Earticle

This study was investigated to optimize the efficiency of cloning and to produce the clone mice. Somatic cell nucleus transfer (SCNT) embryos were produced by injecting cumulus cell nucleus into enucleated B6D2F matured oocyte. Mouse chimeras can be also successfully produced with tetraploid host embryos. The fusion of mouse 2- cell embryos can be produced tetraploid embryos. Thus, we utilized the tetraploid embryos to increase the clone efficiency by aggregation with SCNT embryos of adult cumulus cells. SCNT embryos were found to be optimized for 6 hours activation with strontium (SrCl2). The cytochalasin B (CB) concentration (5 ug/ml) in the enucleation was evaluated in the efficiency of implantation sites and fetus offspring. Trichostatin A (TSA), histonedeacetylase inhibitor (HDACi), is recommended to production of clone mice, continuously exposed in 5～50 nM for 10 hours. And we also improved the SCNT implantation rate/ live offspring by co-transfer with parthenogenic embryo in uterus of the other site. Next, the aggregated SCNT were constructed by aggregation of SCNT embryo with tetraploid embryos to reduce epigenetic error in placenta. The pregnancy and implantation rates of aggregated SCNT were significantly higher than SCNT alone. The full term developmental rate in aggregated embryo was slightly higher than that of SCNT (3.57 vs 1.16). The placental weight of SCNT clone was significantly higher than that of in vitro fertilization as shown in previously reported. However, the placenta weight was almost reduced to IVF group in the aggregated SCNT. It was shown to be typical hyperplastic histology of mouse clones. But the aggregated SCNT method was useful to significantly reduce the placental weight known as general problems in cloned mice.

Amino acids have been regarded as beneficial for the development of preimplantation embryos and acted critically in the in vitro culture of porcine embryos. However, some reviews of emerging literatures show that supplementation of essential amino acids (EAAs) to in vitro culture medium was improved less than usually expected. The present study was designed to investigate the effects of EAAs on the in vitro development of porcine preimplantation embryos after parthenogenetic activation. The results showed that supplementation of EAAs to porcine zygote medium-3 (PZM-3) with 1:50 significantly decreased the blastocyst rate of parthenogenetic embryos (12.8±4.0 vs. 25.6±4.2) although cleavage rate was not different (p<0.05). On the other hand, the distribution pattern of total cell number of the blastocysts varied widely while the average number has not shown significant difference. Real-time qPCR analysis revealed that EAAs have turned over transcription pattern of imprinted genes: the relative abundance of H19 transcript was enhanced and level of IGF2r transcript was decreased significantly (p<0.05). Interestingly, gene expression of mTOR, activated by some essential amino acids, was significantly up-regulated (p<0.05). In conclusion, these results indicate that the presence of essential amino acids in the in vitro culture medium might impair embryo development due to mRNA turnover of imprinted genes, but could possibly play an important role at the implantation stage because development of protrusive trophoblast activity requires some amino acids-dependent mTOR signaling in early blastocysts.

Sloan-Kettering virus gene product of a cellular protooncogene c-Ski is an unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. Ski protein is implicated in proliferation/differentiation in a variety of cells. The aim of the present study was to locate Ski protein in rat ovaries in order to predict the possible involvement of Ski in follicular development, atresia and luteinization. First, expression of c-Ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the shape and size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in rats having atretic follicles with double-staining. These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles, and suggest that Ski plays a role in apoptosis of granulosa cells during follicular atresia. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of corpus luteum (CL). Together, our data indicate that Ski is expressed in a cell- specific manner during ovarian follicular development, atresia, and luteinization and that Ski might play essential roles in these physiological processes.

It is generally accepted that chronic stress impairs female reproduction. It negatively affect ovarian function and the number of ovulated oocytes. Chronic stress lower the number of retrieved oocytes. Ovarian follicular development is regulated by both pituitary-derived gonadotropins and intraovarian regulatory factors. The main corticosteroids are cortisol, cortisone, 11-deoxycortisol and corticosterone, cortisol being one of the most commonly used welfare and stress physiological indicator. In this study, we investigated the effect of cortisol level on progesterone patterns and the number of ovulated oocytes in the dog. Blood samples were collected and progesterone levels were analyzed for determining ovulation time via radioimmunoassay. At the same time, serum cortisol level was measured. The day of ovulation was considered as the day when serum progesterone level was 6.0～8.0 ng/ml. In vivo matured dog oocytes were collected using flushing oviducts of mixed-breed bitches at three days after ovulation. We classified dogs as having higher cortisol level (over 8 μg/dL), medium cortisol level (4～7 μg/dL), and lower corisol level (1～3 μg/dL). The patterns of progesterone was not different in all cortisol groups. The average numbers of retrieved oocytes was not different in all cortisol groups. 9 in higher cortisol level (over 8 μg/dL), 9.5 in medium cortisol level (4～7 μg/dL), and 9.6 in lower corisol level (1～3 μg/dL). The effects of high cortisol on the competence of oocytes and steroid hormone patterns will be studied.