Dedifferentiation of differentiated cells such as fibroblasts into pluripotent stem cells, so-called iPS cells, was first reported by Yamanaka et al., who successfully employed retroviral gene delivery of four stem-cell-specific transcription factors (Oct-3/4, Klf4, Sox2 and c-myc). Despite the mouse system in which an Oct-3/4 or Nanog promoter-based reporter system has already been established, there is no useful system in pigs for reporting the reprogramming state of gene-engineered cells. In this study, we constructed a pOEIN plasmid carrying a ca. 5.4-kb mouse Oct-3/4 promoter linked to the EGFP cDNA and neomycin expression unit and produced a porcine embryonic cell line stably incorporating it in the genome. Cell fusion with mouse embryonal carcinoma cell line F9 resulted in generation of colonies with distinct EGFP-derived fluorescence around 14 days after fusion. RT-PCR using these colonies also confirmed expression of endogenous porcine pluripotency-specific Oct-3/4, Sox2 and Stat3 mRNA. These findings suggest that mouse-derived components are sufficient to induce dedifferentiation of differentiated pig cells and also that reprogramming proceeds gradually. The present non-invasive reporter system will be useful to better define the reprogramming mechanism and/or to identify novel reprogramming molecules in the pig.

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In the adult rat, the superior spermatic nerve (SSN) and inferior spermatic nerve (ISN) are involved in regulating testosterone secretion and spermatogenesis, in addition to endocrine control mechanisms. However, there are currently few data on how the testis nerve supply regulates testicular development and related mechanisms. The present study was thus designed to investigate the regulating effects of testis nerve supply to testicular maturation, spermatogenesis and the involved mechanisms from prepuberty to adulthood in rats. We transected the SSNs and ISNs of rats on postnatal day (PD) 30 and then analyzed changes in testicular morphology and cauda epididymal sperm content, cell proliferation and apoptosis and primary spermatocyte meiosis on PD60 and PD90. The results demonstrated that testicular denervation significantly reduced testis mass, cauda epididymal sperm counts and serum testosterone concentrations. Proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 immunohistochemistry staining proved that the denervation had no influence on the proliferation of spermatogonia and primary spermatocytes, but obviously promoted the apoptosis of round spermatids and Leydig cells. It is novel that denervation reduced the meiotic activation of zygotene and pachytene spermatocytes through the expression of synaptonemal complex protein 3 (SCP3)-a marker of meiosis. In addition, RT-PCR showed that testis denervation significantly decreased testis 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1) and luteinizing hormone receptor (LHR) mRNA levels, but had no obvious influence on testis follicle stimulating hormone receptor (FSHR) mRNA expression. These results suggest that the testicular nerve supply plays an important role in supporting seminiferous tubule development and spermatogenesis from prepuberty to adulthood.

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Histone H3 trimethylation on lysine 27 is one of the histone modifications associated with chromatin of silenced regions. H3K27me3 labeling is initially asymmetrical between pronuclei in mammalian embryos, and then it is remodeled during early development. However, in mouse embryos obtained after somatic cell nuclear transfer (SCNT), H3K27me3 histones inherited from the somatic female cell and associated with X chromosome inactivation have been reported to escape remodeling. Using immunostaining, we investigated the remodeling of H3K27me3 in Bos taurus embryos obtained after in vitro fertilization (IVF) and SCNT. In this species, transfer-induced chromatin remodeling can be clearly separated from embryonic genome activation (EGA), which occurs at the 8-16-cell stage, and cloning by SCNT is 10 times more successful than in the mouse. In early IVF bovine embryos, dense H3K27me3 labeling was localized in the pericentric heterochromatin as recently described in the mouse. Labeling was however unevenly distributed up to the 8-cell stage, suggesting that the parental genomes partitioned before EGA. In female IVF blastocysts, a somatic-like female profile appeared in 21% of the trophoblast cells. This profile, which had one major nuclear H3K27me3 patch, the putative inactive X chromosome (Xi), was absent in male blastocysts. In contrast, the somatic-like female H3K27me3 profile was observed in the majority of the nuclei of female bovine SCNT embryos before EGA. At the 8-16-cell stage, this profile was transiently replaced by pericentric-like labeling in most nuclei. Immunostaining of mitotic chromosomes suggested that the ratio of H3K27me3 labeling in pericentric heterochromatin vs. euchromatin was then rapidly altered. Finally, Xi-like H3K27me3 staining appeared again in trophoblast cells in female SCNT blastocysts. These results suggest a role for EGA in H3K27me3 remodeling, which affects the heterochromatin inherited from the donor cell or produced during development.

