Transcriptomic analysis of ovarian follicles uncovers the crucial genes relevant to follicle selection and preovulatory hierarchy in hens.

Follicle selection and preovulatory hierarchy of hen ovaries were important stages of follicle development and crucially determining egg-laying performance. The selected follicles with a higher expression level of follicle-stimulating hormone receptor (FSHR) mRNA that facilitates response to FSH, and rapidly develops into preovulatory follicles with distinctive characteristics of granulosa cells (GCs) proliferation and differentiation. Identification of the key genes involved in these developmental events is helpful for elucidation of the molecular mechanism underlying egg-laying traits in chicken and other domestic fowl. Herein, the comparative transcriptomic analysis of ovarian prehierarchical follicles before selection (BSF), follicles at selection stage (ASF), and hierarchical follicles (HF) were implemented in the Jilin Black chicken (JB) and Lohmann Brown layer (LB) with the divergences in their egg-laying performance by RNA-sequencing. The results showed that nine deferentially expressed genes (DEGs), including STMN4, FABP3, ROBO2, RSPO4, and DMRT1 were revealed between follicles BSF and ASF; and seventeen DEGs, such as SLC6A15, SLITRK3, PRKG2 and TMC3 were mined between ASF and HF. These two group DEGs being co-expressed between BSF and ASF, and between ASF and HF were compared and substantiated in the JB and LB layers, respectively. Furthermore, 10 signaling pathways, such as cAMP signaling, PPAR signaling pathway, AMPK(Adenosine 5'-monophosphate (AMP)-activated protein kinase) pathway, and estrogen signaling pathway were also identified. Moreover, the roles of two representative candidates ROBO2 and PRKG2 genes presented as downregulated mRNA expression pattern in the transcriptomic profiles were further verified in vitro. The results demonstrated that downregulation of ROBO2 or PRKG2 significantly increased the expression levels of FSHR mRNA and protein with the boosted expression of CCND1, STAR, and BCL-2, whereas remarkably inhibited the expression of Caspase-3, consequently, brought about the decrease of GC apoptosis in the ovarian follicles, but increase of GC proliferation and differentiation serving as the hallmarks for follicle selection. It indicated that ROBO2 and PRKG2 may play indispensable roles in follicle selection and preovulatory hierarchy of hen ovaries separately. Our findings provided a comparative transcriptomic evidence for clarifying the molecular mechanism of the follicle development underlying egg-laying traits in chicken.

Chicken ovarian follicle development undergoes follicle recruitment, prehierarchy, follicle selection, preovulatory/hierarchy, and finally ovulation. The follicle selection and preovulatory hierarchy play a vital role in egg production of hens. However, underlying the mechanism of the key genes involved in these developmental events remains largely unknown. Herein, to explore the promising genes potentially involved in follicle selection and hierarchical development of hen ovary, a comparative transcriptome analysis of the ovarian follicles before and after selection was performed by using Jilin Black (JB) chicken, an indigenous Chinese breed with a low egg-laying rate and strong broodiness, and which was substantiated by using Lohmann Brown (LB) layer, a commercial egg-laying breed, being characterized by a high rate of egg production. As a result, a total of nine critical differentially expressed genes (DEGs) that co-expressed in the ovarian follicles before selection compared with follicles at selection stage (ASF), and 17 DEGs in the ASF follicles compared with hierarchical follicles that developed shortly after the time of follicle selection were identified in the JB hens as well as in the LB layers, respectively. Moreover, the exact roles of two representative candidates ROBO2 and PRKG2 of the DEGs were further verified in regulation of the follicular granulosa cell proliferation and differentiation which are the major characteristics of follicle selection. And 10 signaling pathways that implicated in follicle selection and hierarchy were also enriched. The objectives aim to provide molecular evidence for underlying the regulatory mechanism of follicle development and egg production in chicken.

Differential regulation on C5 expression in goose versus mammals by glucose/palmitate provides a potential protection for goose fatty liver.

