Bone morphogenetic protein 4 (BMP4) has an important role in regulating cellular proliferation, differentiation and apoptosis. It, however, is still unclear as to the mechanisms by which BMP4 regulates the apoptosis of granulosa cells (GCs) in geese. In the present study, there was cloning of the full-length coding sequence of goose BMP4 gene, which consisted of 1212 nucleotides encoding 403 amino acids. Its deduced amino acid sequence comprised one signal peptide, one TGFβ pro-peptide and one mature peptide domain. Results from conducting the quantitative real-time PCR (qPCR) indicated the relative abundances of BMP4 mRNA in geese GCs increased gradually from the relative abundances in pre-hierarchical follicles that were 4 to 6 mm in diameter to that in the fifth largest (F5) follicle and then relative abundances of BMP4 mRNA decreased with further development as the largest (F1) follicle. Results from use of the TUNEL assay indicated that overexpression of the goose BMP4 gene suppressed GC apoptosis and this was confirmed when relative abundances of the CAD, Caspase-9 and Caspase-3 proteins were determined using western blotting. In addition, overexpression of the BMP4 gene induced phosphorylation of AKT, which was inhibited with use of the PI3K inhibitor, LY294002. Co-transfection of BMP4 and LY294002 resulted in increased relative abundances of Caspase-9 and CAD proteins but had no effect on that of Caspase-3. Taken together, these results suggested that expression of the BMP4 gene resulted in a reduction in Caspase-9 protein leading to inhibition of GC apoptosis via the PI3K/AKT signaling pathway in geese.

Very low density lipoprotein receptor (VLDLR)-mediated endocytosis of plasma lipoproteins into the ovary is essential for ovarian follicle development. Two splice variants of VLDLR have been identified in several species, yet little is known about their distinctive roles in ovarian developing follicles. In the present study, the full-length cDNAs of two splice isoforms of VLDLR were obtained from geese (Anser cygnoide) ovaries using the RACE method. The longer isoform (TypeI VLDLR) is 3141bp and contains five conserved structural domains, while the other (TypeII VLDLR) lacks 90bp encoding for the O-linked sugar domain. TypeII VLDLR was predominantly expressed in the ovary, with greater amounts of mRNA in theca and granulosa cells from early stages of follicle development but decreased during vitellogenesis. However, there was minimal expression of the TypeI VLDLR gene in theca cells and expression was almost undetectable in granulosa cells throughout follicle development. Yolk VLDL concentrations decreased as stage of development advanced while yolk triglyceride and cholesterol concentrations increased in a follicular size-dependent manner. The significant correlations between transcripts of TypeII VLDLR and yolk lipids supported its important role on yolk lipid deposition. In addition, in vitro experiments suggested that exogenous cholesterol, 25-hydroxycholesterol and mevinolin (a highly potent competitive inhibitor of HMG-CoA) treatment could significantly alter TypeII VLDLR gene expression in granulosa cells from both pre-hierarchical and pre-ovulatory follicles. Collectively, data from the present study indicate that TypeII VLDLR is more important for the transport of plasma lipoproteins into developing follicles than TypeI VLDLR, and provide new evidence about the influence of steroids in modulating VLDLR gene expression in ovarian cells.