Transforming growth factor-β (TGF-β) has been shown to have a positive effect on in vitro fertilization (IVF) and has been reported to stimulate meiosis at follicular level in variety of species. The study was designed to determine the expression patterns of TGF-β1, TGF-β receptors type Ⅰ, Ⅱ and Smads gene in bovine oocytes and embryos. TGF-β1 and their receptors were observed in the unfertilized oocytes. TGF-β1 and type Ⅱ receptor were not expressed at the blastocyst stage, however, only type I receptor was exclusively observed at the same stage. The blastocyst stage, in particular, showed high levels of mRNA expression patterns containing a TGF-β type Ⅰ receptor. The mRNA expression pattern of Smad 2 at all stages of embryonic development was similar in all respect with TGF-β1 type I receptor. On the contrary, Smad 3 and 4 were expressed with high and low level mRNA at the blastocyst stage. In conclusion, it is suggested that TGF-β signaling may be regarded as an important entity during the preimplantation embryo development.

Our previous research has identified granulin (grn) and p130 genes as sex steroidinducible genes in the rat hypothalamus, which might be involved in sexual differentiation of the brain. Phthalate esters that are used as plasticizers and also found at low levels in foods such as dairy products are often mentioned as suspected endocrine disrupters. The purpose of the present study is to elucidate whether perinatal exposure to dinbutyl phthalate (DBP), diisononyl phthalate (DINP) and di2ethylhexyl adipate (DEHA) affects hypothalamic sex steroidinducible genes. The present study assessed the effects of perinatal exposure to DBP, DINP and DEHA on sex steroid hormones levels and hypothalamic grn and p130 mRNA expressions at postnatal day (PND) 3 and 7. Pregnant rats were fed a soyfree diet containing 20, 200, 2,000 and 10,000 ppm of DBP, 40, 400, 4,000 and 20,000 ppm of DINP, or 480, 2,400 and 12,000 ppm of DEHA from gestational day (GD) 15 to GD 3 or 7. At PND 3 and 7, perinatal exposure to these chemicals did not substantially affect serum concentrations of testosterone and estradiol. At PND 3, the expression of grn mRNA levels in males was decreased by DEHA, and that of p130 was decreased by DBP, DINP and DEHA, though the effects were not dosedependent. At PND 7, the expression of grn gene in female pups was increased by higher doses of DBP and all the doses, except for 4,000 ppm, of DINP, while that in male pups decreased by 480 and 12,000 ppm of DEHA. Hypothalamic expression of p130 mRNA in males was increased by lower doses of DBP and all the doses of DINP, whereas that of females was decreased by 480 and 2,400 ppm of DEHA. These results suggest that these chemicals may affect the expression of grn and p130 genes by directly acting on the hypothalamus, thus leading to inappropriate expression of these genes.

The possibility of producing transgenic embryos expressing the green fluorescence protein (GFP) gene have been evaluated after transfer of exogenous gene into the porcine zygote cytoplasm using the intracytoplasm sperm injection (ICSI) as gene delivery method. For DNA binding to sperm heads, 0.05% Triton X-100 or Lipofectin was used. After injection of the sperm bound to DNA by means of Lipofectin or Triton X-100 triturate, the blastocyst formation rates on day 6 were not significantly different from that of ICSI only group (18.8, 19.2 and 25.3%). In terms of GFP expression, more embryos were in GFP form in Triton X-100 group than in Lipofectin group (40.6 vs 36.4%), while percentage of non-mosaic embryos expressing the GFP gene in all blastomere was higher (P<0.05) in Lipofectin group than in Triton X-100 group (4.2 vs 0.9%). ICSI embryos derived from sperm treated with Lipofectin/DNA complex was transferred into 3 recipients and were collected by uterine flushing on days 5, 7 and 15 after embryo transfer, and then GFP expression was observed by a fluorescence microscopy. Over 26% of the collected embryos were normally expressed GFP gene. These results suggest that foreign gene transfer method with DNA bound sperm caused minimal damage to structure of oocytes that can result to full development of porcine embryos. This was confirmed in this study when the embryos that were transferred after ISCI of DNA bound sperm had a normal development and gene expression until preimplantation.

This study was conducted to examine the effect of IRES controlled reporter gene on screening and production of recombinant human erythropoietin (EPO) proteins from cultured CHO cells. The cDNA was cloned for EPO from human liver cDNA. Using site-directed mutagenesis, we generated recombinant human EPO (rhEPO) with two additional N-glycosylations (Novel erythropoiesis-stimulating protein: NESP). Wild type hEPO and NESP were cloned into expression vectors with GFP reporter gene under regulatory control of CMV promoter and IRES so that the vectors could express both rhEPO and GFP. The expression vectors were transfected to cultured CHO-K1 cells. Under microscopy, expression of GFP was visible. Using supernatant of the culture, ELISA assay, immunocytochemistry and in vitro assay using EPO dependant cell line were performed to estimate biological activity to compare the production characteristics (secretion levels, etc.) between rhEPO and NESP. The activity of NESP protein, obtained by mutagenesis, was described and compared with its rhEPO counterpart produced under same conditions. Although NESP had less secretion level in CHO cell line, the biological activity of NESP was greater than that of rhEPO. These results are consistent with previous researches. We also demonstrated that rhEPO and GFP proteins expressed simultaneously from transfected CHO cell line. Therefore we conclude that use of GFP reporter gene under IRES control could be used to screen and produce rhEPO in cultured CHO cells.