FOXL2 regulates RhoA expression to change actin cytoskeleton rearrangement in granulosa cells of chicken pre-ovulatory follicles†.

Forkhead box L2 (FOXL2) is an indispensable key regulator of female follicular development, and it plays important roles in the morphogenesis, proliferation, and differentiation of follicle granulosa cells, such as establishing normal estradiol signaling and regulating steroid hormone synthesis. Nevertheless, the effects of FOXL2 on granulosa cell morphology and the underlying mechanism remain unknown. Using FOXL2 ChIP-seq analysis, we found that FOXL2 target genes were significantly enriched in the actin cytoskeleton-related pathways. We confirmed that FOXL2 inhibited the expression of RhoA, a key gene for actin cytoskeleton rearrangement, by binding to TCATCCATCTCT in RhoA promoter region. In addition, FOXL2 overexpression in granulosa cells induced the depolymerization of F-actin and disordered the actin filaments, resulting in a slowdown in the expansion of granulosa cells, while FOXL2 silencing inhibited F-actin depolymerization and stabilized the actin filaments, thereby accelerating granulosa cell expansion. RhoA/ROCK pathway inhibitor Y-27632 exhibited similar effects to FOXL2 overexpression, even reversed the actin polymerization in FOXL2 silencing granulosa cells. This study revealed for the first time that FOXL2 regulated granulosa cell actin cytoskeleton by RhoA/ROCK pathway, thus affecting granulosa cell expansion. Our findings provide new insights for constructing the regulatory network of FOXL2 and propose a potential mechanism for facilitating rapid follicle expansion, thereby laying a foundation for further understanding follicular development.

1α,25-Dihydroxyvitamin D3 Improves Follicular Development and Steroid Hormone Biosynthesis by Regulating Vitamin D Receptor in the Layers Model.

1α,25-Dihydroxyvitamin D3 (VitD3) is the active form of vitamin D, and it regulates gene expression and protein synthesis in mammalian follicle development. However, the function of VitD3 in the follicular development of layers remains unclear. This study investigated, through in vivo and in vitro experiments, the effects of VitD3 on follicle development and steroid hormone biosynthesis in young layers. In vivo, ninety 18-week-old Hy-Line Brown laying hens were randomly divided into three groups for different treatments of VitD3 (0, 10, and 100 µg/kg). VitD3 supplementation promoted follicle development, increasing the number of small yellow follicles (SYFs) and large yellow follicles (LYFs) and the thickness of the granulosa layer (GL) of SYFs. Transcriptome analysis revealed that VitD3 supplementation altered gene expression in the ovarian steroidogenesis, cholesterol metabolism, and glycerolipid metabolism signaling pathways. Steroid hormone-targeted metabolomics profiling identified 20 steroid hormones altered by VitD3 treatment, with 5 being significantly different among the groups. In vitro, it was found that VitD3 increased cell proliferation, promoted cell-cycle progression, regulated the expression of cell-cycle-related genes, and inhibited the apoptosis of granulosa cells from pre-hierarchical follicles (phGCs) and theca cells from prehierarchical follicles (phTCs). In addition, the steroid hormone biosynthesis-related genes, estradiol (E2) and progesterone (P4) concentrations, and vitamin D receptor (VDR) expression level was significantly altered by VitD3. Our findings identified that VitD3 altered the gene expression related to steroid metabolism and the production of testosterone, estradiol, and progesterone in the pre-hierarchical follicles (PHFs), resulting in positive effects on poultry follicular development.

Key parameters of physiological responses to acute heat stress in two commercial layers determined by Fisher discriminant analyses.

