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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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Gremlin genes are known members of the DAN family of bone morphogenetic protein (BMP) antagonists, but their functions and regulatory mechanisms in ovarian follicular development of chicken remain unknown. The current study was designed to investigate the mRNA expression patterns of gremlin1 gene (GREM1) and its protein location in the follicles sampled, and to explore the biological effect of GREM1 on the prehierarchical follicular development. This work revealed that chicken GREM1 mRNA exhibits a constant expression level across all the prehierarchical follicles (PFs) from 1-4 mm to 7-8 mm in diameter, and the preovulatory follicles (from F6 to F1) by using RT-qPCR (P > 0.05). The GREM1 protein is predominantly expressed in the oocytes and granulosa cells (GCs) of the PFs by immunohistochemistry. Furthermore, our data demonstrated that siRNA-mediated knockdown of GREM1 in the GCs resulted in a significant reduction in cell proliferation (P < 0.001); conversely, overexpression of GREM1 in the GCs led to a remarkable increase in cell proliferation (P < 0.001). Interestingly, the expression levels of proliferating cell nuclear antigen (PCNA) and cyclin D2 (CCND2) mRNA and proteins were notably increased when GREM1 expression was upregulated in the GCs (P < 0.01), however, the expression levels of CYP11A1 and StAR were markedly downregulated (P < 0.01). The current results showed that GREM1 gene plays a stimulatory role in GC proliferation during growth and development of the prehierarchical follicles in vitro but an inhibitory role in GC differentiation and steroidogenesis of the hen ovary follicles., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The SLIT/ROBO pathway has been implicated in prehierarchical follicular development of hen ovary by an intrafollicular autocrine and/or paracrine fashion. SLIT3, one of the key components of the SLIT/ROBO family, serves as a ligand that potentially interacts with the four receptors, ROBO1, ROBO2, ROBO3 and ROBO4. But the exact roles and regulatory mechanism of SLIT3 in chicken ovarian follicle development remain largely unclear. The present study was conducted to investigate the potential roles and molecular regulation of SLIT3 in granulosa cell (GC) proliferation, differentiation and follicle selection within the prehierarchical follicles of hen ovary. We found that SLIT3 interacts physically with the four ROBO receptors, but the expression of the ROBO1 and ROBO2 genes are more susceptible to the regulation of SLIT3 ligand than that of the ROBO3 and ROBO4 genes. Moreover, the siRNA-mediated knockdown of SLIT3 in the follicular GCs leads to a significant increase in cell proliferation. Conversely, overexpression of SLIT3 results in a remarkable reduction in GC proliferation. Furthermore, the overexpressed SLIT3 has notably decreased the mRNA and protein expression levels of follicle-stimulating hormone (FSHR), growth and differentiation factor 9 (GDF9), steroidogenic acute regulatory protein (STAR) and cytochrome P450 11A1 (CYP11A1) in the GCs. These results indicated that SLIT3 may play an inhibitory effect on GC proliferation, differentiation and follicle selection, and these suppressive actions of SLIT3 in the GC proliferation can be prohibited by the siRNA-mediated knockdown of ROBO1 and ROBO2 receptors. The current data provide a basis for further investigation of molecular mechanisms of SLIT3-ROBO1/2 pathway in controlling the prehierarchical follicle development of the hen ovary., (© 2018 International Federation for Cell Biology.)

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.

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: The growth and development of ovary follicles is an intricate, highly organized process involving many local intra-ovarian factors. Ras-related C3 botulinum toxin substrate1 (RAC1) is speculated to be associated with prehierarchical follicle development of hen ovaries. The current study initially revealed RAC1 mRNA to be expressed in varied-size follicles and stroma and its expression levels in the prehierarchical follicles of 1.0–3.9 mm, 6.0–6.9 mm and 7.0–8.0 mm in diameter were remarkably higher than the other groups. Moreover, RAC1 protein was mainly expressed in the oocytes and granulosa cells (GC), as well as in stromal tissues of the follicles. To understand the exact roles of the RAC1 gene in regulation of follicular GC proliferation and differentiation, siRNA interference and overexpression of the RAC1 gene were conducted. Our experiments demonstrated that the RAC1 gene can significantly promote the expression of mRNA and proteins of FSHR, CCND2, CYP11A1, PCNA and StAR genes in GC and directly elevate the proliferation of GC in vitro. These results indicated RAC1 played a crucial role in regulation of GC proliferation and differentiation and steroidogenesis during the development of prehierarchical follicles. This study provided a base for elucidating the molecular mechanisms underlying the biological effect of RAC1 on the hen ovary follicle growth and development. 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. [ABSTRACT FROM AUTHOR]

Transcription cofactors Vestigial like family (VGLL) members consisting of four homologs (VGLL1-4) are associated with cell growth and metastasis in mammals, among which VGLL1 gene has been documented to possess tumorigenic functions in various types of tumor, and VGLL4 acts as a new tumor suppressor; likewise several studies indicated that they potentially play a role in the regulation of ovary growth and function. However, the biological effects of chicken VGLL1 and VGLL4 on the proliferation, apoptosis, and steroidogenesis of the granulosa cells (GCs) during ovarian follicle development remain unknown now. This study found that VGLL1 and VGLL4 genes present divergent expression patterns of the transcripts in the GCs of various sized prehierarchical follicles (PFs) before follicle selection. Specific small interfering RNA (siRNA) was employed to elucidate the exact roles of VGLL1 and VGLL4 in regulating the PF development of the hen ovary. The results demonstrated that the mRNA expression levels of the steroidogenic-related enzyme steroidogenic acute regulatory protein (STAR) gene and the cell proliferation-related factors B-cell lymphoma-2 (BCL2), and cyclin D1 (CCND1) genes were significantly down-regulated in the cells with VGLL1 silence but remarkably up-regulated in the cells lacking VGLL4. Whereas the expression level of the cell apoptosis biomarker caspase-3 (CASP3) transcript was noticeably enhanced in the GCs without VGLL1 but significantly decreased in the GCs deprived of VGLL4. Further results showed that the siRNA-mediated silence of VGLL1 caused a significant increase in apoptosis with a reduction in the proliferation of GCs. Nevertheless, knockdown of VGLL4 resulted in a remarkable decrement in apoptosis but a memorable augment in proliferation of the GCs. Taken together, this study proved that VGLL1 promotes cell proliferation and steroidogenesis but inhibits apoptosis. In contrast, VGLL4 stimulates GC apoptosis while suppressing the GC proliferation and steroidogenesis in the hen ovarian follicles. We conluded that VGLL1 and VGLL4 affect oppositely the ovarian prehierarchical follicle development by the different regulatory manner in the GC proliferation and apoptosis of chicken ovary. • The VGLL1 and VGLL4 mRNA present divergent expression patterns in the follicular GCs. • VGLL1 promotes the GC proliferation and steroidogenesis but inhibits its apoptosis. • VGLL4 enhances the GC apoptosis but represses the proliferation and steroidogenesis. • VGLL1 and VGLL4 play an opposite role in ovary prehierarchical follicle development. [ABSTRACT FROM AUTHOR]