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Transforming growth factor beta 1 (TGF-β1) modulates male reproductive function. Genetically modified mice overexpressing α/β subunits of hCG (hCG+) show Leydig cell hyperplasia/hypertrophy at prepuberty that disappears as the mice approach adulthood. In this study we analyzed the gene expression of TGF-β1, its specific receptors, type II (TGF-βRII) and type I (activin receptor-like kinase 1 and 5: ALK1 and ALK5), and co-receptor endoglin (CD105) in purified Leydig cells from hCG+ and wild-type mice at 3 and 8 weeks of age and the occurrence of TGF-β1, ALK1 and ALK5 by immunohistochemistry. The expression of TGF-β1 was higher in hCG+ mice at both ages studied, and no changes were observed in TGF-βRII. ALK5 diminished with age in wild-type mice, whereas ALK1 decreased in hCG+ mice at 8 weeks of age. Endoglin expression showed a marked increase in 3-week-old hCG+ animals. In vitro incubation of Leydig cells from wild-type animals with hCG (10 IU/ml) increased TGF-β1 and ALK5 expression. Progesterone (10^-6 M) induced endoglin expression. These studies provide novel evidence for differential gene and protein expression of ALK1 and ALK5 at different ages and endoglin expression and hormonal, in purified Leydig cells.

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The aim of this study was to investigate whether seasonal changes affected in vitro developmental competence of porcine oocytes. The relationship between atmospheric temperature and embryonic development of in vitro matured porcine oocytes following intracytoplasmic sperm injection was examined throughout the year. The blastocyst rate (31.1%) in winter (mean atmospheric temperature during December to February: -3.8 C) was significantly higher (P<0.05) than those of other seasons in 2008/2009 (19.7-23.5%; 6.3-17.5 C). The monthly mean blastocyst rates were negatively correlated with the temperatures (r=-0.5944, P<0.05). The results of the present study suggest that porcine embryos could be produced throughout the year, but the in vitro production efficiency was significantly affected by season, i.e., atmospheric temperatures. Furthermore, the results showed that winter is a favorable season for blastocyst production in the region of Obihiro, Hokkaido, Japan.

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Several reports indicate that transforming growth factor β1 (TGF-β1) participates in the regulation of cell cycle progression. In this work, we analyzed the in vitro effect of TGF-β1 on Leydig cell proliferation markers and the in vivo effect of this cytokine in Leydig cell hyperplasia/hypertrophy. The in vitro effect of TGF-β1 (1 ng/ml) plus progesterone (10^-6 M) on purified Leydig cells from 3 week-old mice increased the immunocytochemically detected PCNA and stimulated the phosphorylation of Smad 1/5. Progesterone (10^-6 M) in the presence or absence of TGF-β1 diminished the ratio Bax/Bcl-2. Morphometric testicular studies of mice treated with progesterone (s.c.) plus TGF-β1 (intratesticular), showed an increase in interstitial volume and a decrease in tubular volume. Furthermore, the cytoplasmic volume of Leydig cells showed an increment in this experimental group with a diminution in nuclear volume. Thus, it turned out that the administration of progesterone and TGF-β1 augmented the volume of Leydig cells. These results indicate a clear effect of TGF-β1 in the hypertrophy/hyperplasia of Leydig cells.

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This study was performed to examine the distribution of single nucleotide polymorphisms (SNPs) and estimated haplotypes in the canine estrogen receptor (ER) α gene (ESR1) and the association of them with different phenotypes of cryptorchidism (CO) in Miniature Dachshunds and Chihuahuas. Forty CO and 68 normal dogs were used, and CO was classified into unilateral (UCO; n=33) and bilateral CO (BCO; n=5) or into abdominal (ACO; n=16) and inguinal CO (ICO; n=22). Thirteen DNA fragments located in the 70-kb region at the 3' end of ESR1 were amplified by PCR and sequenced to examine 13 SNPs (#1-#13) reported in a canine SNP database. Ten SNPs (#1-#4, #7, #8, #10-#13) were not polymorphic, and 5 new SNPs (#14-#18) were discovered. A common haplotype block in normal, CO and CO phenotypes was identified for an approximately 20-kb region encompassing 4 SNPs (#14-#17). Allele, genotype and haplotype frequencies in CO without classification by phenotype and also in UCO, ACO and ICO phenotypes were not statistically different from the normal group. Significant differences in genotype frequencies and homozygosity for the estimated GTTG haplotype within the block were observed in BCO compared with the normal group, although the number of BCO animals was small. Our results demonstrate that the examined SNPs and haplotypes in the 3' end of canine ESR1 are not associated with unilateral, abdominal and inguinal CO phenotypes and CO per se in Miniature Dachshunds and Chihuahuas. Further studies are necessary to suggest a clear association between the ESR1 SNPs and bilateral CO in dogs.