Given the escalating global warming and the intense nature of modern poultry production, layers are becoming increasingly susceptible to heat stress. This stress disrupts the physiological processes of layers, which leads to reduced productivity and welfare. To address this issue, it is crucial to first evaluate the stress response systematically. However, such evaluations are still lacking in this field. The objective of this study was to accurately monitor the impact of thermal stress and identify common and key indicators that would support decision-making to maintain layer welfare and productivity under stress. We constructed two heat stress models to reflect moderate (32 °C) to severe (36 °C) stress effects and obtained a comprehensive profile of blood physiological parameters associated with the layers' responses to heat stress. We found that genetic differences had limited influence on their physiological responses to heat stress after 32 °C heat challenges. Using 8 selected and significantly changed parameters, layers' physiological status under heat stress could be accurately determined (judgmental accuracy of 98%). As ambient temperature increased to 36 °C, birds suffered more severe challenges that parameters changed in larger percentages. Additionally, breed variations of the physiological responses became apparent, a Fisher discriminant function based on 5 selected parameters could distinguish heat stress effects at 32 °C or 36 °C with 80% accuracy. The results obtained from this study provide two discriminant models for assessing heat stress and shed lights on developing effective and widely applicable heat stress mitigation strategies targeting these indicators.

FOXL2 regulates the expression of the Col4a1 collagen gene in chicken granulosa cells.

Forkhead box L2 (FOXL2), one member in the superfamily of forkhead transcription factors, is a core transcription factor specifically expressed in ovarian granulosa cells and is essential for the development of follicles. FOXL2 has been shown to regulate the transcription of genes encoding enzymes that synthesize steroid hormones and estrogen receptors and regulate the expression of collagen genes in granulosa cells. This study explored the effect of FOXL2 on collagen gene expression in granulosa cells by overexpressing Foxl2 in pregranulosa cells, prehierarchical follicles and preovulation follicle granulosa cells. The results showed that FOXL2 regulated the expression of several genes encoding collagens in chicken granulosa cells and that overexpression of Foxl2 significantly reduced the messenger RNA and protein levels of Col4a1 in different granulosa cells. Moreover, luciferase reporter and chromatin immunoprecipitation assays were performed to study how FOXL2 regulates the expression of collagen genes, and the results showed that FOXL2 directly regulated the expression of Col4a1 by binding to the motif of CAGCAGCACCAGCAG between -640 and -625 bp upstream of the coding region. The results indicated that FOXL2 could regulate the components of the extracellular matrix; however, the biological significance of this regulation needs further clarification.

Transcriptional regulation of CYP19A1 expression in chickens: ESR1, ESR2 and NR5A2 form a functional network.

Aromatase, encoded by CYP19A1, is responsible for the conversion of androgen to estrogen, which plays a vital role in the development and function of the ovary and functions in many other physiological processes in both sexes. Instead of being expressed in ovarian granulosa cells, as in mammals, CYP19A1 is expressed in chickens in the theca cells of ovarian follicles, and the mechanism of CYP19A1 expression regulation remains unknown. Here, using immunofluorescence and western blotting assay, we first confirmed that CYP19A1 and FOXL2 (Forkheadbox L2) were coexpressed in pre-granulosa cells of female chicken embryonic gonads, while FOXL2 did not affect aromatase expression at embryonic stages. Second, our research showed that CYP19A1, ESR1 (estrogen receptor alpha), ESR2 (estrogen receptor beta) and NR5A2 (liver receptor homologue-1) were coexpressed in the theca cell layers of chicken small yellow follicles. There was cross-talk between CYP19A1 and candidate transcription factors (ESR1, ESR2 and NR5A2), which was identified by generating a reliable theca cell culture model. Using luciferase assays in theca cells and chicken embryonic fibroblast (DF-1) cells, the results suggested that ESR1 and NR5A2 had potential effects on CYP19A1 promoter activity in chickens. Overexpression of ESR1, ESR2 and NR5A2 in chicken embryonic fibroblast (DF-1) cells upregulated the protein expression of CYP19A1, mutually restricted each other and formed a potential regulatory network to coordinate the expression of CYP19A1. To conclude, our results indicated that FOXL2 cannot regulate the expression of CYP19A1 at chicken embryonic stages and after sexual maturity, ESR1, ESR2 and NR5A2 form a functional network to affect the expression of CYP19A1. These results laid a foundation for further research on the transcriptional regulation of chicken aromatase.

Characterization and proteomics of chicken seminal plasma extracellular vesicles.

