Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cell (iPS) experiments have generally demonstrated that a differentiated cell directly converts into a undifferentiated or pluripotent state. In SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor cell nuclei to the recipient cytoplasm of matured oocytes. Although nuclear reprogramming of cells by the ex-ovo methods is not always consistent or efficient, it has been suggested that a combination of nuclear reprogramming technique may improve the efficiency or frequency of normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from GV stage sturgeon's oocytes prior to their use as nuclear donors for SCNT will improve subsequent development. We reported a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte exteact (SOE) to porcine fetal fibroblast cell nuclei ex ovo. Porcine fibroblasts were permeabilized by 4 μg/ml of digitonin for 2 min at 4℃ and then incubated in SOE for 7h at 15 18℃ followed by resealing of cell membrane. As results, no difference was observed in the number of fused couplets or the number of fused couplets that cleaved between the extract treated or control group. However, there was a significantly decrease in the percentage of fused couplets that developed to the blastocyst stage in the SOE treated group (p<0.05). Histone acetylation status was determined using an antibody to acetylation at lysine 9 on histone 3 (H3K9Ac). The intensity of H3K9Ac staining in 1-cell stage NT embryos was significantly increased when treated with the SOE (p<0.05), similar to that in 1-cell stage IVF embryos. In addition, porcine NT embryos reconstructed by using donor cell exposed to SOE prior to cell fusion significantly decreased developmental competence to the blastocyst stage but increased pluripotent gene expressions (Sox2, Nanog and Oct3/4) when compared with those in normal NT embryos (p<0.05).

S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP3 receptor- binding protein released with IP3 (IRBIT), is a member of the AHCY-like protein family. AHCYL1 protein regulates IP3-induced Ca2+ release in the cytoplasm of cells and, therefore, is likely to be an important gene regulating various biological processes in the oviduct of chickens. Inmammals, expression is greatest during activation of dendritic cells which are antigen presenting cells associated with immunoregulatory processes in blood and skin. However, the identification of the AHCYL1 gene in chickens has not been investigated. In the present study, we first used RT-PCR to demonstrate AHCYL1 gene expression in adult chicken organs and oviducts of immature chickens treated with DES (diethylstilbesterol, a synthetic estrogen agonist). The results indicated that AHCYL1 mRNA is expressed in chicken reproductive organs (testis, ovary and oviduct). Inaddition, expression of AHCYL1 mRNA increased in response to DES-treated immature oviducts compared to the non-treated control immature oviducts of chickens. Interestingly, AHCYL1 was abundant in the cytoplasm of luminal and glandular epithelia, but not in other cell-types such as stroma and connective tissues, of the chicken oviduct. These results suggest that AHCYL1 is a novel estrogen-stimulated gene associated with development of the chicken oviduct, as well as functions of oviductal glandular and luminal epithelia that may include activation of resident immune cells, such as dendritic cells.

Serpins are a superfamily of related protease inhibitors with common structural features and inhibitory mechanisms. However, SERPINA 14 in mammals does not have inhibitory activity against most known proteases. Rather, it may have an immunoregulatory role in mammals to prevent rejection of the fetal allograft by inhibiting lymphocyte proliferation and natural killer cell function. In the pig, SERPINA14 is involved in iron transport to the fetus by binding to and stabilizing the iron-binding protein uteroferrin (ACP5). In chickens, these very little known about serpins in chickens. Therefore, we investigated the expression patterns of serpin genes in the oviduct of adult hens and in the oviduct of 37-day-old chicks treated with an estrogen analogue, diethylstilbestrol (DES). Results indicated that SERPINB3 and SERPINB11 genes were highly expressed in oviducts of DES-treated chicks, but not in oviducts of control chicks. Both SERPINB3 and SERPINB11 transcripts were localized specifically to the gland-like areas of oviducts of DES-treated chicks. Immunohistochemical analyses confirmed that SERPINB3 and SERPINB11 proteins were present in the gland-like area and luminal epithelium of the oviducts of DES-treated chicks. Collectively, the results suggest that SERPINB3 and SERPINB11 are expressed in response to estrogens and they have distinct functions related to development and differentiation of the mature oviduct in hens.