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We have previously documented synthesis of lysophosphatidic acid (LPA) in the bovine endometrium and the increased presence of LPA receptor mRNA expression during pregnancy. Therefore, LPA could contribute to early pregnancy establishment in the cow. In the present study, we investigated the effect of intravaginally administered LPA on pregnancy rates and on the plasma levels of progesterone (P4) and prostaglandins (PGs) in heifers. Animals were inseminated and from day 15 to 18 after estrus were treated intravaginally with saline, LPA (1 mg) or LPA receptor blocker (VPC32183; 1 mg). Blood samples were collected on days 0, 6, 12, 15, 16, 17, 18 and 21 after insemination. Pregnancy was confirmed by ultrasonography and per rectum examination on days 30 and 49-50 after insemination. Intravaginal LPA administration increased the plasma P4 and PGE₂ concentrations compared with saline and VPC32183-treated heifers. In the saline and LPA-treated groups, 6 out of 8 heifers were pregnant (75%), whereas the pregnancy rate in the VPC32183-treated heifers was only 37%. We also examined the effects of LPA on PG secretion and PG synthase mRNA expression in stromal and epithelial cells of the bovine endometrium on days 16-18 of pregnancy and the estrous cycle. LPA increased PGE₂ production and PGE₂ synthase (PGES) mRNA expression in stromal cells during the estrous cycle and pregnancy. On Days 16-18 of pregnancy, LPA inhibited PGF_2α production and PGFS mRNA expression in epithelial cells. The results suggest that LPA serves as a luteotropic factor during the estrous cycle and pregnancy, stimulating P4 secretion in vivo and PGE₂ secretion in vitro through activation of PGES mRNA expression in stromal cells. Moreover, during the early pregnancy, LPA decreases PGF_2α synthesis and mRNA expression for PGFS in epithelial cells of the bovine endometrium.

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The aims of this study were to determine whether a single treatment of estradiol dipropionate (EDP) could induce pseudopregnancy in gilts and to determine the effectiveness of PGF_2α treatment on estrus synchronization in EDP-induced pseudopregnant gilts. In experiment 1, gilts were treated with 20 mg of EDP (n=11) or vehicle (n=5) on Day 12 (Day 0=onset of estrus). Establishment of pseudopregnancy was defined as a lack of estrus and maintenance of the plasma progesterone concentration above 1 ng/ml between Days 12 and 36. Nine of 11 gilts (82%) treated with EDP became pseudopregnant. The plasma estradiol-17β level was significantly higher in the EDP-treated gilts than in the control gilts until Day 29. In experiment 2, PGF_2α was administered twice with a 24-h interval from Day 36 in pseudopregnant gilts (n=6) or Day 10 in cyclic gilts (control; n=5). Estrus after PGF_2α treatment was observed in 83% of the pseudopregnant gilts. The interval from the day of the first PGF_2α treatment to the onset of estrus and the peak of the LH surge was significantly shorter in the pseudopregnant gilts than in the control gilts. In experiment 3, six pseudopregnant gilts were bred by artificial insemination at the estrus after PGF_2α treatment. The farrowing rate and average litter size did not differ between the PGF_2α-treated pseudopregnant and cyclic gilts. These results indicate that a single treatment of EDP on Day 12 of the estrous cycle can induce pseudopregnancy in pigs and that a convenient protocol for administering PGF_2α to EDP-induced pseudopregnant pigs is available for estrus synchronization programs in cyclic pigs.

