Transcriptome profiling of in vitro-matured oocytes from a korean native cow (hanwoo) after cysteamine supplementation.

This study elucidated the molecular markers that decrease oocyte quality during in vitro culture, restricting optimal developmental potential. Here, we evaluated the transcriptomic differences between cysteamine-treated and non-treated bovine cumulus oocyte complexes (COCs) after 22 h of co-culture in the maturation media using RNA sequencing. In total, 39,014 transcripts were sequenced between cysteamine-treated and non-treated mature COCs. We evaluated the relative expression of 21,472 genes, with 59 genes showing differential expression between the two COC groups. The cysteamine-treated group had 36 up-regulated gene transcripts and 23 down-regulated gene transcripts. Moreover, gene ontology (GO) enrichment analysis revealed that multiple biological processes were significantly enriched after cysteamine supplementation. Differentially expressed genes appeared to maintain normal oocyte physiology, regulation of apoptosis, differentiation, ossification or bone formation, cardiac and muscle physiology, hormonal secretion, and membrane construction for further embryonic development. In conclusion, cysteamine affects the mRNA level of COCs during oocyte maturation by upregulating potential molecular markers and downregulating genes that affect further embryonic development.

Estimating genetic diversity and population structure of 22 chicken breeds in Asia using microsatellite markers.

OBJECTIVE: Estimating the genetic diversity and structures, both within and among chicken breeds, is critical for the identification and conservation of valuable genetic resources. In chickens, microsatellite (MS) marker polymorphisms have previously been widely used to evaluate these distinctions. Our objective was to analyze the genetic diversity and relationships among 22 chicken breeds in Asia based on allelic frequencies. METHODS: We used 469 genomic DNA samples from 22 chicken breeds from eight Asian countries (South Korea, KNG, KNB, KNR, KNW, KNY, KNO; Laos, LYO, LCH, LBB, LOU; Indonesia, INK, INS, ING; Vietnam, VTN, VNH; Mongolia, MGN; Kyrgyzstan, KGPS; Nepal, NPS; Sri Lanka, SBC) and three imported breeds (RIR, Rhode Island Red; WLG, White Leghorn; CON, Cornish). Their genetic diversity and phylogenetic relationships were analyzed using 20 MS markers. RESULTS: In total, 193 alleles were observed across all 20 MS markers, and the number of alleles ranged from 3 (MCW0103) to 20 (LEI0192) with a mean of 9.7 overall. The NPS breed had the highest expected heterozygosity (Hexp, 0.718±0.027) and polymorphism information content (PIC, 0.663±0.030). Additionally, the observed heterozygosity (Hobs) was highest in LCH (0.690±0.039), whereas WLG showed the lowest Hexp (0.372±0.055), Hobs (0.384±0.019), and PIC (0.325±0.049). Nei's DA genetic distance was the closest between VTN and VNH (0.086), and farthest between KNG and MGN (0.503). Principal coordinate analysis showed similar results to the phylogenetic analysis, and three axes explained 56.2% of the variance (axis 1, 19.17%; 2, 18.92%; 3, 18.11%). STRUCTURE analysis revealed that the 22 chicken breeds should be divided into 20 clusters, based on the highest ΔK value (46.92). CONCLUSION: This study provides a basis for future genetic variation studies and the development of conservation strategies for 22 chicken breeds in Asia.

Localization and quantitative analysis of Cx43 in porcine oocytes during in vitro maturation.

Many studies of the main gap junction protein, Cx43, have been conducted in porcine oocyte research, but they have been limited to investigations of cumulus-oocyte complexes (COCs). In this study, we verified Cx43 not in COCs, but in porcine oocytes during maturation, and conducted a quantitative time course analysis. The location and dynamics of Cx43 were examined by immunocytochemistry and western blotting, respectively. COCs were cultured in NCSU23 medium and processed for immunocytochemistry and western blotting at 0, 14, 28, and 42 h after denuding. A Cx43 signal was detected on oolemmas, transzonal projections and the surface of zona pellucidae. Western blotting showed that Cx43 band density increased from 0 to 14 h, and gradually decreased thereafter. Our results clarified that Cx43 is localized in the ooplasmic membrane through zona pellucidae and its level changes over time during culture in porcine oocytes.

