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.

The opposite effects of VGLL1 and VGLL4 genes on granulosa cell proliferation and apoptosis of hen ovarian prehierarchical follicles.

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.

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.

Novel insights into the regulation of LATS2 kinase in prehierarchical follicle development via the Hippo pathway in hen ovary.

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.

Transcriptome Analysis of Ovarian Follicles Reveals Potential Pivotal Genes Associated With Increased and Decreased Rates of Chicken Egg Production.

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.

Effects of RAC1 on Proliferation of Hen Ovarian Prehierarchical Follicle Granulosa Cells.

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.

Determination of the roles of GREM1 gene in granulosa cell proliferation and steroidogenesis of hen ovarian prehierarchical follicles.

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.

Synergistic inhibition of csal1 and csal3 in granulosa cell proliferation and steroidogenesis of hen ovarian prehierarchical development†.

SALL1 and SALL3 are transcription factors that play an essential role in regulating developmental processes and organogenesis in many species. However, the functional role of SALL1 and SALL3 in chicken prehierarchical follicle development is unknown. This study aimed to explore the potential role and mechanism of csal1 and csal3 in granulosa cell proliferation, differentiation, and follicle selection within the prehierarchical follicles of hen ovary. Our data demonstrated that the csal1 and csal3 transcriptions were highly expressed in granulosa cells of prehierarchical follicles, and their proteins were mainly localized in the cytoplasm of granulosa cells and oocytes as well as in the ovarian stroma and epithelium. It initially revealed that both csal1 and csal3 may be involved in chicken prehierarchical follicle development via a translocation mechanism. Furthermore, our results showed an abundance of CCND1, Bcat, StAR, CYP11A1, and FSHR mRNA in granulosa cells, and the proliferation levels of granulosa cells from the prehierarchical follicles were significantly increased by siRNA-mediated knockdown of csal1 or/and csal3. Conversely, the overexpression of csal1 or/and csal3 in the granulosa cells led to a remarkably decreased of them. Moreover, csal1 and csal3 together exert a much stronger effect on the regulation than any of csal1 or csal3. These results indicated that csal1 and csal3 play synergistic inhibitory roles on granulosa cell proliferation, differentiation, and steroidogenesis during prehierarchical follicle development in vitro. The current data provide a basis of molecular mechanisms of csal1 and csal3 in controlling the prehierarchical follicle development and growth of hen ovary in vivo.

Differential Transcriptome Analysis of Early Postnatal Developing Longissimus Dorsi Muscle from Two Pig Breeds Characterized in Divergent Myofiber Traits and Fatness.

Meat quality traits (MQTs) are very important in the porcine industry, which are mainly determined by skeletal muscle fiber composition, extra-muscular and/or intramuscular fat content. To identify the differentially expressed candidate genes affecting the meat quality traits, first we compared the MQTs and skeletal muscle fiber characteristics in the longissimus dorsi muscle (LDM) of the Northeast Min pig (NM) and the Changbaishan wild boar (CW) with their body weight approaching 90 kg. The significant divergences in the skeletal muscle fiber phenotypes and fatness traits between the two porcine breeds established an ideal model system for further identifying potential key functional genes that dominated MQTs. Further, a transcriptome profile analysis was performed using the Illumina sequencing method in early postnatal developing LDM from the two breeds at the ages of 42 days. Comparative analysis between these two cDNA libraries showed that there were 17,653 and 22,049 unambiguous tag-mapped sense transcripts detected from NM and CW, respectively. 4522 differentially expressed genes (DEGs) were revealed between the two tissue samples, of them, 4176 genes were found as having been upregulated and 346 genes were identified as having been downregulated in the NM library. By pathway enrichment analysis, a set of significantly enriched pathways were identified for the DEGs, which are potentially involved in myofiber development, differentiation and growth, lipogenesis and lipolysis in porcine skeletal muscle. The expression levels of 30 out of the DEGs were validated by real-time quantitative reverse transcriptase PCR (qRT-PCR) and the observed result was consistent noticeably with the Illumina transcriptome profiles. The findings from this study can contribute to future investigations of skeletal muscle growth and development mechanism and to establishing molecular approaches to improve meat quality traits in pig breeding.

Implication of SLIT3-ROBO1/ROBO2 in granulosa cell proliferation, differentiation and follicle selection in the prehierarchical follicles of hen ovary.