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Administration of prostaglandin (PG) F_2α in cattle during the mid-luteal phase (Days 8-12 of the estrous cycle) drastically reduces the plasma progesterone concentrations and the volume of the corpus luteum (CL). However, PGF_2α does not induce luteolysis during the early luteal phase (up to Day 5 of the estrous cycle). To characterize the possible distinct difference in acute response to a luteolytic dose of PGF_2α administration, we determined various mRNA expressions in the early and mid CL relating to angiogenesis, vasoactivation and PG-related factors at 30 min after PGF_2α injection in cyclic cows. The experiments were conducted on Day 4 (early CL) and Days 10-12 (mid CL). Cows were either injected with 500 μg PGF_2α analogue or saline as the control (early CL control, n=5; early CL PGF_2α treated, n=5; mid CL control, n=5; mid CL PGF_2α treated, n=7). Thirty min after injection of PGF_2α or saline, the cows were ovariectomized transvaginally, and the CL tissues were collected from regions designated as the periphery and center of the CL. Administration of PGF_2α up-regulated the mRNA expressions of angiogenic-related factors such as vascular endothelial growth factors, vasohibin, fibroblast growth factor 2 and insulin-like growth factor-II in the early CL, whereas PGF_2α down-regulated these mRNA expressions in the mid CL. In the vasoactive factors, PGF_2α stimulated the mRNA expressions of endothelin-1, angiotensin converting enzyme, endothelial nitric oxide synthase (NOS) and inducible NOS in the periphery area of the mid CL, but not in the early CL. However, PGF_2α drastically down-regulated PGF_2α receptor mRNA expression in both regions of the early and mid CL. The results indicated a clear difference in the acute action of PGF_2α depending not only on the luteal phase (immature vs. mature) but also the region (periphery vs. center) within the CL at 30 min after PGF_2α injection in the cow.

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Motility and fertilizing ability are known to be two important physiological attributes of a mature sperm, yet the mechanism by which spermatozoa mature and become motile remains largely unknown. It has been shown that nerve growth factor (NGF) is a protein essential for the development, maintenance and survival of the peripheral and central nervous systems. However, the presence of high levels of NGF protein and mRNA do not correlate with the innervations by NGF sensitive fibers in tissues such as the testis, prostate and seminal vesicles. These observations have shifted the attention of research to the role of NGF outside of the nervous system. Here, we demonstrate that NGF and its receptors TrkA and p75 are widely expressed in the testis, accessory reproductive organ, and the epididymal sperms. We also show that NGF stimulates two important aspects of sperm functions, motility and the acrosome reaction, in a time- and dose-dependent manner. NGF activated the sperm cell acrosome reaction, while addition of inhibitors specific for MAPK kinase significantly blocked the sperm acrosome reaction. Taken together, our findings suggest that NGF plays an integral role in sperm motility and the acrosome reaction through, at least in part, the MAPK signalling pathway.

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This study was conducted to determine uterine echogenicity by grey-scale analysis (GSA) and transcutaneous ultrasonography in pregnant sows (P-sows; n=16) and gilts (P-gilts; n=13) vs. cyclic gilts (C-gilts; n=9) between days 8 and 16 post ovulation (po) with the aims of testing for feasibility of uterine GSA and of gathering reference data. Estruses and ovulations were hormonally synchronized and the animals artificially inseminated. Ovulation was monitored by ultrasound. The equipment used was a HS 2000 ultrasound unit and a 5 MHz linear probe. Unit settings were standardized for all GSA scanning sessions and the animals crated during scanning. For GSA, cross-sections of the uterine horns were imaged, entirely defined as regions of interest, and pixel analyses done. A total of 342 scanning sessions were performed, 341 GSA accomplished, and 1-13 cross-sections analyzed per session. Comparison of coefficients of variation suggests that analysis of two cross-sections per session is sufficient for a reliable GSA per animal. P-sows and P-gilts were similar in their echogenicity course, but differed from C-gilts. Most noticeable, echogenicity declined in pregnant animals on day 12 po, while it increased in cyclic gilts. In conclusion, the study demonstrates that GSA using transcutaneous ultrasound is a feasible procedure for the determination of uterine echogenicity in the pig, and that pregnant and cyclic pigs differ in the uterine echogenicity, particularly during the time when maternal recognition of pregnancy occurs.

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The aim of this study was to evaluate the prevalence of follicles with more than one oocyte (polyovular follicles, POFs) in mouse lines selected for high fecundity. The ovaries of 18 mice, 6 each from 3 different lines, were examined to evaluate the number of POFs and the follicular histology. Polyovular follicles were observed in the two high fecundity breeds, FL1 and FL2, as well as in the unselected control line, DUKsi. The highest number of POFs per ovary (27.0 ± 7.2) was found in the FL1 line. The FL2 and DUKsi lines had 1.9 ± 0.7 and 0.6 ± 0.3 polyovular follicles per ovary, respectively. Most of the POFs contained 2 oocytes (>80%), but occasionally follicles containing up to 7 oocytes were observed. Follicles with more than 2 oocytes were observed in the FL1 line only.