In mammals, seminal plasma extracellular vesicles (SPEVs) can regulate sperm motility and capacitation. The characteristics and functions of SPEVs in avians have been rarely reported. In this study, chicken SPEVs were isolated and characterized by transmission and scanning electron microscopy (TEM/SEM) and nanoparticle tracking analysis (NTA); furthermore, seven extracellular vesicle (EVs) marker proteins were detected by Western blot (WB). TEM revealed that chicken SPEVs had a classic bilayer membrane structure. NTA confirmed that the size of SPEVs was 30-250 nm, and concentration ranged from 8.0 E + 11-8.5 E + 11 particles/ml. There were 3073 SPEVs proteins identified by deep sequencing, including 2794 intracellular proteins and 279 extracellular proteins. The overlap rate of proteomes between chicken SPEVs and vesicles reported in the Vesiclepedia database reached 86%, and 360 new proteins that had not been reported by the ExoCarta and Vesiclepedia databases were identified in chicken SPEV proteomes. Gene Ontology (GO) analysis revealed that chicken SPEV proteins were mainly enriched in supplying energy and transporting protein. There were 4 IFT family proteins speculated to play an important role in sperm composition and function. Our data were compared with two previously published studies on the proteomics of chicken seminal plasma (SP) and hen uterine fluid, and some overlapping proteins described in chicken SPEVs had been identified in hen uterine fluid (545) and chicken SP (284). In conclusion, these findings will increase our understanding of the content and composition of proteome in SPEVs and provide new insights into the important role of the SPEV regulation in sperm functions.

Analysis of deep sequencing exosome-microRNA expression profile derived from CP-II reveals potential role of gga-miRNA-451 in inflammation.

Mycoplasma gallisepticum (MG) can cause chronic respiratory disease (CRD) in chickens. While several studies have reported the inflammatory functions of microRNAs during MG infection, the mechanism by which exosomal miRNAs regulate MG-induced inflammation remains to be elucidated. The expression of exosome-microRNA derived from MG-infected chicken type II pneumocytes (CP-II) was screened, and the target genes and function of differentially expressed miRNAs (DEGs) were predicted. To verify the role of exosomal gga-miR-451, Western blot, ELISA and RT-qPCR were used in this study. The results showed that a total of 722 miRNAs were identified from the two exosomal small RNA (sRNA) libraries, and 30 miRNAs (9 up-regulated and 21 down-regulated) were significantly differentially expressed. The target miRNAs were significantly enriched in the treatment group, such as cell cycle, Toll-like receptor signalling pathway and MAPK signalling pathway. The results have also confirmed that gga-miR-451-absent exosomes derived from MG-infected CP-II cells increased inflammatory cytokine production in chicken fibroblast cells (DF-1), and wild-type CP-II cell-derived exosomes displayed protective effects. Collectively, our work suggests that exosomes from MG-infected CP-II cells alter the dynamics of the DF-1 cells, and may contribute to pathology of the MG infection via exosomal gga-miR-451 targeting YWHAZ involving in inflammation.

Effects of husbandry systems and Chinese indigenous chicken strain on cecum microbial diversity.

OBJECTIVE: This study was to evaluate the effect of husbandry systems and strains on cecum microbial diversity of Jingyang chickens under the same dietary conditions. METHODS: A total of 320 laying hens (body weight, 1.70±0.15 kg; 47 weeks old) were randomly allocated to one of the four treatments: i) Silver-feathered hens in enrichment cages (SEC) with an individual cage (70×60×75 cm), ii) Silver-feathered hens in free range (SFR) with the stocking density of 1.5 chickens per ten square meters, iii) Gold-feathered hens in enrichment cages (GEC), iv) Gold-feathered hens in free range (GFR). The experiment lasted 8 weeks and the cecum fecal samples were collected for 16S rDNA high throughput sequencing at the end of experiment. RESULTS: i) The core microbiota was composed of Bacteroidetes (49% to 60%), Firmicutes (21% to 32%) and Proteobacteria (2% to 4%) at the phylum level. ii) The core bacteria were Bacteroides (26% to 31%), Rikenellaceae (9% to 16%), Parabacteroides (2% to 5%) and Lachnoclostridium (2% to 6%) at the genus level. iii) The indexes of operational taxonomic unit, Shannon, Simpson and observed species were all higher in SFR group than in SEC group while in GEC group than in GFR group, with SFR group showing the greatest diversity of cecum microorganisms among the four groups. iv) The clustering result was consistent with the strain classification, with a similar composition of cecum bacteria in the two strains of laying hens. CONCLUSION: The core microbiota were not altered by husbandry systems or strains. The free-range system increased the diversity of cecal microbes only for silver feathered hens. However, the cecum microbial composition was similar in two strain treatments under the same dietary conditions.