Goose fatty liver is a specific type of nonalcoholic fatty liver that is protected from harmful effects associated with severe steatosis. Our previous findings suggest that suppression of the complement C5 may be relevant, but the mechanism is unclear. Therefore, in this study, we first verified the expression pattern of complement genes (including C5) during goose fatty liver formation and then determined the liver fat content and fatty acid composition by high-performance liquid chromatography (HPLC), followed by selecting the differential metabolites to treat HepG2, goose and mouse primary hepatocytes, aiming to explore the mechanism of C5 and inflammation suppression in goose fatty liver. The data confirmed the suppression of complement genes (including C5) in goose fatty livers. Moreover, fat content was significantly higher in fatty liver versus normal ones, with oleic acid and palmitic acid dominantly accounting for the difference. In line with this, high concentration of palmitate led to down regulation of C5 expression in goose primary hepatocytes whereas upregulation in mouse primary hepatocytes and HepG2 cells. In conclusion, regulation on C5 expression by fatty liver related factors including high level of palmitic acid may contribute to the protection of goose liver from severe hepatic steatosis.

Transcriptome comparative analysis of ovarian follicles reveals the key genes and signaling pathways implicated in hen egg production.

BACKGROUND: Ovarian follicle development plays an important role in determination of poultry egg production. The follicles at the various developmental stages possess their own distinct molecular genetic characteristics and have different biological roles in chicken ovary development and function. In the each stage, several genes of follicle-specific expression and biological pathways are involved in the vary-sized follicular development and physiological events. Identification of the pivotal genes and signaling pathways that control the follicular development is helpful for understanding their exact regulatory functions and molecular mechanisms underlying egg-laying traits of laying hens. RESULTS: The comparative mRNA transcriptomic analysis of ovarian follicles at three key developmental stages including slow growing white follicles (GWF), small yellow follicles (SYF) of recruitment into the hierarchy, and differentiated large yellow follicles (LYF), was accomplished in the layers with lower and higher egg production. Totally, 137, 447, and 229 of up-regulated differentially expressed genes (DEGs), and 99, 97, and 157 of down-regulated DEGs in the GWF, SYF and LYF follicles, including VIPR1, VIPR2, ADRB2, and HSD17B1 were identified, respectively. Moreover, NDUFAB1 and GABRA1 genes, two most promising candidates potentially associated with egg-laying performance were screened out from the 13 co-expressed DEGs in the GWF, SYF and LYF samples. We further investigated the biological effects of NDUFAB1 and GABRA1 on ovarian follicular development and found that NDUFAB1 promotes follicle development by stimulating granulosa cell (GC) proliferation and decreasing cell apoptosis, increases the expression of CCND1 and BCL-2 but attenuates the expression of caspase-3, and facilitates steroidogenesis by enhancing the expression of STAR and CYP11A1. In contrast, GABRA1 inhibits GC proliferation and stimulates cell apoptosis, decreases the expression of CCND1, BCL-2, STAR, and CYP11A1 but elevates the expression of caspase-3. Furthermore, the three crucial signaling pathways such as PPAR signaling pathway, cAMP signaling pathway and neuroactive ligand-receptor interaction were significantly enriched, which may play essential roles in ovarian follicle growth, differentiation, follicle selection, and maturation. CONCLUSIONS: The current study provided new molecular data for insight into the regulatory mechanism underlying ovarian follicle development associated with egg production in chicken.

Maintaining intestinal structural integrity is a potential protective mechanism against inflammation in goose fatty liver.

Overfeeding causes severe steatosis but not inflammation in goose liver, suggesting existence of protective components. Previous studies have shown that some intestinal microbes and their metabolites damage intestinal structural integrity and function, thus causing inflammation in the development of human and mouse nonalcoholic fatty liver disease. Therefore, this study hypothesizes that intestinal structural integrity of goose is maintained during overfeeding, which may provide goose fatty liver a protective mechanism against inflammation. To test this hypothesis, 48 seventy-day-old healthy Landes male geese were overfed (as overfeeding group) or normally fed (as control group). Blood and intestine (jejunum, ileum, and cecum) samples were harvested on the 12th and 24th d of overfeeding. Data showed that goose fatty liver was successfully induced by 24 d of overfeeding. Hematoxylin-eosin staining analysis indicated that the arrangement of villi and crypts in the intestine was orderly, and the intestinal structure was intact with no pathological symptoms in the 2 groups. Enzyme-linked immunosorbent assay and quantitative PCR analysis indicated no significant differences in the expression of tight junction and inflammation-related genes as well as plasma lipopolysaccharide concentration between the groups. Ileal hypertrophy and cecal atrophy were observed in the overfed vs. control geese, probably because of change of sphingolipid metabolism. Activation of apoptotic pathway may help cecum avoid necrosis-induced inflammation. In conclusion, healthy and intact intestine provides a layer of protection for goose fatty liver against inflammation. Sphingolipid metabolism may be involved in the adaptation of ileum and cecum to overfeeding. The hypertrophy of ileum makes it an important contributor to the development of goose fatty liver. The atrophy and decline in the function of cecum may be caused by apoptosis induced by overfeeding.

