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.

The large tumor suppressor homolog 2 (LATS2), one of the central regulators of the Hippo/MST signaling pathway, plays an inhibitory role in ovarian function and different organ development and growth in mammals. However, the exact roles and molecular regulatory mechanisms of LATS2 in chicken granulosa cell (GC) proliferation, differentiation, and steroidogenesis required for ovarian follicle growth, development, and follicular selection remain poorly understood. This study demonstrated that the LATS2 protein was predominantly localized in the oocytes and undifferentiated GCs of various-sized prehierarchical follicles of the hen ovary. Expression levels of LATS2 mRNA were significantly higher in the smaller follicles (from 1 mm to 5.9 mm in diameter) and the GCs than in the larger follicles (6–6.9 mm in diameter up to F1). Moreover, we found that high levels of LATS2 suppressed the GC proliferation and the mRNA and protein expression of the genes serving as the biomarkers of follicle selection, GC differentiation, and steroidogenesis in the GCs, including FSHR, STAR, CYP11A1, ESR1, and ESR2. Interestingly, the LATS2 significantly downregulated SAV1 and YAP1 transcripts but upregulated the expression of STK3, STK4, TEAD1, and TEAD3 mRNA. Our study provided evidences that STK3/4-LATS2-YAP1 not only acts as a suppressor of cell proliferation and follicle selection but also LATS2 may serve as an enhancer in cell proliferation and follicle selection through the YAP1-LATS2 and the LATS2-STK3/4 feedback loops by promoting the expression of TEAD1/3 but inhibiting the expression of SAV1 transcripts in the prehierarchical follicle development of hen ovary. Taken together, the present study initially revealed the pivotal role and molecular mechanism of LATS2 in the regulation of hen prehierarchical follicle development by controlling GC proliferation, differentiation, steroidogenesis, and follicle selection via the Hippo/MST signaling pathway.

RAC1 belongs to the small G protein Rho subfamily and is implicated in regulating gene expression, cell proliferation and differentiation in mammals and humans; nevertheless, the function of RAC1 in growth and development of hen ovarian follicles is still unclear. This study sought to understand the biological effects of RAC1 on granulosa cell (GC) proliferation and differentiation of hen ovarian prehierarchical follicles. Firstly, our results showed expression levels of RAC1 mRNA in the follicles with diameters of 7.0–8.0 mm, 6.0–6.9 mm and 1.0–3.9 mm were greater than other follicles (p < 0.05). The RAC1 protein was mainly expressed in oocyte and its around GCs and stromal tissues of the prehierarchical follicles by immunohistochemistry. Further investigation revealed the RAC1 gene remarkably enhanced the mRNA and protein expression levels of FSHR (a marker of follicle selection), CCND2 (a marker of cell-cycle progression and GC differentiation), PCNA (a marker of GC proliferation), StAR and CYP11A1 (markers of GC differentiation and steroidogenesis) (p < 0.05). Furthermore, our data demonstrated siRNA interference of RAC1 significantly reduced GC proliferation (p < 0.05), while RAC1 gene overexpression enhanced GC proliferation in vitro (p < 0.05). Collectively, this study provided new evidence that the biological effects of RAC1 on GC proliferation, differentiation and steroidogenesis of chicken ovary follicles.

Follicle selection is a pivotal process that determines which dominant prehierarchical follicle will enter the preovulatory hierarchy in the hen ovary and directly affects egg-laying productivity, in which granulosa cells (GCs) are characterized by active proliferation and significantly enhanced FSHR mRNA expression. Increasing evidence has shown that the PI3K/Akt signaling pathway and its important target and effector FOXO1, which promotes GC apoptosis, play crucial roles in ovarian follicular development in mammals. To investigate the molecular mechanism by which follicle-stimulating hormone (FSH)-mediated forkhead box O1 (FOXO1) participates in follicle selection, we treated granulosa cells from 6–8 mm prehierarchical follicles of chickens with FSH and leptomycin B (LMB). The results showed that under FSH and/or LMB treatment, the expression levels of FSHR, FOXO1, and its phosphorylated forms (p-FOXO1) at the predicted protein kinase B (PKB/Akt) phosphorylation sites Thr²⁴, Ser²⁴⁸, and Ser³¹¹ were differentially regulated. The subcellular localization of p-FOXO1 in hen ovarian GCs was determined by Western blotting and immunofluorescence staining (IF) analysis. And the expression of FOXO1 was significantly reduced, whereas the expression of p-FOXO1 corresponding to the PKB phosphorylation sites Ser²⁴⁸ and Ser³¹¹ was noticeably boosted in cultured GCs induced by FSH, accompanied by exclusion of FOXO1 from the nucleus to the cytoplasm. Subsequently, the effects of the PI3K/Akt signaling pathway on phosphorylation levels and nuclear exclusion of p-FOXO1 at the sites Ser²⁴⁸ and Ser³¹¹ were examined. The results indicate that the PI3K/Akt-dependent phosphorylation at these sites directly resulted in nuclear exclusion of FOXO1 in ovarian GCs, in which the Ser²⁴⁸ site is more essential than the Ser³¹¹ site. Subsequently, the FSH-induced acetylation of FOXO1 mediated by the cAMP/PKA pathway can enhance the phosphorylation level of FOXO1 at the Ser²⁴⁸ site. In summary, our findings demonstrate that FSH induces FOXO1 phosphorylation, nuclear exclusion, and functional inactivation by activating the PI3K/Akt signaling pathway. Moreover, during follicular development and selection, FOXO1 acts as a pivotal mediator linking the PI3K/Akt and P62/Keap1/Nrf2 signaling pathways to regulate granulosa cell proliferation and apoptosis, thereby exerting a central regulatory role.