The Sertoli cell marker FOXD1 regulates testis development and function in the chicken.

FOXD1, one of the transcription factors of the FOX family, has been shown to be important for mammalian reproduction but little is known about its function in avian species. In the present study, we identified the expression pattern and location of FOXD1 in chicken tissues and testis by performing quantitative polymerase chain reaction, immunohistochemistry and immunofluorescence, and further investigated the regulatory relationship of FOXD1 with genes involved in testis development by RNA interference. Our results showed that FOXD1 is confirmed to be significantly male-biased expressed in the brain, kidney and testis of adults as well as in embryonic gonads, and it is localised in the testicular Sertoli cell in chicken, consistent with its localisation in mammals. After knock-down of FOXD1 in chicken Sertoli cells, the expression of anti-Müllerian hormone (AMH), sex-determining region Y-box 9 (SOX9) and PKA regulatory subunits type I α (RIα) was significantly downregulated, expression of androgen receptor (AR) was notably increased whereas double-sex and MAB-3-related transcription factor 1 (DMRT1) showed no obvious change in expression. These results suggest that FOXD1 is an essential marker for Sertoli cells upstream of SOX9 expression and a potential regulator of embryonic testis differentiation and development and of normal testis function in the chicken.

Growth differentiation factor 9 promotes follicle-stimulating hormone-induced progesterone production in chicken follicular granulosa cells.

The function of oocyte-derived growth differentiation factor 9 (GDF9) in ovarian follicles has thus far been poorly defined in avian species compared with the defined function in mammals. Our aim here is to investigate the effects of GDF9 on steroidogenesis and on chicken ovarian granulosa cell (GC) mitosis. Primary GCs from both prehierarchical (6-8 mm in diameter, phGCs) and preovulatory follicles (F1-F5, poGCs) were cultured in the presence or absence of the GDF9 protein. The progesterone (P4) levels in the culture medium were then measured by radioimmunoassay (RIA), and the expression levels of steroidogenesis genes were detected by quantitative PCR. We found that GDF9 alone showed no significant effect on the P4 levels by regulating the expression of steroidogenesis genes, such as STAR, CYP11A1 and HSD3B. Further experiments indicated that GDF9 promoted follicle-stimulating hormone (FSH)-induced P4 production and STAR expression. GDF9 also rescued the FSH-induced decrease of FSH receptor (FSHR) expression but had no effect on the forskolin-induced P4, STAR and forskolin-inhibited FSHR expression levels, suggesting that GDF9 might achieve its regulatory role of P4 by enhancing FSHR and STAR expression. In addition, GDF9 also promoted GC cell cycle progression, regulated the gene transcription of related genes, potentiated DNA replication and inhibited apoptosis. Interestingly, these effects differed between the phGCs and the poGCs. To our knowledge, this is the first report that illustrates the function of GDF9 on chicken GCs and the effects on ovarian steroidogenesis. Our findings highlight the regulation of central oocytes on the surrounding granulosa cells and emphasize the interaction between paracrine signals and endocrine hormones on ovarian progesterone production; these findings contribute to the understanding of the development of avian ovarian follicles.

Transcription of CYP19A1 is directly regulated by SF-1 in the theca cells of ovary follicles in chicken.

Many studies have suggested the important role of estrogen in ovarian differentiation and development of vertebrates including chicken. Cytochrome P450 aromatase, encoded by CYP19A1, is a key enzyme in estrogen synthesis, but the mechanism of CYP19A1 regulation in chicken remains unknown. Here, we found that CYP19A1 was only expressed in the theca cell layers of chicken ovary follicles. Steroidogenic factor 1 (SF-1, also named as nuclear receptor subfamily 5 group A member 1, NR5A1), a potential regulators, was expressed in both the theca cell layers and granulosa cell layers. Forkheadbox L2 (FOXL2), another potential regulator, was only expressed in the granulosa cell layers. Using luciferase assays in vitro, we found that SF-1 could activate the promoter of CYP19A1 by binding to the nuclear receptor half-site (5'-TCAAGGTCA-3') from -280 to -271 base pairs. FOXL2 did not activate the promoter of chicken CYP19A1 gene in either 293T or DF-1 cells. Overexpression of SF-1 in DF-1 cells upregulated aromatase expression, but FOXL2 could not. Taken together, our results indicated that SF-1 activates CYP19A1 mRNA expression via a conserved binding site in chicken ovary, but FOXL2 may not affect the expression of CYP19A1.