Relationship between Sloan-Kettering virus expression and mammalian follicular development.

Sloan-Kettering virus gene, a product of a cellular proto-oncogene c-Ski is a unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. The aim of the present study was to locate Ski protein in rat ovaries in order to find insights into the possible involvement of Ski in follicular development. 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). RT-PCR and in situ hybridization revealed that c-Ski mRNA was expressed in the ovaries of the adult rat on the day of estrous and localized mainly in the granulose cells. 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. Based on the present findings, Ski may play a role in the apoptosis of granulosa cells during follicular atresia.

Protective effect of centipedegrass against Aß oligomerization and Aß-mediated cell death in PC12 cells.

CONTEXT: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the abnormal accumulation of ß-amyloid (Aß). Multiple Aß-aggregated species have been identified, and neurotoxicity appears to be correlated with the amount of non-fibrillar oligomers. Potent inhibitors of Aß oligomer formation or Aß-induced cell toxicity have emerged as attractive means of therapeutic intervention. Eremochloa ophiuroide Hack. (Poaceae), also known as centipedegrass (CG), originates from China and South America and is reported to contain several C-glycosyl flavones and phenolic constituents. OBJECTIVE: We investigated whether CG could suppress Aß aggregation, BACE1 activity, and toxicity at neuronal cell. MATERIALS AND METHODS: The inhibitory effect of CG extracts toward aggregation of Aß42 was investigated in the absence and presence of 50 µg/mL CG. We investigated the inhibitory effects of CG (0-5 µg/mL) on BACE1 using fluorescence resonance energy transfer (FRET)-based assay. The effects of CG (0-75 µg/mL) on Aß42-induced neurotoxicity were examined in PC12 cells in the presence or absence of maysin and its derivatives of CG. RESULTS: We isolated EA-CG fraction (70% MeOH fraction from EtOAc extracts) from methanol extracts of CG, which contained approximately 60% maysin and its derivatives. In the present studies, we found that several Aß oligomeric forms such as the monomer, dimer, trimer, and highly aggregated oligomeric forms were remarkably inhibited in the presence of 50 µg/mL of EA-CG. EA-CG also inhibited BACE1 enzyme activity in a dose-dependent manner. EA-CG treatment generated approximately 50% or 85% inhibition to the control at the tested concentrations of 1 or 5 µg/mL, respectively. Moreover, the neurotoxicity induced by Aß42 was significantly reduced by treatment of EA-CG, and the 75 µg/mL EA-CG treatment significantly increased cell viability up to 82.5%. DISCUSSION AND CONCLUSION: These results suggested that the anti-Alzheimer's effects of CG occurred through inhibition of neuronal cell death by intervening with oligomeric Aß formation and reducing BACE1 activity. Maysin in CG could be an excellent therapeutic candidate for the prevention of AD.

Assessment of Genetic Diversity, Relationships and Structure among Korean Native Cattle Breeds Using Microsatellite Markers.

Four Korean native cattle (KNC) breeds-Hanwoo, Chikso, Heugu, and Jeju black-are entered in the Domestic Animal Diversity Information System of the United Nations Food and Agriculture Organization (FAO). The objective of this study was to assess the genetic diversity, phylogenetic relationships and population structure of these KNC breeds (n = 120) and exotic breeds (Holstein and Charolais, n = 56). Thirty microsatellite loci recommended by the International Society for Animal Genetics/FAO were genotyped. These genotypes were used to determine the allele frequencies, allelic richness, heterozygosity and polymorphism information content per locus and breed. Genetic diversity was lower in Heugu and Jeju black breeds. Phylogenetic analysis, Factorial Correspondence Analysis and genetic clustering grouped each breed in its own cluster, which supported the genetic uniqueness of the KNC breeds. These results will be useful for conservation and management of KNC breeds as animal genetic resources.

Genetic diversity and relationships of korean chicken breeds based on 30 microsatellite markers.

