Animal cloning technology has been developed to produce progenies genetically identical to a given donor cell. However, in nuclear transfer protocols, the recipient oocytes contribute a heritable mitochondrial genomic (mtDNA) background to the progeny. Additionally, a small amount of donor cell-derived mitochondria accompanies the transferred nucleus in the process; hence, the mtDNAs of two origins are mixed in the cytoplasm (heteroplasmy) of the reconstituted oocyte. Herein, I would like to introduce some of our previous results concerning five key considerations associated with animal cloning, including: mtDNA heteroplasmy in somatic cell nuclear transferred (SCNT) animals, the variation in the transmission of mtDNA heteroplasmy to subsequent generations SCNT cows and pigs, the influence of mtDNA sequence differences on mitochondrial proteins in SCNT cows and pigs, the effects of the introduction of mitochondria derived from somatic cells into recipient oocytes on embryonic development, and alterations of mtDNA heteroplasmy in inter/intraspecies nuclear transfer embryos.

The incidence of male reproductive system disorders, especially hypospadias, has been increasing in developed countries since the latter half of the 20th century. Endocrine-disrupting chemicals from the environment are considered to be involved in hypospadias onset through epigenetic alterations. This pilot study aimed to explore disease-specific methylated CpGs in human patient samples using the methylated-site display-amplified fragment length polymorphism (MSD-AFLP) technique developed by our research group [1]. We compared clinical samples from hypospadias and phimosis patients. Foreskin and blood samples were collected from one- to two-year-old patients with hypospadias (N = 3) and phimosis (N = 3) during surgical treatment. MSD-AFLP analysis showed significantly decreased CpG-methylation levels of genes such as MYH11 and increased CpG-methylation levels of genes such as PLA2G15 in hypospadias patients. Hierarchical clustering analysis showed that genes with significantly altered CpG levels were more markedly altered in DNA from blood than from foreskin. Because of the small number of samples, further investigation is necessary to elucidate the association between variations in CpG levels in foreskin and blood DNA and male genital abnormalities. However, our MSD-AFLP method appears to be a useful tool for exploring disease-specific methylated-CpGs in human epidemiological studies.

L-carnitine (LC) is well known for its antioxidant activity. In this study, we explored the potential mechanistic effects of LC supplementation on aged bovine oocytes in vitro. We showed that in-vitro maturation could enhance the subsequent developmental capacity of aging oocytes, when supplemented with LC. After in vitro fertilization, the blastocyst formation rate in the aged oocytes post-LC treatment significantly increased compared to that in untreated aged oocytes (29.23 ± 2.20% vs. 20.90 ± 3.05%). Furthermore, after LC treatment, the level of intracellular reactive oxygen species in aged oocytes significantly decreased, and glutathione levels significantly increased, compared to those in untreated aged oocytes. Mitochondrial membrane potential, the percentage of early apoptotic oocytes, and caspase-3 activity were significantly reduced in LC-treated aged oocytes compared to those in untreated aged oocytes. Furthermore, during in vitro aging, the mRNA levels of the anti-apoptotic genes, Bcl-xl and survivin in LC-treated aged oocytes were significantly higher than those in untreated aged oocytes. Overall, these results indicate that at least in in vitro conditions, LC can prevent the aging of bovine oocytes and improve the developmental capacity of bovine embryo.

This study assessed the effects of two hormones, human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone (GnRH), on ovulatory responses during different diestrous stages in lactating dairy cows. Estrous cycles of 21 cows were synchronized and were enrolled in stage 1 of the experiment. The cows were treated with a prostaglandin (PG) F_2α analog either 9 to 10 days [mid-diestrus (MD) group] or 5.5 to 6.5 days [early-diestrus (ED) group] after synchronized ovulation (day 0 = first PGF_2α administration). On day 2, the cows were administrated 250 μg GnRH or 3000 IU hCG. Ovulation was determined every 2 h from 24 to 36 h after GnRH or hCG administration, and then every 4 h up to 72 h until ovulation. Cows in stage 2 were administered these treatments in the reverse order. The results indicated that average ovulation times in cows treated with GnRH in the MD group (GnRH-MD group) and cows treated with GnRH in the ED group (GnRH-ED group) were 30.0 ± 1.0 h and 28.8 ± 0.4 h, respectively. However, ovulation times for cows treated with hCG in the MD group (hCG-MD group) and cows treated with hCG in the ED group (hCG-ED group) were 35.8 ± 4.6 h and 32.8 ± 2.2 h, respectively, and ovulation occurred significantly later in the hCG-treated groups than in the GnRH-treated groups. In summary, we found that hCG-induced ovulation occurred later than GnRH-induced ovulation regardless of different diestrous peroids; however, the two treatments did not differ in terms of percentage of ovulation.

Prediction of parturition is essential for sustainable production in beef and dairy cattle, yet the present methods are limited by their high invasiveness and low utility. Here we compared prepartum changes in ventral tail base surface temperature (ST) with changes in vaginal temperature (VT) and behavioral indices. We analyzed 22 parturitions from 22 beef cows. Changes in daily values of ST, VT, and behavioral indices over the 7 days before parturition were investigated. Hourly values were calculated as the actual values minus the mean values for the same hour over a 3-day period, and the changes in hourly values over the 48 h before parturition were investigated. To test the effect of ambient temperature, tested cows were assigned to two season-groups based on the ambient temperature to which they were exposed (warm: n = 13; cool: n = 9), and the daily and hourly values of the indices were compared between seasons. A decrease in ST occurred approximately 30 h before parturition, which was similar to the time of the decrease in VT and earlier than the increase of behavioral indices. In addition, a unique fluctuation of ST observed in the last few hours before parturition indicates that ST could provide a sign for parturition not only in the long-term like VT, but also in the short-term like behavioral indices. Although ST was more sensitive to ambient temperature than VT or the behavioral indices, the day of parturition could be predicted from ST in both the warm and cool seasons.