Aromatase inhibitors can improve the semen quality of aged roosters by up regulating genes related to steroid hormone synthesis.

Excessive aromatase can reduce reproductive performance in aged roosters. Aromatase inhibitors (AI) can inhibit the aromatase activity and improve the semen quality of aged roosters. However, relevant molecular mechanism is still unclear. The purpose of this study was to explore the regulatory mechanism of AI letrozole improving semen quality in aged roosters by transcriptomic and proteomic sequencing. In this study, 56-week-old roosters were reared in separate cages on a standard basice diet and oral letrozole 42 days (D) at a daily dose 0.25 mg/kg. Semen quality and serum hormone were measured before (0 D) and after (42 D) letrozole administration. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected, respectively. The results indicated that semen volume, sperm motility, sperm density, MMP, testosterone (T) and gonadotropin releasing hormone (GnRH) in letrozole treatment group (LET) were significantly increased than those in control group (CN) (P<0.05); estradiol (E2) and ROS in LET were significantly lower than those in CN (P<0.05). Through transcriptomic and proteomic analysis, we identified 189 differently expressed genes (DEGs) and 64 differentially expressed proteins (DEPs) in the comparison of LET and CN. DEGs and DEPs Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) items are mainly enriched in steroid biosynthetic process, cell differentiation and proliferation, lipid metabolic process, oxidation-reduction process and electron transfer activity. Furthermore, 8 genes including STAR, CYP17A1, NSDHL, SULT1E1, EHF, NRNPA1, PLIN2 and SDHA were identified as key genes for letrozole to regulate semen quality in aged roosters. These results indicate that letrozole can up-regulate the expression of genes related to steroid hormone synthesis, cell differentiation and proliferation, electron transfer activity, and enhance mitochondrial activity, increase testicular weight, and ultimately improve the semen quality of aged roosters.

Duck pan-genome reveals two transposon insertions caused bodyweight enlarging and white plumage phenotype formation during evolution.

Structural variations (SVs) are a major source of domestication and improvement traits. We present the first duck pan-genome constructed using five genome assemblies capturing ∼40.98 Mb new sequences. This pan-genome together with high-depth sequencing data (∼46.5×) identified 101,041 SVs, of which substantial proportions were derived from transposable element (TE) activity. Many TE-derived SVs anchoring in a gene body or regulatory region are linked to duck's domestication and improvement. By combining quantitative genetics with molecular experiments, we, for the first time, unraveled a 6945 bp Gypsy insertion as a functional mutation of the major gene IGF2BP1 associated with duck bodyweight. This Gypsy insertion, to our knowledge, explains the largest effect on bodyweight among avian species (27.61% of phenotypic variation). In addition, we also examined another 6634 bp Gypsy insertion in MITF intron, which triggers a novel transcript of MITF, thereby contributing to the development of white plumage. Our findings highlight the importance of using a pan-genome as a reference in genomics studies and illuminate the impact of transposons in trait formation and livestock breeding.

Non-synonymous SNPs in MC1R gene are associated with the extended black variant in domestic ducks (Anas platyrhynchos).

In this study, we performed a sequence characterization of the duck melanocortin 1 receptor (alpha-melanocyte stimulating hormone receptor) (MC1R) gene to analyze the relationship between MC1R polymorphism and the extended black variant in domestic ducks based on the extended black (E) and non-extended black (e(+) ) allele hypothesis of the duck MC1R gene. Both c.52G>A and c.376G>A substitutions are highly associated with the duck extended black variant (P < 0.01), but the novel c.52G>A substitution is more of a fit for the allele hypothesis of the duck MC1R gene.