The effective management of endangered animal genetic resources is one of the most important concerns of modern breeding. Evaluation of genetic diversity and relationship of local breeds is an important factor towards the identification of unique and valuable genetic resources. This study aimed to analyze the genetic diversity and population structure of six Korean native chicken breeds (n = 300), which were compared with three imported breeds in Korea (n = 150). For the analysis of genetic diversity, 30 microsatellite markers from FAO/ISAG recommended diversity panel or previously reported microsatellite markers were used. The number of alleles ranged from 2 to 15 per locus, with a mean of 8.13. The average observed heterozygosity within native breeds varied between 0.46 and 0.59. The overall heterozygote deficiency (F IT) in native chicken was 0.234±0.025. Over 30.7% of F IT was contributed by within-population deficiency (F IS). Bayesian clustering analysis, using the STRUCTURE software suggested 9 clusters. This study may provide the background for future studies to identify the genetic uniqueness of the Korean native chicken breeds.

Siberian Sturgeon Oocyte Extract Induces Epigenetic Modifications of Porcine Somatic Cells and Improves Developmental Competence of SCNT Embryos.

Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from the germinal vesicle (GV) stage Siberian sturgeon oocytes prior to their use as nuclear donor for SCNT would improve in vitro development. A reversible permeability protocol with 4 µg/mL of digitonin for 2 min at 4°C in order to deliver Siberian sturgeon oocyte extract (SOE) to porcine fetal fibroblasts (PFFs) was carried out. As results, the intensity of H3K9ac staining in PFFs following treatment of SOE for 7 h at 18°C was significantly increased but the intensity of H3K9me3 staining in PFFs was significantly decreased as compared with the control (p<0.05). Additionally, the level of histone acetylation in SCNT embryos at the zygote stage was significantly increased when reconstructed using SOE-treated cells (p<0.05), similar to that of IVF embryos at the zygote stage. The number of apoptotic cells was significantly decreased and pluripotency markers (Nanog, Oct4 and Sox2) were highly expressed in the blastocyst stage of SCNT embryos reconstructed using SOE-treated cells as nuclear donor (p<0.05). And there was observed a better development to the blastocyst stage in the SOE-treated group (p<0.05). Our results suggested that pre-treatment of cells with SOE could improve epigenetic reprogramming and the quality of porcine SCNT embryos.

Altered protein profiles in human umbilical cords with preterm and full-term delivery.

Several biomarkers are routinely used clinically for predicting preterm labor; however, these factors are either nonspecific or detected too late. Here, we performed protein profiles in preterm- and term-derived human umbilical cord by using 2DE. Approximately 200 different proteins were identified between preterm- and term-delivered umbilical cords. Among them, 48 proteins were identified. A comparison of preterm proteome to that of term proteome revealed potential candidates for biomarkers, such as hypoxia-inducible proteins, phosphorylated heat-shock protein 27 (HSP27), transgelin, vimentin, and transferrin that are specific to preterm umbilical cords. Especially, HSP27 in preterm-derived umbilical cords shows a significant increase in the mono- and tetra-phosphorylation. The real importance of all of HSP27 phosphorylation as well as hypoxia-inducible factor 1alpha, and glyceraldehyde 3-phosphate dehydrogenase require further validation in vitro and in vivo; nevertheless, we believe that they could represent promising diagnostic targets for detection of sudden early delivery. In conclusion, the results of the current study may provide important insights into the molecular mechanisms underlying umbilical cord development.

Dynamic methylation pattern of the methyltransferase1o (Dnmt1o) 5'-flanking region during mouse oogenesis and spermatogenesis.

DNA methyltransferase1o (Dnmt1o), which is specific to oocyte and preimplantation embryo, plays a role in maintaining DNA methylation in mammalian cells. Here, we investigated the methylation status of CpGs sites in the Dnmt1o 5'-flanking region in germ cells at different stages of oogenesis or spermatogenesis. The methylation levels of the CpG sites at the 5'-flanking regions were hypermethylated in growing oocytes of all follicular stages, while the oocytes in meiotic metaphase II (MII) were demethylated. The methylation pattern within the CpGs sites in the 5'-flanking region, however, was dramatically changed during spermatogenesis. We observed that there was significant non-CpG methylation both in MII oocytes and spermatocytes. Although a low methylation level in non-CpG sites was observed in primary and secondary oocytes, the CpA site of position 25 and CpT site of position 29 within the no-CpG region in the 5'-flanking region of Dnmt1o was highly methylated in MII oocytes. During spermatogenesis, the low degree of methylation at CpG sites in spermatocytes increased to a higher degree in sperm, while the high ratio of methylation in non-CpG sites in spermatocytes decreased. Together, germ cells showed inverted methylation patterns between CpG and non-CpG sites in the Dnmt1o 5'-upstream region, and the methylation pattern during oogenesis did not drastically change, remaining generally hypomethylated at the MII stage.

