has been involved in the production of sex preselected offspring. More than 30,000 bovine offspring have been produced using AI and other means using spermatozoa separated by flow cytometer. Flow cytometric sperm sorting based on differences in their DNA content is the best method for separation of X- and Y-chromosome bearing spermatozoa. At first, flow cytometers were modified for DNA confirmation and sorting of sperm with high resolution. The beveled insertion needle can regulate orientation of flat-shaped bull sperm heads. The forward fluorescence detector is essential for measuring the DNA content of sperm. Recently, high-speed sperm sorting with orienting nozzles has resulted in production of 90% pure X- and Y-sperm at rate of 15-20 million sperm per hour. Application of this new technique will enable conduct of more conventional technologies for both artificial insemination and cryopreservation in the bovine and in other farm animals using X- or Y-sperm.

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We reviewed recent in vivo studies of the real-time changes in the vasculature of the follicle wall during selection of the dominant follicle as well as during ovulation in cows. Changes in follicle diameter and vascularity were determined by transrectal ultrasonography. Blood flow within the walls of the two largest follicles was detected at the time of wave emergence (largest follicle=5 mm in diameter). Before selection of a follicle (largest follicle <8.5 mm in diameter), the degrees of vascularity of the two largest follicles were not significantly different. After the largest follicle reached a diameter of 10 mm, the vascularity of the largest (dominant) follicle was higher than that of the second largest (subordinate) follicle. In the preovulatory follicle, follicular vascularity gradually increased, and as ovulation approached, the LH-surge induced an increase in blood flow within the follicle wall. The above results suggest that maintenance of follicular vasculature and appropriate blood supplies to follicles are essential for establishment of follicular dominance. Consequently, only a dominant follicle with high vascularity may have a chance to reach final maturation and acquire ovulatory capacity.

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Estradiol and progesterone may play a role in controlling leptin secretion by utilizing their receptors in adipocytes and the genomic mechanisms of the leptin gene. This study was conducted to evaluate the effects of exogenous sex steroids on the blood leptin concentrations in ewes in the non-breeding season. Multiparous ewes were fed to maintenance level for their live weights. Blood samples were collected at 12-h intervals from Days -3 to -1 to determine the basal leptin levels (pre-injection period). From Day 0 to Day 5 (injection period), blood sampling continued at 12-h intervals, and the ewes were injected intramuscularly at 24-h intervals with oil, 50 mg progesterone in oil, 1 mg of estradiol in oil, or both steroids in oil. Leptin was measured using a sensitive and specific radioimmunoassay based on recombinant bovine leptin. Overall, plasma concentrations of leptin were not affected by any of the steroid treatments, and there were no differences in the value of leptin between the pre-injection and injection periods among the 4 groups. Therefore, the exogenous estrogen and progesterone used in this study do not have a strong effect on the blood leptin concentrations of ewes in the non-breeding season.

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The aims of the present study were to establish a culture system for goat skeletal muscle stem cells and to examine their myogenic and adipogenic properties in vitro. Cells were isolated from the skeletal muscle of the Shiba goat and cultured in vitro. Most of the cells were positive for myogenic markers, such as Pax7, MyoD, and desmin, and immunocytochemistry revealed they differentiated to form myotubes expressing myosin heavy chain, indicating they were highly myogenic. Myogenic differentiation was strongly suppressed by the addition of basic fibroblast growth factor, while proliferation was unaffected. When the cells were cultured in adipogenic differentiation medium, some of the cells differentiated into mature adipocytes that stained with Oil Red-O. These cells were immunocytochemically positive for adipogenic markers, including peroxisome proliferator-activated receptor-γ (PPAR γ) and CCAAT/enhancer-binding protein-α (C/EBP α). These results clearly demonstrate the presence of both myogenic and adipogenic stem cells in goat skeletal muscle.

