The present study assessed the effect of FSH and LH on oocyte meiotic, cytoplasmic maturation and on the expression level and polyadenylation status of several maternal genes. Cumulus-oocyte complexes were cultured in the presence of FSH, LH, or the combination of FSH and LH. Significant cumulus expansion and nuclear maturation was observed upon exposure to FSH alone and to the combination of FSH and LH. The combination of FSH and LH during entire IVM increased the mRNA level of four maternal genes, C-mos, Cyclin B1, Gdf9 and Bmp15, at 28 h. Supplemented with FSH or LH significantly enhanced the polyadenylation of Gdf9 and Bmp15; and altered the expression level of Gdf9 and Bmp15. Following parthenogenesis, the exposure of oocytes to combination of FSH and LH during IVM significantly increased cleavage rate, blastocyst formation rate and total cell number, and decreased apoptosis. In addition, FSH and LH down-regulated the autophagy gene Atg6 and upregulated the apoptosis gene Bcl-xL at the mRNA level in blastocysts. These data suggest that the FSH and LH enhance meiotic and cytoplasmic maturation, possibly through the regulation of maternal gene expression and polyadenylation. Overall, we show here that FSH and LH inhibit apoptosis and autophagy and improve parthenogenetic embryo competence and development.

Among laboratory animals, pigs are anatomically and physiologically closer to human. Transgenic (TG) pigs can be widely applied as models of human diseases. Many researchers created TG pigs which have specific modified genome under a constitutively active promoter. A constitutively active promoter is effective to express a target gene, but the uncontrollable expression often results in unwanted outcomes. In this study, as a way to solve these problems, we tried to regulate the expression of target genes by tetracycline (Tet) on/off system. We tested the operation of Tet on/off system in TG donor cells. Miniature porcine fetal fibroblasts were transfected with universal doxycycline- inducible vector and an enhanced green fluorescent protein (eGFP) was used as the target gene. The induced transgene expression by doxycycline was detected on fluorescence microscopy. On one day after 1 μg/ml doxycycline treatment, the fluorescence intensity for TG cells was increased. And we then performed Somatic Cell Nuclear Transfer (SCNT) to confirm the working of Tet on/off system in the porcine SCNT-TG embryos. Total 649 transgenic porcine SCNT embryos were made. From these, 64 of SCNT embryos used in invitroculturewith1 μg/mldoxycycline. Among these porcine SCNT-TG embryos, 39 embryos (60.9%) were cleaved. Finally, 15 transgenic porcine SCNT embryos developed blastocyst. Induced transgene expression was observed all of cleaved embryos and blastocysts. The remaining 585 embryos were transferred to 6 surrogates. On 25 days after embryo transfer, two surrogates were diagnosed as pregnant (pregnancy rate =33.3%). On day 113 (one day prior to delivery), we obtained six cloned TG piglets from first pregnant surrogate. Unfortunately, all TG piglets died because their surrogate died suddenly at delivery time. However, we could obtain the TG cell lines from the cloned TG piglets. Being analyzed by PCR, all piglets were found to be eGFP gene targeted. Now, second pregnant surrogate have maintained at 80 days after embryo transfer and shown more than three embryonic sacs. This data suggested that, Tet on/off system can control target gene expression in transgenic porcine SCNT embryos. This result has presented new possibilities of regulation of target gene expression in cloned TG pigs by Tet on/off system. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.

