The Notch and transforming growth factor (TGF)-β signalling pathways play an important role in granulosa cell proliferation. However, the mechanisms underlying the cross-talk between these two signalling pathways are unknown. Herein we demonstrated a functional synergism between Notch and TGF-β signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-β signalling increased hairy/enhancer-of-split related with YRPW motif 2 gene (Hey2) expression (one of the target genes of the Notch pathway) in PAGCs, and suppression of TGF-β signalling by Smad3 knockdown reduced Hey2 expression. Inhibition of the proliferation of PAGCs by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT), an inhibitor of Notch signalling, was rescued by both the addition of ActA and overexpression of Smad3, indicating an interaction between the TGF-β and Notch signalling pathways. Co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assays were performed to identify the point of interaction between the two signalling pathways. CoIP showed direct protein-protein interaction between Smad3 and Notch2 intracellular domain (NICD2), whereas ChIP showed that Smad3 could be recruited to the promoter regions of Notch target genes as a transcription factor. Therefore, the findings of the present study support the idea that nuclear Smad3 protein can integrate with NICD2 to form a complex that acts as a transcription factor to bind specific DNA motifs in Notch target genes, such as Hey1 and Hey2, and thus participates in the transcriptional regulation of Notch target genes, as well as regulation of the proliferation of PAGCs.

The fatty acid dehydrogenase I (FatI) is able to express in mammalian cells and convert n-6 polyunsaturated fatty acids (PUFAs) to n-3 PUFAs. n-3 PUFA is an important component of the cell membrane and plays an important role in the prevention and control of a variety of human diseases. However, n-3 PUFAs cannot be endogenously synthesized by mammals because they lack the dehydrogenase that converts n-6 to n-3 PUFA. For the time being, gradually matured transgenic technology makes it possible to produce transgenic animals that are able to synthesize n-3 PUFAs by themselves. However, the transgenic technology itself may bring negative impacts. In this study, the eukaryotic expression vector pcDNA3.1-FatI was introduced into the genome of Boer goat fetal fibroblasts cultured in vitro, and the influence of biological characteristics of the fetal fibroblast was studied via overexpression of FatI. The results showed that the proliferation and apoptosis of cultured fetal fibroblast were not affected significantly by the overexpression of FatI using BrdU and TUNEL staining methods, respectively. Moreover, the overexpression of FatI significantly inhibited the senescence of somatic cells compared with enhanced green fluorescent protein (EGFP) transgenic cells (P < 0.01). Quantitative PCR revealed that the mRNA expression of P16 and P53 in the FatI transgenic cell group was significantly lower than that in the EGFP transgenic cell group (P < 0.01). In conclusion, the senescence of goat somatic cells was inhibited by the overexpression of the FatI gene.

Currently, n-3 polyunsaturated fatty acids (n-3 PUFAs) have attracted great attention because of their biological significance to organisms. In addition, PUFAs show an obvious impact on prevention and treatment of various diseases. Because n-3 PUFAs cannot be endogenously synthesized by mammals, mammals have to rely on a dietary supplement for sufficient supply. The finding and application of the fatty acid dehydrogenase I (FatI) gene are expected to change the current situation because it can convert n-6 polyunsaturated fatty acids (n-6 PUFAs) to n-3 PUFAs. Meanwhile, the gradual maturation of transgenic technology makes it possible to produce transgenic animals that can synthesize n-3 PUFAs by themselves. In this study, the DNA coding sequence of FatI was synthesized by a chemical method after codon optimization according to the mammal's codon bias. The synthesized DNA sequence was introduced into Boer goat fetal fibroblasts by the constructed recombinant eukaryotic expression vector pcDNA3.1(+)-FatI. Boer goat fetal fibroblasts were transfected by electroporation, and the stable transfected cell lines were obtained by G418 selection. Genomic DNA PCR and Southern blot were applied to verify that the foreign gene FatI was integrated into the genome of the Boer goat fibroblasts. RT-PCR results showed the expression of FatI gene at the mRNA level. The fatty acid profile of cells carrying the FatI gene revealed an increase in total n-3 PUFAs (from 0.61 to 0.95), but a decrease in n-6 PUFAs (from 10.34 to 9.85), resulting in a remarkable increase in the n-3:n-6 ratio (from 0.059 to 0.096). The n-3:n-6 ratio had a 63.49 percent increase, which is a precursor of the response of n-3 desaturase activity of the FatI gene. The study may provide a practical tool for producing transgenic animals that can produce n-3 PUFAs by themselves, and we hope that the application will lay the foundation for animals producing n-3 PUFAs, which will benefit human nutrition and wellness.