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.

Inhibitory effect of SLIT2 on granulosa cell proliferation mediated by the CDC42-PAKs-ERK1/2 MAPK pathway in the prehierarchical follicles of the chicken ovary.

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.

Genetic effects of the transcription factors-sal-like 1 and spalt-like transcription factor 3 on egg production-related traits in Chinese Dagu hens.

Transcription factors (TFs) encoded by SALL1 and SALL3 genes play central roles in the regulation of ovarian development in hens. The present study aimed to examine polymorphisms of these two genes in Chinese Dagu chickens, and to identify the effects of TFs on the laying performance. Among the population, two novel single-nucleotide polymorphisms (SNPs) were identified by single-strand conformation polymorphism (SSCP) in the amplicons of the candidate genes. The effect of the SNP (729C > A) in exon 2 of SALL1 gene on egg production at 43, 57, and 66 weeks and EW at 30 and 43 weeks were the most significant in the 360 samples (P < 0.05). Moreover, for the SNP 1014T > A (in exon 2 of SALL3), the TT genotype was significantly correlated with higher egg production and EW (P < 0.05). Furthermore, four combined genotypes were reconstructed based on the two SNPs. The combined genotype TATT was correlated with the highest egg production at 43-66 weeks and with higher EW at 30, 43 weeks (P < 0.05). The polymorphisms of the two TFs studied are potential molecular genetic markers for chicken breeding, which might help in understanding the genetic structure of laying performance and improving these traits directly by marker-assisted selection (MAS).

Sequence variations in estrogen receptor 1 and 2 genes and their association with egg production traits in Chinese Dagu chickens.

Estrogen receptors α (ESR1) and ß (ESR2) play central roles in folliculogenesis and therefore in reproductive biology. In the present study, two single nucleotide polymorphisms (SNPs) were identified in the ESR1 and ESR2 genes using PCR-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing. One of the identified SNPs, a T1101C transition located within exon 4 of the ESR1 gene, was significantly associated with hen-housed egg production (HHEP) at 30, 43, 57 and 66 weeks of age (P<0.05), and egg weight (EW) at 30 weeks (P<0.05). Another SNP, a G1755A transition leading to a non-synonymous substitution (valine 459-to-isoleucine) located within exon 8 of the ESR2 gene, was also markedly correlated with the HHEP at 30, 43, 57 and 66 weeks of age (P<0.05), and EW at 30 weeks (P<0.05). A greater proportion of the additive variance was explained by the SNPs for most of the associated egg production traits (>1%). Furthermore, the results of the combined genotype-based association analysis supported the finding that the two SNPs were associated with the traits under a study. Taken together, our findings suggest that the two sequence variations in the ESR1 and ESR2 genes may provide promising genetic markers for the early selection and prediction of advantageous phenotypes in chicken breeding.

The Hippo/MST Pathway Member SAV1 Plays a Suppressive Role in Development of the Prehierarchical Follicles in Hen 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.

Associations of the Novel Polymorphisms of Periostin and Platelet-Derived Growth Factor Receptor-Like Genes with Egg Production Traits in Local Chinese Dagu Hens.

The periostin (POSTN) and platelet-derived growth factor receptor-like (PDGFRL) genes are implicated in regulation of hen ovarian development. In the present study, these genes were explored as possible molecular markers associated with egg production, egg weight and body weight in Chinese Dagu hens. Samples were analyzed using the PCR-single strand conformation polymorphism (PCR-SSCP) method, followed by sequencing analysis, and three novel single nucleotide polymorphisms (SNPs) were identified within the candidate genes. Among them, an A/T transversion at base position 2727 in intron 2 of POSTN gene was found to be polymorphic and named SNP A2727T; and two transitions, G/A at position 6761 and A/G at base 6839 in exon 2 of PDGFRL gene were detected and named SNPs G6761A and A6839G, respectively. For the SNP A2727T, a total of 360 Dagu hens were classified as AA and AB genotypes, allele A was found present at a higher frequency. Moreover, the AA genotype was significantly correlated with higher hen-housed egg production (HHEP) at 43, 57, and 66 weeks (wks) of age and with a higher egg weight (EW) at 30 wks (P < 0.05). For the two linked SNPs (G6761A and A6839G) in the PDGFRL fragment, the hens were typed into TT, TC and CC genotypes, with the T allele shown to be dominant. The TT genotype was correlated with higher HHEP at 57 and 66 wks of age; genotype CC associated with the highest body weight and EW at 30 and 43 wks (P < 0.05), while it was correlated with the lowest HHEP at 57 and 66 wks of age (P < 0.05). Furthermore, five haplotypes were reconstructed based on these SNPs, with the AATT haplotype associated with the highest HHEP at 43 to 66 wks of age and higher EW at 30 wks (P < 0.05). Collectively, these SNPs identified in this study might be used as a potential molecular marker favorable to genetic improvement of egg productivity in chicken breeding.

