The SLIT2 ligand and ROBO receptors of the SLIT/ROBO pathway are expressed in hen ovarian follicles and have been shown to play critical roles in ovary development, cell proliferation and apoptosis in mammals. However, the exact roles of SLIT2 and the molecular mechanisms of chicken follicle development remain poorly understood. Here, we discovered that high levels of SLIT2 suppress FSHR, GDF9, STAR and CYP11A1 mRNA and protein expression in granulosa cells (GCs) and cell proliferation (p < 0.01). However, these inhibitory effects can be abolished by the siRNA-mediated knockdown of the ROBO1 and ROBO2 receptors. Furthermore, the activity of CDC42, which is a key Rho GTPase in the SLIT/ROBO pathway, is regulated by the ligand SLIT2 because the intrinsic GTPase activation activity of CDC42 is activated or repressed by regulating SRGAP1 expression (p < 0.01). The effects of the SLIT2 overexpression on GC proliferation and phosphorylation of the B-RAF, RAF1 and ERK1/2 kinases were completely abrogated by knocking down endogenous PAK1 and partially abrogated by the knockdown of PAK2 and PAK3 in the GCs. Collectively, our findings indicate that SLIT2 suppresses GC proliferation, differentiation and follicle selection mainly by a mechanism involving ROBO1 and ROBO2 and that this suppression is mediated by the CDC42-PAKs-ERK1/2 MAPK signaling cascade in the prehierarchical follicles of the chicken ovary.

The Hippo/MST signaling pathway is a critical player in controlling cell proliferation, self-renewal, differentiation, and apoptosis of most tissues and organs in diverse species. Previous studies have shown that Salvador homolog 1 (SAV1), a scaffolding protein which functions in the signaling system is expressed in mammalian ovaries and play a vital role in governing the follicle development. But the exact biological effects of chicken SAV1 in prehierarchical follicle development remain poorly understood. In the present study, we demonstrated that the SAV1 protein is predominantly expressed in the oocytes and undifferentiated granulosa cells in the various sized prehierarchical follicles of hen ovary, and the endogenous expression level of SAV1 mRNA appears down-regulated from the primordial follicles to the largest preovulatory follicles (F2-F1) by immunohistochemistry and real-time RT-PCR, respectively. Moreover, we found the intracellular SAV1 physically interacts with each of the pathway members, including STK4/MST1, STK3/MST2, LATS1 and MOB2 using western blotting. And SAV1 significantly promotes the phosphorylation of LATS1 induced by the kinase of STK4 or STK3 in vitro. Furthermore, SAV1 knockdown by small interfering RNA (siRNA) significantly increased proliferation of granulosa cells from the prehierarchical follicles (6–8 mm in diameter) by BrdU-incorporation assay, in which the expression levels of GDF9, StAR and FSHR mRNA was notably enhanced. Meanwhile, these findings were consolidated by the data of SAV1 overexpression. Taken together, the present results revealed that SAV1 can inhibit proliferation of the granulosa cells whereby the expression levels of GDF9, StAR and FSHR mRNA were negatively regulated. Accordingly, SAV1, as a member of the hippo/MST signaling pathway plays a suppressive role in ovarian follicle development by promoting phosphorylation and activity of the downstream LATS1, may consequently lead to prevention of the follicle selection during ovary development.

Egg production is an important economic trait in the commercial poultry industry. Ovarian follicle development plays a pivotal role in regulation of laying hen performance and reproductive physiology. However, the key genes and signaling pathways involved in the various-stages of laying hen follicular development remain poorly understood. In this study, transcriptomes of ovarian follicles at three developmental stages, the large white follicle (LWF), small yellow follicle (SYF), and large yellow follicle (LYF), were comparatively analyzed in hens with high (HR) and low (LR) egg-laying rates by RNA-sequencing. Eighteen cDNA libraries were constructed and a total of 236, 544, and 386 unigenes were significantly differentially expressed in the LWF, SYF, and LYF follicles of HR and LR hens, respectively. Among them, 47 co-transcribed differentially expressed genes (DEGs) in LWF and SYF, 68 co-expressed DEGs in SYF and LYF, and 54 co-expressed DEGs in LWF and LYF were mined. Thirteen co-expressed DEGs were found in LWF, SYF, and LYF follicles. Eighteen candidate genes, including P2RX1, CAB39L, BLK, CSMD3, GPR65, ADRB2, CSMD1, PLPP4, ATF3, PRLL, STMN3, RORB, PIK3R1, PERP1, ACSBG1, MRTO4, CDKN1A, and EDA2R were identified to be potentially related to egg production. Furthermore, Kyoto Encyclopedia of Genes and Genomes analysis indicated neuroactive ligand-receptor interaction, cell adhesion molecules, peroxisome proliferator-activated receptor pathway, and cAMP signaling pathway might elicit an important role in formation of egg-laying traits by influencing ovarian follicle development. This study represents the first transcriptome analysis of various-sized follicles between HR and LR hens. These results provide useful molecular evidence for elucidating the genetic mechanism underlying ovarian follicle development associated with egg production in chicken.

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.

Forkhead box L2 (FOXL2) is a member of the forkhead nuclear factor 3 gene family and plays an essential role in ovarian growth and maturation in mammals. However, its potential effects and regulative mechanism in development of chicken ovarian prehierarchical follicles remain unexplored. In this study, the cooperative effects of FOXL2 with activin A, growth differentiation factor-9 (GDF9) and follistatin, three members of the transforming growth factor beta (TGF-β) superfamily that were previously suggested to exert a critical role in follicle development was investigated. We demonstrated herein, using in-situ hybridization, Northern blot and immunohistochemical analyses of oocytes and granulosa cells in various sizes of prehierarchical follicles that both FOXL2 transcripts and FOXL2 proteins are predominantly expressed in a highly similar expression pattern to that of GDF9 gene. In addition, the FOXL2 transcript was found at lower levels in theca cells in the absence of GDF9. Furthermore, culture of granulosa cells (GCs) from the prehierarchical follicles (6–8 mm) in conditioned medium revealed that in the pcDNA3.0-FOXL2 transfected GCs, there was a more dramatic increase in FSHR mRNA expression after treatment with activin A (10 ng/ml) or GDF9 (100 ng/ml) for 24 h which caused a stimulatory effect on the GC proliferation. In contrast, a significant decrease of FSHR mRNA was detected after treatment with follistatin (50 ng/ml) and resulted in an inhibitory effect on the cell proliferation. The results of this suggested that FOXL2 plays a bidirectional modulating role involved in the intracellular FSHR transcription and GC proliferation via an autocrine regulatory mechanism in a positive or negative manner through cooperation with activin A and/or GDF9, and follistatin in the hen follicle development. This cooperative action may be mediated by the examined Smad signals and simultaneously implicated in modulation of the StAR, CCND2, and CYP11A1 expression.

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.