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Interleukin-18 (IL-18) is a proinflammatory cytokine involved in chronic inflammation, autoimmune diseases, and a variety of cancers, and is expressed in mouse uteri. Our previous study suggested that IL-18 acts as a paracrine factor, regulating endometrial function. To elucidate the physiological roles of IL-18 in the mouse endometrium, the expression of the IL-18 receptor (IL-18R) α subunit was analyzed. IL-18Rα mRNA was expressed in several mouse organs in addition to the endometrium. In situ hybridization analysis using a biotin-labeled mouse IL-18Rα riboprobe demonstrated that IL-18Rα mRNA expression was detected in glandular epithelial cells, stromal cells around uterine glands, and myometrial cells in the mouse uterus, suggesting that these cells are targets for IL-18. The uterine IL-18Rα mRNA expression level changed with the estrous cycle. The uterine IL-18Rα mRNA levels of estrous mice were higher than those of diestrous mice. In addition, the IL-18Rα mRNA levels in uteri at 3 and 14 days after ovariectomy were higher than those at diestrus and decreased following treatment with estradiol-17β or progesterone. These findings suggest that IL-18Rα gene expression is regulated by estrogen and progesterone and that the uterine IL-18 system is involved in the regulation of uterine functions in a paracrine manner.

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The placenta is a highly differentiated organ essential for embryonic growth and development. In order to search for key molecules that are associated with mouse placental lactogen II (mPL-II) gene expression, we applied mouse cDNA microarray analysis to RNAs extracted from placentae on days 10, 12, 14, 16 and 18 of pregnancy. Changes in gene expression were categorized between days 10 and 12, 12 and 14, 14 and 16 and 16 and 18 of pregnancy. After microarray analysis, which had a minimum detectable fold change for differential expression of 2, we selected 10 genes, Apoa2, Apoc2, Ceacam14, Creg1, Fmo1, Igf2, Slc2a1, Spink3, Spi1-1 and Tpbpa, exhibiting a expression pattern similar to the mPL-II gene. Furthermore, we performed real-time PCR analysis and in situ hybridization (ISH) to find correlative expression genes for the mPL-II gene. From these results, we identified a resemblance in gene expression between mPL-II and Igf2 and selected these genes for performance of double-fluorescence immunohistochemical staining. We colocalized these proteins in labyrinthine trophoblast cells. These results strongly suggest that the expression of mPL-II and Igf2 is highly related to placental development in mice. This large-scale identification of genes regulated during placentogenesis assists in further elucidation of the molecular basis of extraembryonic development and function.

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The objective of this study was to determine changes in the transcription of insulin-like growth factor (IGF)-related genes in blastocyst (BC)- and elongated (EL)-stage embryos produced by nuclear transfer using somatic cells (NT-SC). Bovine BC (day 7)- and EL (day 15)-stage embryos were obtained from NT-SC or in vivo production (Vivo). The relative abundance of mRNA was examined by RT- real-time PCR. The transcript of IGF-II was only detected at the EL stage in both the NT-SC and Vivo embryos. The level of transcription of the IGF-I receptor (r) in the NT-SC embryos was decreased at the EL stage and was significantly (P<0.05) lower than at the BC stage. In contrast, the IGF-IIr levels did not differ significantly between the NT-SC and Vivo embryos, regardless of the developmental stage. IGF-binding protein (IGFBP)-2 levels were markedly decreased in the NT-SC and Vivo embryos at the EL stage (P<0.05). The IGFBP-3 level in Vivo was significantly (P<0.05) increased at the EL stage compared with at the BC stage. However, the IGFBP-3 levels in NT-SC embryos were unchanged and lower (P<0.05) than in the Vivo embryos at the EL stage. These results suggest that there are differences in the levels and changes in the transcription of IGF-related genes in bovine embryos produced by NT-SC compared with those produced by Vivo.

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Danazol, which has been used as a medicine for endometriosis, has a valid effect in pretreatment of patients receiving in vitro fertilization and embryo transfer, although its reproductive mechanism remains unclear. BALB/c mice were subcutaneously injected with danazol for 2 weeks. Blood and uteri were collected and cytokines were assayed. Following danazol treatment, an increase in pregnancy ratio was evident that was accompanied by up-regulation in serum macrophage-colony stimulating factor (M-CSF). RT-PCR analysis revealed that expression of M-CSF and Ly49, a phenotypic marker of natural killer (NK) cells, was up-regulated in the uteri of the danazol-treated mice. In immunohistochemical analysis, M-CSF and Ly49, together with α5 integrin, were clearly detected in the endometrium of the danazol-treated mice with very similar pattern of localization. These results suggest that danazol has an effect to promote pregnancy that induces recruitment of NK cells and a concomitant increase in the expression of M-CSF and α5 integrin in the uterus.

