Transcription factor forkhead box L2 (FOXL2) and growth differentiation factor-9 (GDF9) genes have critical roles in the regulation of hen ovarian development. In the present study, these genes were explored as possible molecular markers associated with BW, hen-housed egg production, and egg weight in Chinese Dagu hens. Samples were analyzed using the PCR-single strand conformation polymorphism (PCR-SSCP) technique followed by sequencing analysis, and two novel single nucleotide polymorphisms (SNPs) were identified within these candidate genes. Among them, an A/G transition at base position 238 in the coding region of the FOXL2 gene and a G/T transversion at base position 1609 in exon 2 of the GDF9 gene were found to be polymorphic and named SNPs A238G and G1609T, respectively. The SNP A238G (FOXL2) leads to a nonsynonymous substitution (isoleucine77-to-valine), and when the 360 Dagu hen samples were divided into genotypes AA and AB, allele A was found to be present at a higher frequency. Furthermore, the AA genotype correlated with significantly higher hen-housed egg production at 30, 43, 57, and 66 wk of age and with a higher egg weight at 43 wk (P<0.05). For the SNP G1609T (GDF9), the hens were typed into TT and TC genotypes, with the T allele shown to be dominant. The TC genotype was also markedly correlated with higher hen-housed egg production and a higher egg weight (P<0.05). Moreover, four haplotypes were reconstructed based on these two SNPs, with the AATC haplotype found to be correlated with the highest hen-housed egg production at 30 to 66 wk of age and with higher egg weights at 43 wk (P<0.05). Collectively, the two SNPs identified in this study might be used as possible genetic molecular markers to aid in the improvement of egg production traits in chicken breeding.

The SLIT/Roundabout (ROBO) pathway is involved in follicle development of mammalian ovary, and 2 secreted hormones activin A and inhibin A have potential roles in modulation of the SLIT/ROBO system, but the related actions remain poorly understood in bird. The aims of the present study were to examine the spatial and temporal expression of the SLIT ligand genes (SLIT1, SLIT2, and SLIT3) and their receptor ROBO1, ROBO2, ROBO3, and ROBO4 genes in various-sized prehierarchical follicles during hen ovary development and the effects of activin A and inhibin A on the expression of these genes in the cultured hen follicles. Our result demonstrated that the transcripts of the 3 SLIT genes were highly expressed in the developing follicles and expression patterns of the SLIT transcripts were different from those of ROBO genes detected by real-time quantitative reverse transcriptase PCR. Both SLIT and ROBO transcripts were predominantly expressed in oocytes and granulosa cells from the prehierarchichal follicles examined by in situ hybridization. The localization for SLIT and ROBO proteins was revealed by immunohistochemistry similar to the spatial distribution of their transcript. In cultured follicles (4 to 8 mm in diameter), the expression levels of SLIT and ROBO members are hormonally regulated by activin A (10 ng/mL) and/or inhibin A (20 ng/mL) after treatment for 24 h. However, the expression of only SLIT2, SLIT3, and ROBO3 mRNA presented a directly opposite response to activin A and inhibin A hormones. These results indicate that SLIT/ROBO pathway is implicated in the prehierarchical follicular development of the hen ovary by an intrafollicular autocrine and/or paracrine action, and is influenced by activin A and inhibin A hormones.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a master regulator of adipogenesis, and alterations in its function are associated with various pathological processes related to metabolic syndrome. Recently, we found that the chicken PPARγ gene is regulated by three alternative promoters (P1, P2 and P3), producing five different transcript isoforms and two protein isoforms. In this study, the P1 promoter structure was characterized. Bioinformatics identified six putative nuclear respiratory factor 1 (NRF1) binding sites in the P1 promoter, and a reporter assay showed that NRF1 inhibited the activity of the P1 promoter. Of the six putative NRF1 binding sites, individual mutations of three of them abolished the inhibitory effect of NRF1 on P1 promoter activity. Furthermore, a ChIP assay indicated that NRF1 directly bound to the P1 promoter, and real-time quantitative RT-PCR analysis showed that NRF1 mRNA expression was negatively correlated with PPARγ1 expression (Pearson’s r = -0.148, p = 0.033). Further study showed that NRF1 overexpression inhibited the differentiation of the immortalized chicken preadipocyte cell line (ICP1), which was accompanied by reduced PPARγ1 mRNA expression. Taken together, our findings indicated that NRF1 directly negatively regulates the P1 promoter of the chicken PPARγ gene and inhibits adipogenesis.

Peroxisome proliferator-activated receptor γ ( PPARγ ) has 2 protein isoforms ( PPARγ1 and PPARγ2 ) generated by alternative promoter usage and alternative splicing. However, their functional uniqueness and similarity remain unclear. In the study, we investigated the effects of lentivirus-mediated overexpression of PPARγ1 and PPARγ2 on proliferation, apoptosis, and differentiation of the immortalized chicken preadipocytes. Cell Counting Kit–8 assay showed PPARγ1 and PPARγ2 overexpression markedly suppressed cell proliferation, and fluorescence activated cell sorting analysis showed that PPARγ1 and PPARγ2 overexpression caused cell cycle arrest at G0/G1 phase. Cell death detection ELISA analysis showed both PPARγ1 and PPARγ2 overexpression induced cell apoptosis. Oil red O staining and gene expression analysis showed both PPARγ1 and PPARγ2 overexpression promoted preadipocyte differentiation. In the presence of PPARγ ligand, rosiglitazone, PPARγ2 overexpression was more potent in inducing apoptosis, promoting adipogenesis, and suppressing cell proliferation than PPARγ1 overexpression. We further explored the molecular basis for their functional differences. Reporter gene assay showed that under ligand conditions, PPARγ2 overexpression resulted in 1.68-fold increase in transcription activity compared with PPARγ1. Electrophoretic mobility shift assay showed both PPARγ1 and PPARγ2 could bind to PPAR response element ( PPRE ) as heterodimer with retinoid X receptor alpha, and by comparison, PPARγ2 had a higher affinity for PPRE than PPARγ1. Reporter gene assay showed expression PPARγ1 and PPARγ2 similarly induced fatty acid synthase and adipocyte fatty acid–binding protein promoter activity but differentially induced lipoprotein lipase and perilipin 1 promoter activities. Coimmunoprecipitation analysis showed that PPARγ1 and PPARγ2 interacted similarly with the coactivators, Tat-interacting protein 60. Taken together, our results demonstrate that PPARγ1 and PPARγ2 differentially regulate preadipocyte proliferation, apoptosis, and differentiation as a result of their distinct and overlapping molecular functions.