Research Note: Increase of bad bacteria and decrease of good bacteria in the gut of layers with vs. without hepatic steatosis.

Fatty liver hemorrhagic syndrome (FLHS), usually occurring in hens in the late laying period, can lower egg yield and even cause death. Similar to nonalcoholic fatty liver disease in humans, FLHS may begin with simple hepatic steatosis. It is known that gut microbiota is important to the development of nonalcoholic fatty liver disease, but the relationship between FLHS and gut bacteria remains unclear. In this study, bacteria compositions in the ileum and cecum were determined by 16S rDNA sequencing analysis in the groups of hens with vs. without hepatic steatosis (average liver fat contents were 0.34 mmol/g protein or 2.6% vs. 0.84 mmol/g protein or 6.0%). These hens were in the 2 tails of the liver fat content distribution with each tail accounting for 10% of the test population containing 90 66-wk-old healthy Rhode Island Red laying hens raised under routine feeding regimen. The results showed that liver weight but not the weights of the body, heart and abdominal fat were significantly different between the groups. Moreover, bacterial diversity was not significantly different between the groups, but bacterial diversity in the cecum was higher than that in the ileum. Proteobacteria was the most dominant phylum in the ileum, whereas Proteobacteria, Firmicutes, and Bacteroidetes were the most dominant phyla in the cecum. Some bacteria were common to the ileum and cecum, but some were unique. Furthermore, some bacteria were significantly different in abundance between the groups, that is the group without hepatic steatosis had more bacteria inhibiting host energy absorption or benefiting intestinal health, whereas the group with hepatic steatosis had more conditionally pathogenic or harmful bacteria. In conclusion, hepatic steatosis in laying hens is associated with intestinal bacterial composition (bacterial abundance) but not bacterial diversity. The identified common and unique bacteria are related to the functional characteristics of the ileum and cecum. The decrease of beneficial bacteria and the increase of harmful bacteria in the intestine of laying hens may increase FLHS incidence by promoting hepatic steatosis.

Increase of E3 ubiquitin ligase NEDD4 expression leads to degradation of its target proteins PTEN/IGF1R during the formation of goose fatty liver.

Goose fatty liver may have a unique protective mechanism as it does not show a pathological injury even in the case of severe steatosis. Although neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) participates in repair and regeneration of injured liver through its target proteins, its role in nonalcoholic fatty liver disease remains unknown. Using quantitative polymerase chain reaction (PCR) and immunoblot analyses, here, we found that the messenger RNA (mRNA) and protein expressions of NEDD4 were induced in goose fatty liver compared with normal liver. The mRNA expression of the gene of phosphate and tension homology deleted on chromosome ten (PTEN) and insulin-like growth factor 1 receptor (IGF1R) was also induced in goose fatty liver; however, their protein expression was or tended to be suppressed. Moreover, the co-immunoprecipitation analysis indicated that there was a physical association between NEDD4 and PTEN in goose liver, which was consistent with the ubiquitination of PTEN in goose fatty liver. Furthermore, NEDD4 overexpression in goose primary hepatocytes suppressed the PTEN and IGF1R protein levels without a significant effect on their mRNA expression. In conclusion, the increased expression of NEDD4 leads to the degradation of PTEN and IGF1R proteins through ubiquitination in goose fatty liver, suggesting that NEDD4 may protect goose fatty liver from severe steatosis-associated injury via its target proteins during the development of goose fatty liver.