Proteomic analysis of cloned porcine conceptuses during the implantation period.

Differentially regulated proteins within porcine somatic cell nuclear transfer (SCNT)-derived conceptuses were compared with conceptuses that were derived from natural matings on day 14 of pregnancy. Proteins that were expressed prominently on day 14 were identified in SCNT-derived conceptuses using 2-D PAGE and MALDI-TOF MS. Sixty eight proteins were identified as being differentially regulated in the SCNT-derived conceptuses. Among these, 62 were down-regulated whereas the other six proteins were up-regulated. Glycolytic proteins, such as pyruvate dehydrogenase, malate dehydrogenase and lactate dehydrogenase, were down-regulated in the SCNT-derived conceptuses whereas apoptosis-related genes as annexin V, Hsp60, and lamin A were up-regulated. Thus, apoptosis-related genes are expressed at significantly higher levels in the SCNT-derived conceptuses than in the control conceptuses, whereas metabolism-related genes are significantly reduced.

mtDNA Diversity and Phylogenetic State of Korean Cattle Breed, Chikso.

In order to analyze the genetic diversity and phylogenetic status of the Korean Chikso breed, we determined sequences of mtDNA cytochrome b (cyt b) gene and performed phylogenetic analysis using 239 individuals from 5 Chikso populations. Five non-synonymous mutations of a total of 15 polymorphic sites were identified among 239 cyt b coding sequences. Thirteen haplotypes were defined, and haplotype diversity was 0.4709 ranging from 0.2577 to 0.6114. Thirty-five haplotypes (C1-C35) were classified among 9 Asia and 3 European breeds. C2 was a major haplotype that contained 206 sequences (64.6%) from all breeds used. C3-C13 haplotypes were Chikso-specific haplotypes. C1 and C2 haplotypes contained 80.5% of cyt b sequences of Hanwoo, Yanbian, Zaosheng and JB breeds. In phylogenetic analyses, the Chikso breed was contained into B. taurus lineage and was genetically more closely related to two Chinese breeds than to Korean brown cattle, Hanwoo. These results suggest that Chikso and Hanwoo have a genetic difference based on the mtDNA cyt b gene as well as their coat color, sufficient for classification as a separate breed.

Citrus aurantium flavonoids inhibit adipogenesis through the Akt signaling pathway in 3T3-L1 cells.

BACKGROUND: Obesity is a health hazard that is associated with a number of diseases and metabolic abnormalities, such as type-2 diabetes, hypertension, dyslipidemia, and coronary heart disease. In the current study, we investigated the effects of Citrus aurantium flavonoids (CAF) on the inhibition of adipogenesis and adipocyte differentiation in 3T3-L1 cells. METHODS: During adipocyte differentiation, 3T3-L1 cells were treated with 0, 10, and 50 µg/ml CAF, and then the mRNA and protein expression of adipogenesis-related genes was assayed. We examined the effect of CAF on level of phosphorylated Akt in 3T3-L1 cells treated with CAF at various concentrations during adipocyte differentiation. RESULTS: The insulin-induced expression of C/EBPß and PPARγ mRNA and protein were significantly down-regulated in a dose-dependent manner following CAF treatment. CAF also dramatically decreased the expression of C/EBPα, which is essential for the acquisition of insulin sensitivity by adipocytes. Moreover, the expression of the aP2 and FAS genes, which are involved in lipid metabolism, decreased dramatically upon treatment with CAF. Interestingly, CAF diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3ß (Ser9), which may reduce glucose uptake in response to insulin and lipid accumulation. Furthermore, CAF not only inhibited triglyceride accumulation during adipogenesis but also contributed to the lipolysis of adipocytes. CONCLUSIONS: In the present study, we demonstrate that CAF suppressed adipogenesis in 3T3-L1 adipocytes. Our results indicated that CAF down-regulates the expression of C/EBPß and subsequently inhibits the activation of PPARγ and C/EBPα. The anti-adipogenic activity of CAF was mediated by the inhibition of Akt activation and GSK3ß phosphorylation, which induced the down-regulation of lipid accumulation and lipid metabolizing genes, ultimately inhibiting adipocyte differentiation.