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Prostaglandin F_2α (PGF) induces a rapid reduction in progesterone production (functional luteolysis) followed by tissue degeneration and cell death (structural luteolysis). Reactive oxygen species (ROS) including nitric oxide (NO) play crucial roles in the luteolytic action of PGF. The local concentration of intraluteal ROS is controlled by superoxide dismutase (SOD), the main enzyme involved in the control of intraluteal ROS. To clarify the roles of NO in the regulation of SOD in luteolysis, we examined the effects of NO on SOD expression and activity in cultured bovine luteal endothelial cells (LECs) during short-term (2 h, mimicking functional luteolysis) and long-term (24 h, mimicking structural luteolysis) incubation. We also investigated whether NO modulates PGF production by LECs. LECs were isolated from mid-luteal phase CLs, and exposed to NONOate (a NO donor) for 2 or 24 h. SOD mRNA expression was stimulated by NONOate (10-100 μM) at 2 h (P<0.05). Moreover, 10 μM NONOate stimulated SOD protein expression and SOD activity at 2 h (P<0.05), whereas NONOate inhibited SOD mRNA and protein expressions at 24 h (P<0.05). NONOate stimulated PGF biosynthesis at both incubation times. The overall findings suggest that NO differently regulates SOD in cultured LECs, depending on the exposure time. Acute elevation of SOD may represent a response of LECs to protect themselves against oxidative stress induced by PGF during functional luteolysis, whereas a later reduction of SOD levels by NO may facilitate an excess of intraluteal ROS during structural luteolysis.

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In this study, two successive field trials were conducted during the non-breeding season to investigate various factors affecting on fertility of Suffolk ewes after intrauterine insemination with frozen-thawed semen. In the first year (Experiment 1), three sperm numbers per insemination dose (0.25, 0.5 and 1 million sperm) and five sheep farms were used, and in the second year (Experiment 2), parity, age, body weight, body condition score (BCS) and postpartum days were investigated to compare pregnancy and lambing rates. High pregnancy and lambing rates (70.6 and 70.6%, respectively) were obtained with 0.25 million sperm per dose. There were no significant differences in the pregnancy and lambing rates among the five farms, but there was a tendency for one farm to have higher pregnancy (75.8%, P=0.065) and lambing (72.7%, P=0.077) rates than those (46.7-53.3% and 45.2-53.3% for the pregnancy and lambing rates, respectively) of the other farms. In Experiment 2, ewe age significantly affected both the pregnancy and lambing rates. Nulliparous ewes had a higher lambing rate (72.0%) than that (44.2%) of multiparous ewes, but a significant difference was not revealed. Regardless of body weight, BCS tended to be an important factor influencing on fertility of ewes. Body weight and the postpartum days did not affect the fertility of ewes. It was concluded from these results that the fertility of Suffolk ewes after intrauterine insemination with frozen semen was significantly influenced by sperm number per dose and ewe age. Nulliparous ewes at less than three years of age and with a BCS of more than 3.0 are expected to have higher fertility than other ewes.

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During follicular development in mammalian ovaries, the majority of follicles undergo atresia. One of the characteristics of this process is apoptotic cell death in granulosa cells. Several death ligands and receptors, including Fas ligand (FasL) and Fas, have been detected in ovarian follicles and also demonstrated to be capable of inducing apoptosis in follicular cells. Decoy receptor 3 (DcR3) competes with Fas to bind FasL but lacks intracellular death domains, thus inhibiting the induction of apoptosis by the FasL/Fas system. In the present study, we examined the expression of putative porcine DcR3 (pDcR3) mRNA in porcine ovarian follicles. Total RNA was extracted from granulosa cells and thecal layer cells of tertiary follicles at healthy, early atretic and progressed atretic stages, and the expression of pDcR3 mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR). The nucleic acid sequence in the coding region had 80% homology to that of human DcR3, and the deduced amino acid sequence was 73% identical to that of human DcR3. In an in situ hybridization experiment, pDcR3 mRNA expression was confirmed in granulosa and thecal layers, in both healthy and atretic follicles. Quantitative real time RT-PCR analysis showed that the expression of pDcR3 mRNA was weaker in granulosa cells of atretic follicles than those of healthy follicles. No notable changes were seen in the thecal layer cells. These results suggest that DcR3 plays a significant role in the regulation of apoptosis in granulosa cells, but not in thecal layer cells, during atresia.

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We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 μl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle.

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