This study aimed to examine the effect of rapamycin (autophagy inducer) and 3-methyladenine (3-MA, autophagy inhibitor) on the meiotic and developmental competencies of porcine oocytes derived from medium follicles (MF, 3–6 mm in diameter) and small follicles (SF, 1–2 mm in diameter) during in vitro maturation (IVM) process. The presence of 1 nM but not 10 nM rapamycin significantly increased the maturation rate of MF-derived oocytes (P < 0.05). However, the maturation rate of SF-derived oocytes was not affected by rapamycin at both concentrations (1 nM and 10 nM). The maturation rate of MF-derived oocytes decreased significantly (P < 0.05) in the presence of 0.2 mM but not 2 mM 3-MA than non-supplemented control. In contrast, in SF-derived oocytes, 3-MA at both 0.2 and 2 mM concentrations did not affect the maturation rates. The presence of 1 nM rapamycin significantly increased the blastocyst formation rate of MF-derived mature oocytes following parthenogenetic activation (P < 0.05). However, the blastocyst formation rate of SF-derived mature oocytes was not affected by the presence of rapamycin. The presence of 3-MA significantly reduced the blastocyst formation rate of MF-derived mature oocytes but did not change that of SF-derived oocytes. In conclusion, our study results show differences in activity of the autophagy inducer and inhibitor on the meiotic and developmental competencies of MF- and SF-derived porcine oocytes.

Xist is an X-linked ribonucleic acid (RNA) gene responsible for the cis induction of X chromosome inactivation (XCI). In cloned mammalian embryos, Xist is ectopically activated at the morula to blastocyst stage on the X chromosome that is supposed to be active, thus resulting in abnormal XCI. Suppression of erroneous Xist expression by injecting small interfering RNA (siRNA) remarkably increased the developmental efficiency of cloned male mouse embryos by approximately 10-fold. However, injection of anti-Xist siRNA resulted in only a slight increase in the developmental ability of injected cloned male pig embryos because the blocking effect of the injected siRNA was not maintained beyond the morula stage, which is 5 days post-activation. To develop a more effective approach for suppressing the ectopic expression of Xist in cloned pig embryos, we compared the silencing effect of short hairpin RNA (shRNA) and siRNA on Xist expression and the effects of these two Xist knockdown methods on the developmental competence of cloned male pig embryos. Results indicated that an shRNA-based RNA interference (RNAi) has a longer blocking effect on Xist expression than an siRNA-mediated RNAi. Injection of anti-Xist shRNA plasmid into two-cell-stage cloned male pig embryos effectively suppressed Xist expression, rescued XCI at the blastocyst stage, and improved the in vitro developmental ability of injected cloned embryos. These positive effects, however, were not observed in cloned male pig embryos injected with anti-Xist siRNA. This study demonstrates that vector-based rather than siRNA-mediated RNAi of Xist expression can be employed to improve pig cloning efficiency.

In this study, we examined the effects of 5-hydroxytryptamine (5-HT) on the motility and hyperactivation of mouse spermatozoa. In addition, we examined whether 5-HT increases the success of in vitro fertilization (IVF) in mice. Interestingly, 5-HT and agonists of the 5-HT₂, 5-HT₃, 5-HT₄, and 5-HT₇ receptors significantly increased the percentage of hyperactivated spermatozoa but did not affect the percentage of motile spermatozoa. Moreover, agonists of the 5-HT₂, 5-HT₃, and 5-HT₄ receptors significantly affected the velocities, linearity, straightness, wobbler coefficient, amplitude and/or frequency of spermatozoa. In particular, the improvement of hyperactivation by 5-HT was strongly inhibited by antagonists of the receptors 5-HT₄ and 5-HT₇ and was completely inhibited by a mixture of the four 5-HT-receptor antagonists. The increase in hyperactivation by the agonists was significantly inhibited by the corresponding 5-HT-receptor antagonist. Moreover, 5-HT significantly increased the percentage of two-cell embryos. The increase in the IVF success rate by 5-HT was significantly inhibited by a 5-HT₄-receptor antagonist. These results suggest that 5-HT increased hyperactivation through the 5-HT receptors and increased the success of IVF in mice.

Early antral follicle (EAF)-derived porcine oocytes develop more readily on polyacrylamide-gel (PAG) than on plastic plates. Xanthan gum (XG) and locust bean gum (LBG) are edible polysaccharides. We investigated XG-LBG gel supports in the development of EAF-derived porcine oocytes. XG and LBG were mixed in a 1:1 ratio to form a substrate. We cultured oocyte granulosa cell complexes (OGCs) from the EAFs on XG-LBG gels of various concentrations. The oocyte diameters were comparable among the 0.3, 0.5, and 1.0% gels; granulosa cell proliferation was greater on the 1.0% gel. The proliferation and survival rates of the granulosa cells, and the histone H4 at lysine 12 acetylation levels were higher in OGCs cultured on 1.0% XG-LBG than those grown on 0.3% PAG. Development to the blastocyst stage was 13.8% for the XG-LBG gels and 9.4% for PAG. In conclusion, XG-LBG are safe and efficient substrates for in vitro culture of oocytes.