Spent mushroom substrate influences elk (cervus elaphus canadensis) hematological and serum biochemical parameters.

The objective of this study was to evaluate the effect of spent mushroom substrate (SMS) derived from Pleurotus eryngii on the hematological and biochemical blood properties of elk. A total of 18, two and three-year-old elk were fed three different levels of SMS (0, 15 and 20%) in a corn-wheat bran diet for 80 days. The results indicated significantly high levels of blood monocytes, hemoglobin (Hb), and hematocrit (HCT) in elk fed 15% or 20% SMS (p<0.05) compared to control animals. Serum blood urea nitrogen (BUN) and glucose concentrations were also significantly elevated in elk fed both 15% and 20% SMS. The inclusion of SMS in the elk diet did not affect serum total cholesterol, triglyceride, or low density lipoprotein (LDL)-cholesterol concentrations; however, high density lipoprotein (HDL)-cholesterol concentration was significantly increased in SMS-fed groups. In addition, 20% SMS in the diet increased serum iron and testosterone concentrations in elk. These results indicate that adding SMS to the diet of elk can increase their Hgb, serum BUN, glucose, and HDL-cholesterol concentration; therefore, diets containing SMS may enhance the physiologic condition of elk during growth.

Induction of Ski Protein Expression upon Luteinization in Rat Granulosa Cells.

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells; however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinizationto predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rats, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH) surge. 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 the corpus luteum (CL). Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggests that its expression is regulated post-transcriptionally.

Differential expression and localization of tight junction proteins in the goat epididymis.

The blood-epididymis barrier (BEB) forms a unique microenvironment that is crucial for the maturation, protection, transport, and storage of spermatozoa in the epididymis. To characterize the function of tight junctions (TJs), which are constitutive components of the BEB, we determined the expression and localization of TJ proteins such as zonula occludens (ZO)-1, 2, and 3, occludin, and claudin3 (Cldn3) during postnatal development in the goat epididymis. To assess the expression patterns of TJ proteins in immature (3 months of age) and mature (14 months of age) goat epididymides, two different experimental methods were used including immunofluorescence labeling and western blotting. We show that, ZO-1, 2, and 3, and occludin, were strictly expressed and localized to the TJs of the goat epididymis, whereas Cldn3 was present in basolateral membranes as well as TJs. All TJ proteins examined were more highly expressed in the immature epididymis compared to levels in mature tissue. In conclusion, our study indicates that at least five TJ proteins, namely ZO-1, ZO-2, ZO-3, occludin, and Cldn3, are present in TJs, and the expression strength and pattern of TJ proteins tend to be age dependent in the goat epididymis. Together, these data suggest that the distinct expression patterns of TJ proteins are essential for regulating components of the luminal contents in the epididymal epithelium and for forming adequate luminal conditions that are necessary for the maturation, protection, transport, and storage of spermatozoa in the goat epididymis.

Ultrasonic Cutting of Frozen Semen Straws to Optimize the Use of Spermatozoa for In Vitro Fertilization.

The objective of the present study was to establish conditions for using technology that can potentially enhance the efficiency of bovine embryos derived from in vitro fertilization (IVF) with frozen semen. Frozen semen from selected bulls can be stored indefinitely in liquid nitrogen as genetic resources; however, these resources are considered consumable because they cannot be regenerated. Therefore, to optimize the utilization of frozen semen, as many oocytes as possible should be fertilized with one straw. However, a sufficient number of prepared oocytes might not be available for one experiment, which can limit the use of the total spermatozoa population. Thus, an economical method for producing embryos needs to be established by optimizing technology for transplantable embryos. In this study, the utilization of frozen semen was increased by dividing the straw with an ultrasonic cutter. The post-thaw survival rate of uncut straws from Korean Proven Bulls did not differ from that of half cuttings. When ultrasonic cutting was applied to frozen semen, spermatozoa could be prepared for IVF trials at least four times, and blastocysts were produced. Therefore, cutting frozen semen with an ultrasonic cutter represents a potentially useful tool to expand genetic resources from excellent breeding stocks. This approach could also be valuable in the field of IVF of endangered species or rare breeds for their preservation, as well as in ovum pick-up (OPU) techniques.

