Dongjie Zhang

Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (−) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

Myeloid zinc finger 1 (MZF1) is the most important transcriptional factor for porcine follistatin promoter

Follistatin (FS) is a secreted protein, which was originally isolated from porcine follicular fluid. Expression of follistatin is tightly regulated during porcine growth and development. To study the essential transcriptional regions of the porcine FS promoter, ten primer pairs were designed to amplify segments with different lengths of the FS promoter from −1 800 to +16 bp. The products were then inserted into the pGL3-basic vector to analyze the relative luciferase activity. The results showed that the most remarkable changes of promoter activity were observed between constructs (−302/+16 bp)-FS and (−180/+16 bp)-FS (P<0.01). Further research showed that the reconstructed reporter plasmid lacking myeloid zinc finger 1 (MZF1) binding sequence had significantly decreased luciferase activity (P<0.05). Furthermore, the FS protein expression was significantly increased in PK15 cells while the MZF1 was overexpressed, suggesting that the short sequence “TCCCCACC” (the recognition site of transcription factor MZF1) was the most important for FS transcription activation in the porcine.

Impact of the NCOA1, OPN and RBP4 genes on individual weight at birth and individual weight at 30 days in hybrid pig

Abstract The effects of the polymorphism of the nuclear receptor coactivator 1 gene (NCOA1), osteopontin (OPN), and retinol-binding protein 4 (RBP4) genes on individual weight at birth (WTB) and individual weight at 30 days (WT30) were analyzed by Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) in hybrid pig (Landrace ♀ ×Wild boar ♂). The relationship between different genotypes and WTB and WT30 were analyzed. Results showed that: AA genotype of NCOA1 had higher WT30 than BB individuals (P<0.05). AA and AB genotypes of OPN had a higher WTB and WT30 than BB (P>0.05). BB and AB genotypes of RBP4 had a higher WTB and WT30 than AA (P>0.05). The results presented here demonstrate that the NCOA1 gene may be considered as a marker for WT30 trait in pig.

Bovine oocyte competence shows better tolerance to seasonal cold stress in cold areas of Northern China

ABSTRACT Oocyte competence is an important determinant of reproductive success and can be disrupted by heat/cold stress. Information related to seasonally induced cold stress effects in cold areas on oocyte competence in the bovine is still scarce, however. Therefore, we performed experiments to test the hypothesis that seasonally induced cold/heat stress affects bovine oocyte competence in cold areas. Oocytes were collected in the winter, summer, and autumn, and we showed a significantly better survival and growth rate in the cold compared with hot temperatures, as recorded for two-cell and blastocyst stage embryos (Pu2009<u2009.05). As we intend to perform in vitro fertilization-embryo transfer in winter, we observed a satisfactory level of pregnancy and birth rates during this time period, even when compared with other studies in cold and hot weather (Pu2009<u2009.05). Therefore, our results suggest that oocyte competence is not affected by cold stress.

Molecular cloning and functional characterization of myogenin promoter in Min pig

ABSTRACT Myogenin (MyoG) plays an important role in muscle differentiation, which can initiate the formation of the fusion of muscle cells and myofibroblasts. To investigate the transcriptional regulatory mechanism of MyoG gene in Min pig, the promoter fragment spanning from −2085u2005bp to +118u2005bp was amplified with PCR, and the potential transcriptional elements were predicted via bioinformatics analysis. Subsequently, nine promoter segments with different base pair sizes were obtained and ligated into PGL3-Basic vectors, respectively. Lastly, the functional activity of each of the aforementioned promoters was tested separately in transiently transfected C2C12 cells via a dual-luciferase assay system. The results indicated that the sequence −199u2005bp to −31u2005bp may have the basic activity of the promoter. Further analysis demonstrated that the sequence −137u2005bp to −101u2005bp was important for the basic transcriptional activity. Bioinformatics analysis showed that CdxA and MZF1 transcription factors may play an important role in the regulation of MyoG expression.

A comparative study of the effects of long-term cold exposure, and cold resistance in Min Pigs and Large White Pigs

ABSTRACT Min Pig (Sus scrofa) is a northern Chinese domestic pig breed, whose cold resistance is remarkable. However, the cold-resistant phenotype of Min Pig is unclear. Our studies showed that in long-term cold exposure, the death rate of Min Pigs is significantly lower than that of the control Large White (LW) Pigs. The ear tissue freezing rate for Min Pigs is 0%, but that for LW Pigs is 100%. The shivering time and shivering frequency of Min Pigs are both significantly lower than that of LW Pigs at 5 months (Pu2009<u2009.005) and in 80–90u2005kg pigs (Pu2009<u2009.001). Our results indicated that Min Pig is more cold resistant than LW Pig in long-term cold exposure. These findings provided important reference data for the field of cold resistance research in Min Pigs, and for the breeding of cold-resistant pigs.