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In this study, we introduced a co-transfection method for the selection of donor nuclei in somatic cell-mediated nuclear transfer. Two vectors were constructed in our experiment. One was pMSCV-GFP carrying the neomycin-resistant gene (Neo^r) and the green fluorescent protein (GFP) reporter gene; the other was pBC1-GFP carrying the mammary gland-specific promoter and target gene GFP. Ovine adult fibroblasts were co-transfected with pMSCV-GFP and pBC1-GFP. The data from this work demonstrated that the GFP genes in both vectors could successfully co-integrate into the genomes of ovine adult fibroblasts in three of the four transgenic cell clones assayed. Furthermore, PCR analysis of transgenic embryos proved that the GFP genes in both vectors could co-integrate into the genomes of the reconstructed embryos. Subsequently, analysis of the developmental rate of the reconstructed embryos after nuclear transfer indicated that the blastocyst rate from the co-transfected donor cells was similar (approximate 8 percent) to that from individual pMSCV-GFP transfected donor cells. The influence of co-transfection resulting in modification of donor nuclei on development of reconstructed embryos was also investigated. The results of flow cytometric analysis indicated that the co-transfected ovine fibroblasts had similar quiescent characteristics in terms of cell cycle (G0+G1 percent: 73.20 ± 4.04) to the individual pMSCV-GFP transfected fibroblasts (G0+G1 percent: 70.77 ± 1.19) after they were treated with serum starvation for five days. Our results suggest that the co-transfection method can be used for selection of donor cell clones in somatic cell-mediated gene transfer experiments. It can be potentially extended to applications related to expression of functional protein in mammary glands and other transgenic research relevant to nuclear transfer.

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Vascular endothelial growth factor (VEGF) isoforms (VEGF 120 and VEGF 164) secreted by granulosa cells are involved in thecal angiogenesis during follicular development in the bovine ovary. However, whether the transcript of the VEGF120 and VEGF164 isoforms differs during follicular development in the ovary is still unknown. We first examined the gene expression of VEGF120, VEGF164, fms-like tyrosine kinase (Flt-1), and fetal liver kinase (Flk-1) in the granulosa cells (GCs) and theca cells (TCs) of pre-selection and post-selection follicles (PRF and POF respectively) from the bovine ovary. Then we examined the effects of FSH and estradiol (E2) on these factors in cultured bovine GCs. Messenger RNA (mRNA) expression was quantified using real-time PCR methods. The concentrations of E2 and P4 in the follicular fluid (FF) of the PRF and POF were estimated using an enzyme immunoassay (EIA). The concentrations of E2 and P4 in the FF were significantly higher in the POF than in the PRF. The ratio of E2/P4 in PRF and POF was 0.37 and 3.8, respectively. The expression levels of the VEGF120, VEGF164, and Flk-1 mRNAs in the GCs of POF with high E2 concentration were higher than those of PRF. The levels of the Flt-1 and Flk-1 mRNAs in the TCs were not different between PRF and POF. Since E2 in the FF of the POF used in the present study was high compared with the PRF, we examined the effects of E2 and FSH on the expression of the above genes using cultured GCs. Expression of VEGF120 mRNA was induced by a low concentration (1 ng/ml) of E2, whereas the levels of VEGF164 and Flk-1 mRNAs were not affected by E2. FSH stimulated the expression of the VEGF isoforms and Flk-1 genes. Moreover, the expression of those genes was enhanced when low E2 (1 ng/ml) was added to FSH. In conclusion, our data indicates that the VEGF isoforms have a follicle stage-dependent expression pattern. Thus, our results suggest that the expression of VEGF isoforms may be associated with characterization of the preovulatory phenotype during follicle development in the bovine ovary.