Development and Differentiation of Epididymal Epithelial Cells in Korean Native Black Goat.

The acidic luminal environment of the epididymis is regulated by the communication networks among epididymal epithelial cells; it is necessary for sperm maturation and storage. To characterize epididymal epithelial cell differentiation, the localization and expression of hydrogen-pumping vacuolar ATPase (V-ATPase) and cytokeratin 5 (KRT5) in the clear and basal cells, respectively, of immature and mature goat epididymis and vas deferens was examined. The epididymides and vas deferens were obtained from goats aged 1, 2, and 12-14 months. To assess the localization and expression patterns of V-ATPase and KRT5 in the caput, corpus, and cauda of the epididymis and proximal vas deferens, the tissue sections were subjected to immunofluorescence labeling and observed by confocal microscopy. Both clear and basal cells progressively started to differentiate in a retrograde manner. Clear cells disappeared from the cauda region after puberty, and they were maintained only in the caput and corpus regions of the adult goat epididymis. V-ATPase and KRT5 were co-expressed in the differentiated cells located at the base of the epithelium (i.e., basal cells). This cell type-specific differentiation and distribution of the epithelial cells plays a critical role in establishing a unique luminal environment for sperm maturation and storage in the goat epididymis.

Identification of trophoblast-specific binding sites for GATA-2 that are essential for rat placental lactogen-I gene expression.

We identified a 3.4-kb 5'-flanking region of the rPL-I gene and examined its promoter activity using rat trophoblast Rcho-1 cells. A regulatory element between base pairs (bp) -2,487 and -2,310 in the 5'-flanking region was essential for maximum promoter activity of the rPL-I gene. This regulatory element was further characterized between bp -2,443 to -2,415 and -2,374 to -2,345. Electrophoretic mobility shift analysis showed that the interaction of nuclear extract proteins from differentiated Rcho-1 cells was inhibited by competition with a GATA-like sequence in the promoter, but not by a mutated GATA sequence. Moreover, the promoter activity of 2487 eLuc containing two novel GATA sites was significantly elevated by co-transfection of a GATA-2 expression vector in proliferating Rcho-1 cells. Our results demonstrate that GATA-2 is involved in multiple promoter regions to activate the specific expression of the rPL-I gene in placental tissue.

Osmolarity at early culture stage affects development and expression of apoptosis related genes (Bax-alpha and Bcl-xl) in pre-implantation porcine NT embryos.

This study examined whether high osmolarity of culture medium at the early culture stage affects development and expression of apoptosis related genes (Bax-alpha and Bcl-xl) of porcine nuclear transfer (NT) and in vitro fertilization (IVF) embryos. NT and IVF embryos were divided into three groups and the basic medium was PZM-3 (260-270 mOsmol, control group). The control group of embryos was cultured in PZM-3 for whole culture period. Other two groups of embryos were cultured in a modified PZM-3 with 0.05 M sucrose (300-320 mOsmol, sucrose group) or increased NaCl to 138 mM (300-320 mOsmol, NaCl group) for the first 2 days, and then cultured in PZM-3 for 4 days. NT embryos cultured in NaCl group showed a significantly higher developmental rate to the blastocyst stage with a decreased apoptosis rate compared to the control (P < 0.05). There was no difference in blastocyst formation and apoptosis incidence among the three culture treatments for IVF-derived embryos. Bax-alpha mRNA expression was significantly higher in the control than sucrose or NaCl group for both NT and IVF embryos (P < 0.05). Moreover, the relative abundance of Bax-alpha/Bcl-xl was higher in the control than the treatment groups. These results indicate that the higher osmolarity at the early embryonic stage of porcine NT and IVF embryos can improve the in vitro development with reduced apoptosis through regulating the Bax-alpha/Bcl-xl gene expression.