It is well established that mitochondrial genome is strictly maternally inherited in mammalian, despite the fact that paternal mitochondria enter into oocyte during fertilization. To date, although some mechanisms have been extrapolated to interpret the elimination of paternal mitochondria, the exact mechanism still is unclear. Recent studies suggest that autophagy process and the ubiquitin-mediated degradation pathway may be involved in elimination of paternal mitochondria. However, the dynamic profiles of autophagy and ubiquitination associated with paternal mitochondria degradation have not been determined in mouse model. Through immunostaining with specific antibody LC3 and Ubiquitin and confocal microscopy, we investigated the dynamic profiles of LC3 and Ubiquitin signals in mouse embryos during preimplantation development. In addition, embryos were stained with MitoTracker Red for tracking the degradation process of paternal mitochondria. Our results showed that paternal mitochondria gradually degraded during postfertilization development, and sporadic paternal mitochondria were found at least in 16 cell embryos. LC3 and Ubiquitin signals appeared in the midpiece of sperm at 3 h postfertilization, and they were strictly colocalizated with paternal mitochondria from zygote to 2 cell embryo. Nevertheless, the colocalization became loose at 4 cell embryos, and gradually disappeared beyond 4 cell embryos. Our results confirmed that autophagy process and the ubiquitin-mediated degradation pathway may take part in the postfertilization remove of paternal mitochondria.

Superovulation, or ovarian stimulation is a commonly used ART for treatment of human infertility/subfertility. Recent studies suggest that superovulation unaffects methylated imprints acquisition in mouse oocytes during oogenesis, whereas disrupts DNA methylation maintenance in embryos during preimplantation development. However, the mechanisms of defects in methylation maintanence caused by superovulation remain largely unclear. We hypothesized that superovulation may disrupt the expression of DNA methyltransferases (Dnmts), the enzymes which catalyze DNA methylation acquisition and maintenance. The mice were subjected to superovulate with low (6 IU) and high (10 IU) dosage hormone. We examined the global DNA methylation levels in zygotes and DNA methylation of repeated sequences (IAP and Line 1) in blastocyst stage embryos. In addition, we investigated the expression of Dnmts (Dnmt3a, Dnmt3b, Dnmt3l and Dnmt1o) in ovulated oocytes and zygotes. Through staining with antibody 5mC and Di-H3K9 coupled with confocal microscopy, we found that global methylation profiles in zygotes derived from females after low or high dosage hormone treatment were not affected when compared to control counterpart. Moreover, methylation at IAP in blastocysts also was unaffected by superovulation, irrespective of hormone dosage. In contrast, methylation level at Line 1 decreased when the females were administered by high dosage hormone. Furthermore, expression of de novo DNA methyltransferase Dnmt3a, Dnmt3b, Dnmt3L, as well as maintenance Dnmt1o in MII oocytes and zygotes was not disrupted by superovulation. Given superovulation adversely affected methylation maintenance in blastocysts during preimplantation development but with normal expression of Dnmts in oocytes and zygotes, it is indicated that defects of embryonic methylation didn’t originate from abnormal expression of Dnmts.

Although evidences showed that histone deacetylation plays an important role in the mitotic and meiotic cell cycle, but the mechanisms are still unclear. Level of histone acetylation can be easily changed by deacetylase inhibitors (HDACi) i.e trichostatin A (TSA) and valporic acid. In this study, we determined whether the inhibition of histone deacetylation by TSA could affect porcine oocyte maturation and aging process. Our results showed that treated COCs with 100 nM TSA significantly increase the GVBD in each time group than 0, 5, 50 nM but no significantly different from that of higher concentration (200 nm or 300 nM). No significant differences on maturation, blastocyst development, MAPK pattern and expressions of apoptosis gene when treated oocytes with 100 nM TSA for the first 24h of IVM compared with control and 5, 50 nM TSA. However, in the oocytes treated with 200 nM and 300 nM TSA for first 24 h, MAPK significantly decreased and abnormal spindle were observed. But, in prolonged (64 h) of TSA treated group has no significantly different in control. Another data observed that after 24h TSA-treat to prolonged group were significantly decreased of MAPK activation and normal spindle than the other group. We concluded that TSA played a critical role in meiotic progression in porcine oocytes through the regulation of arrest GVBD, which prolonging the in vitro maturation time, but unaffected the subsequent pre-implantation embryo developmental potential and embryonic qualities. Moreover, the histone deacetylase inhibitor TSA may artificially control porcine oocyte maturation time and delay porcine oocyte aging process.