Cooperative Effects of FOXL2 with the Members of TGF-ß Superfamily on FSH Receptor mRNA Expression and Granulosa Cell Proliferation from Hen Prehierarchical Follicles.

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-ß) 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.

Characterization of genetic polymorphism of novel MHC B-LB II alleles in Chinese indigenous chickens.

Genetic polymorphism of the major histocompatibility complex (MHC) B-LB II gene was studied by amplification of exon 2 using PCR, followed by cloning and DNA sequencing in eight indigenous Chinese chicken populations. To reveal the genetic variation of the B-LB II gene, 37 types of patterns detected by PCR-SSCP were investigated first, which would be used to screen novel B-LB II sequences within the breeds. The types of PCR-SSCP patterns and final sequencing allowed for the identification of 31 novel MHC B-LB II alleles from 30 unrelated individuals of Chinese chickens that were sampled. These are the first designators for the alleles of chicken MHC B-LB II gene based on the rule of assignment for novel mammalian alleles. Sequence alignment of the 31 B-LB II alleles revealed a total of 68 variable sites in the fragment of exon 2, of which 51 parsimony informative and 17 singleton variable sites were observed. Among the polymorphic sites, the nucleotide substitutions in the first and second positions of the codons accounted for 36.76% and 35.29%, respectively. The sequence similarities between the alleles were estimated to be 90.6%-99.5%. The relative frequencies of synonymous and nonsynonymous nucleotide substitutions within the region were 2.92%+/-0.94% and 14.64%+/-2.67%, respectively. These results indicated that the genetic variation within exon 2 appeared to have largely arisen by gene recombination and balancing selection. Alignment of the deduced amino acid sequences of the beta1 domain coded by exon 2 revealed 6 synonymous mutations and 27 nonsynonymous substitutions at the 33 disparate sites. In particular, the nonsynonymous substitutions at the putative peptide-binding sites are considered to be associated with immunological specificity of MHC B-LB II molecule in Chinese native chickens. These results can provide a molecular biological basis for the study of disease resistance in chicken breeding.

Genetic variation within exon 2 of the MHC B-LB // gene in Tibetan chicken.

Genetic variation within exon 2 of chicken major histocompatibility complex B-LB // genes was investigated by PCR amplification, cloning and sequencing of a 374 bp fragment of the indigenous Tibetan chicken genomic DNA. Fifteen novel B-LB // alleles were found. Alignment and comparison of 18 allelic sequences from the individuals sampled revealed a total of 62 variable sites (total of 80 mutations) in exon 2, of which 41 were parsimony informative sites. The nucleotide diversity (pi) within the sequence of exon 2 was calculated to be 0.0718. Analysis of nucleotide variation confirmed a lower level of divergence (0.056 +/- 0.008) as estimated by average pairwise distance within the Tibetan chicken population than the five exotic breeds detected. The relative frequencies of synonymous and non-synonymous nucleotide substitutions within the region were 3.25 +/- 0.94% and 15.61 +/- 2.69% , respectively. These results indicated that the genetic variation within exon 2 seemed to have arisen largely by gene recombination and balancing selection. Alignment of the deduced amino acid sequences of beta1 domain coded by exon 2 revealed 11 synonymous mutations and 27 non-synonymous substitutions at the 38 separate sites. Fifty percent (12/24) of the proposed peptide-binding sites were variable within beta1 domain of chicken MHC B-LB // molecules, of which 11 were unique non-synonymous amino acid substitutions. These particular non-synonymous substitutions are considered to be associated with immunological specificity of MHC B-LB // molecule in Tibetan chicken, and they can provide a molecular biological basis for the study of disease resistance in chicken.