Background: Follicle stimulating hormone (FSH) is key regulator for follicular development, differentiation, and maturation, and the effects involve various intra follicular factors, such as members of the forkhead box O (FOXO) subfamily. However, the specific role and mechanism of FOXO3 and FOXO4 in growth and development of hen follicles by affecting granulosa cell (GC) division and FSH response function are still unclear. Method: This study selected GCs from 6–8 mm chicken follicles, and immunofluorescence and Western blot methods were used to detect FSH-induced FOXO3/4 phosphorylation and nuclear exclusion. Quantitative real-time PCR and flow cytometry were used to investigate the regulatory effects of FSH-induced FOXO3/4 phosphorylation and nuclear exclusion on follicular GC proliferation, differentiation, and apoptosis. Results: This study found that the level of p-FOXO3/4 protein significantly increased in cells treated with FSH for 12 h, while the expression level of non-phosphorylated FOXO3/4 significantly decreased. After co-treatment with 10 ng/mL Leptomycin B (LMB), FOXO3/4 phosphorylation was effectively prevented. The immunofluorescence results showed that FOXO3 and FOXO4 were originally distributed in the GC nucleus and cytoplasm, whereas they were almost accumulated in cytoplasm when treated with FSH for 12 h. Conversely, FOXO3/4 nuclear translocation was blocked by LMB. Moreover, RT-qPCR and flow cytometry results showed that FSH treatment significantly increased proliferation and differentiation of cells but significantly reduced GCs apoptosis. However, LMB also eliminated these stimulating or inhibitory effects on cell proliferation. Conclusion: These findings provide new evidence that FSH-induced FOXO3/4 nuclear exclusion promotes GCs proliferation and reduces GCs apoptosis during hen follicular development.

Introduction: Indigenous chickens are very important to households for income and protein. However, their performance is usually poor, especially under small-scale farmer management, despite their potential to perform better. The performance of these chickens can be improved by selective breeding. However, for this to be a success, there is a need to understand the phenotypic and production characteristics of these chickens fully. Hence, this study aimed to characterize the phenotypes of these chickens and their production system among small-scale farmers.Method: A structured questionnaire was administered to 177 small-scale farmers. A total of 538 chickens whose mean weight was 1.66 kg were individually phenotyped in Luapula, Muchinga, and Northern provinces of Zambia.Results: Ownership of the indigenous chickens was dominated by females (65.37%), with most (64.31%) having attained primary education. Most housed their chickens in family houses (42.03%). All the farmers let their chickens scavenge for their feed, with 45.58% of them providing basic supplementation. Most (84.10%) farmers bought their breed stock from within their community and had a mean flock size of 12.5 chickens/household, which they mostly (78.09%) kept as free-range. The majority (77.39%) practiced culling, with low productivity being the most common reason for culling (84.45%). Only 59.01% of farmers practiced selective breeding, while 86.22% practiced uncontrolled mating. The age at first mating for cocks and hens was 6.8 months and 6.34 months, respectively, with 6.73 months being the age at first egg. It takes 15.43 days to reach a mean clutch size of 13 eggs. The hatchability and mortality at 8 weeks were 83.44% and 67.57%, respectively. All chickens were sold as live chickens, and the majority (51.59%) of the farmers sold their chickens within the community at 7.23 months. Diseases and predators were the most common challenges affecting farmers in the study area. Consultations with veterinarians, vaccinations, and deworming were uncommon while treating sick chickens mostly using ethnoveterinary medicines was common. The most common qualitative traits were brown (27.88%) and mixed (26.77%) plumage color, white skins (91.45%) and shanks (48.70%), single comb type (91.08%), red earlobe (55.76%), and orange eyes (78.07%). All linear body measurements positively and significantly correlated with the body weight averaging 1.66kgs, an indicator that selection for any of them would result in a corresponding increase in body weight. Principal Component Analysis extracted two components with 69.38% of the total variation.Discussion: The diversity in phenotypes of these chickens and their production systems indicate huge potential for improvement by implementing breeding programs.