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Metabolic hormones affect ovarian function in the cow. However, the relationship between metabolic factors and ovarian function is not clear in the postpartum primiparous cow because they are still growing. The aim of the present study was to investigate in detail the time-dependent profile of the metabolic hormones, metabolites, and milk yields of ovulatory and anovulatory primiparous cows during the first follicular wave postpartum. We used 16 primiparous Holstein cows and obtained blood samples for the profiles of metabolites (glucose; non-esterified fatty acid, NEFA; ketone body; total cholesterol; and aspartate aminotransferase), metabolic hormones (growth hormone, GH; insulin-like growth factor-I, IGF-1; and insulin), and progesterone every other day from 1 to 21 days postpartum. In addition, all ovaries were observed using ultrasound. Dairy milk yield was recorded during the experimental period. In all cows, the first follicular wave postpartum was observed and 6 of the cows ovulated. The plasma glucose (P<0.0001) and IGF-1 (P<0.001) concentrations were lower and the plasma NEFA (P<0.0001) and ketone bodies (P<0.0001) concentrations and daily milk yield (P<0.0001) were higher in the anovulatory cows compared to the ovulatory cows. However, the GH levels, which enhance lipolysis for milk production, insulin and other metabolites did not differ between the two groups. In conclusion, the present study suggests that anovulation of the dominant follicle during the first follicular wave postpartum in primiparous cows is induced by low IGF-1 levels that are similar to those of multiparous cows. In addition, anovulatory cows are likely to mobilize body fat stores for milk production more easily than ovulatory cows.

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Stress due to summer heat has adverse effects on reproduction in Holstein dairy cattle. Summer suppression of reproduction of Holsteins can pose an important economic problem, even in Hokkaido prefecture located in the northern region of Japan. Hokkaido is one of the most important dairy farming areas of Japan. This study is an attempt to clarify the seasonal differences in the parameters of luteinizing hormone (LH) response to exogenous gonadotropin releasing hormone (GnRH) in Sapporo, Hokkaido, Japan. A total of 12 prepubertal heifers received an injection with GnRH analogue intramuscularly in either May (n=4, May group), July (n=4, July group), or November (n=4, November group), and serial blood samples were collected to analyze the parameters of the LH response curve after GnRH injection. The parameters were as follows: the basal LH concentration, peak LH concentration, duration from the time of GnRH injection to the time of the peak LH concentration, and area under the LH response curve (AUC). There were no significant differences in the basal and peak LH concentrations or the AUC among the three groups. The July group reached the LH peak significantly (P<0.05) faster than the May group, but there was no significant difference with the November group. Therefore, the results of the present study do not demonstrate an effect of summer heat on the LH response to the exogenous GnRH in Holstein heifers.

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Cyclic adenosine 3',5'-monophosphate (cAMP) signaling regulates the expression of fertilizing ability in mammalian spermatozoa. Many articles indicate that this signaling is mediated mainly via protein kinase A. Recently, a guanine nucleotide exchange factor for small G protein Rap1 (an exchange protein directly activated by cAMP: Epac) was discovered as a new mediator of cAMP signaling in somatic cells. The aim of this study was to reveal the existence of cAMP-Epac signaling in mouse spermatozoa. Northern blot analysis and in situ hybridization suggested that Epac1 and Epac2 mRNAs were transcribed in the seminiferous epithelia of the testis. This shows that expression of Epac mRNAs is present in mouse testicular germ cells. Indirect immunofluorescence with specific polyclonal antibodies suggested possible co-localization of Epac1 and Rap1 proteins in the heads of epididymal spermatozoa. Moreover, treatment of epididymal spermatozoa with an Epac-specific cAMP analog, 8-pMeOPT-2'-O-Me-cAMP, induced activation of Rap1, as revealed with a commercial kit for pull-down assay. These results indicate the existence of cAMP-Epac signaling in the heads of mouse epididymal spermatozoa.

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In the present study, the growth performance of a calf produced by mating a somatic cell cloned dam and sire was compared with that of its full siblings produced by mating the cattle used as nuclear donors for the cloned animals. The somatic cell cloned dam and sire were derived from cultured cumulus cells and ear cells, respectively. The cloned dam was artificially inseminated with semen from the cloned sire. A female calf was produced that was reared under general group feeding conditions. The calf was subjected to a clinical examination and to hematology, serum biochemistry, and telomere length analyses; all of these tests indicated that the calf was normal. The growth characteristics (body weight and shoulder height) of the calf fell within the range of the full siblings of the same sex produced by mating the animals used as the nuclear donors of clones. These findings suggest that the same breeding performance is expected from mating a cloned dam and sire as from mating the animals used as nuclear donors for the clones.

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The aim of the present study was to establish a simple method to monitor ovarian activity and non-invasively diagnose pregnancy in okapi (Okapia johnstoni). The feces of a female okapi were collected daily or every 3 days for 28 months. Steroids in lyophilized feces were extracted with 80% methanol, and the fecal levels of immunoreactive progestagens (progesterone and pregnanediol-glucuronide), androgen (testosterone), and estrogens (estradiol-17β and estrone) were determined by enzyme immunoassays with commercially available antisera. Using the progesterone profiles, the durations of the luteal phase, follicular phase, and estrous cycle were determined to be 11.1 ± 0.4, 5.3 ± 0.6, and 16.5 ± 0.7 days (n=22), respectively. Fecal levels of immunoreactive progesterone, pregnanediol glucuronide, and testosterone gradually increased from early pregnancy and peaked several months before parturition. More pregnanediol glucuronide was excreted in feces than progesterone during late pregnancy, but not during the estrous cycle. Although the fecal concentrations of immunoreactive estradiol-17β and estrone change a little throughout pregnancy and non-pregnancy, they rose sharply and temporarily on the day following parturition. The present study indicates that fecal assays with commercial antisera for progesterone and pregnanediol glucuronide are useful for evaluating luteal activity and diagnosing pregnancy and indicates that estrogens might have some role as a trigger of parturition.

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F-spondin/vascular smooth muscle cell growth-promoting factor (VSGP), purified from the follicular fluid of adult bovine ovaries, has been identified as a promoter of neuronal differentiation and vascular smooth muscle growth. The objectives of the present study were (1) to clarify whether F-spondin is also produced in the testis, which is ontogenically equivalent to the ovary, and (2) to examine whether production of this protein changes with testicular growth. To isolate F-spondin from the testis, testicular homogenates obtained from 8-week-old boars were sequentially subjected to heparin-Sepharose chromatography, diethylaminoethyl (DEAE)-Sepharose chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The isolated protein had a molecular mass of approximately 110 kDa and was cross-reactive with anti-F-spondin antibody by Western blotting. The purified protein was further characterized by amino acid sequence analysis of its internal peptide. The sequence obtained was GEQCNIVPDN VD, and a homology search indicated that the purified protein is a homologue of rat, human, and bovine F-spondin. By fractionation of the same amounts of testis tissue obtained from 1-, 8-, 16-, and 40-week-old boars, we analyzed age-related production of F-spondin in the testis. Western blotting of the fractions obtained from RP-HPLC revealed the presence of a band at approximately 110 kDa, corresponding to F-spondin, in the testes obtained from boars between 1 and 16 weeks old, but this band was not detected at 40 weeks. These results clearly indicate that (1) the porcine testis produces F-spondin and that (2) production of this protein is evident in the immature porcine testis, but not the adult testis.

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The present study was carried out to measure fecal progestagen and estrone concentrations during pregnancy in a giraffe and examine the possibility of utilizing this assay system for pregnancy diagnosis. Fecal samples were collected from a giraffe during her third and fourth parities and mixed with methanol to prepare a fecal solution. Diluted fecal solution was used for direct enzyme immunoassay for progestagen and estrone. The newborn calf from the third parity was viable, although that from the fourth parity died 5 days after calving. In the third parity, the giraffe's progestagen and estrone concentrations increased transiently from days 30 to 120 of pregnancy. Then, they decreased and remained low until day 330. This was followed by a drastic rise in both concentrations as parturition approached. Parturition caused a reduction in the progestagen and estrone concentrations of the feces. In the fourth parity, the progestagen concentration increased gradually after mating until day 320. This was followed by a reduction in the concentration until parturition. However, the estrone concentration fluctuated, and the duration and extent of the prepartum rise in concentration were shorter and lower than those of the third parity. The hormone dynamics of the third parity suggest the possibility of early pregnancy diagnosis by measuring progestagen or estrone between days 30 and 120 after mating.

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Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.

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