Comparative Lipidomics and Metabolomics Reveal the Underlying Mechanisms of Taurine in the Alleviation of Nonalcoholic Fatty Liver Disease Using the Aged Laying Hen Model.

SCOPE: Aged laying hen is recently suggested as a more attractive animal model than rodent for studying nonalcoholic fatty liver disease (NAFLD) of humans. This study aims to reveal effects and metabolic regulation mechanisms of taurine alleviating NAFLD by using the aged laying hen model. METHODS AND RESULTS: Liver histomorphology and biochemical indices show 0.02% taurine effectively alleviated fat deposition and liver damage. Comparative liver lipidomics and gene expressions analyses reveal taurine promoted lipolysis, fatty acids oxidation, lipids transport, and reduced oxidative stress in liver. Furthermore, comparative serum metabolomics screen six core metabolites negatively correlated with NAFLD, including linoleic acid, gamma-linolenic acid, pantothenate, L-methionine, 2-methylbutyroylcarnitine, L-carnitine; and two core metabolites positively correlated with NAFLD, including lysophosphatidylcholine (14:0/0:0) and lysophosphatidylcholine (16:0/0:0). Metabolic pathway analysis reveals taurine mainly regulated linoleic acid metabolism, cysteine and methionine metabolism, carnitine metabolism, pantothenic acid and coenzyme A biosynthesis metabolism, and glycerophospholipid metabolism to up-adjust levels of six negatively correlated metabolites and down-adjust two positively correlated metabolites for alleviating NAFLD of aged hens. CONCLUSION: This study firstly reveals underlying metabolic mechanisms of taurine alleviating NAFLD using the aged hen model, thereby laying the foundation for taurine's application in the prevention of NAFLD in both human and poultry.

Characterization of clutch traits and egg production in six chicken breeds.

OBJECTIVE: The better understanding of laying pattern of birds is crucial for developing breed-specific proper breeding scheme and management. METHODS: Daily egg production until 50 wk of age of six chicken breeds including one layer (White Leghorn, WL), three dual-purpose (Rhode Island Red, RIR; Columbian Plymouth Rock, CR; and Barred Plymouth Rock, BR), one synthetic dwarf (DY), and one indigenous (Beijing-You Chicken, BYC) were used to characterize their clutch traits and egg production. The age at first egg, egg number, average and maximum clutch length, pause length, and number of clutches and pauses were calculated accordingly. RESULTS: The egg number and average clutch length in WL, RIR, CR, and BR were higher than those in DY and BYC (p<0.01). The numbers of clutches and pauses, and pause length in WL, RIR, CR, and BR were lower than those in DY and BYC (p<0.01). The coefficient variations of clutch length in WL, RIR, CR, and BR (57.66%, 66.49%, 64.22%, and 55.35%, respectively) were higher than DY (41.84%) and BYC (36.29%), while the coefficient variations of egg number in WL, RIR, CR, and BR (9.10%, 9.97%, 10.82%, and 9.92%) were lower than DY (15.84%) and BYC (16.85%). The clutch length was positively correlated with egg number (r = 0.51 to 0.66; p<0.01), but not correlated with age at first egg in all breeds. CONCLUSION: The six breeds showed significant different clutch and egg production traits. Due to the selection history, the high and median productive layer breeds had higher clutch length than those of the less productive indigenous BYC. The clutch length is a proper selection criterion for further progress in egg production. The age at first egg, which is independent of clutch traits, is especially encouraged to be improved by selection in the BYC breed.

Molecular cloning, expression profile, polymorphism and the genetic effects of the dopamine D1 receptor gene on duck reproductive traits.

The dopamine D1 receptor (DRD1), a member of the dopamine receptor (DR) gene family, participates in the regulation of reproductive behaviors in birds. In this study, a 1,390 bp fragment covering the complete coding region (CDS) of duck DRD1 gene was obtained. The cDNA (GenBank: JQ346726) contains a 1,353 bp CDS and a 37 bp 3'- UTR including a TGA termination codon (nucleotides 1,354-1,356 bp). The duck DRD1 shares about 76-96 % nucleic acid identity and 82-98 % amino acid identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences displays that duck DRD1 protein is closely related with those of chicken and zebra finch. The quantitative real-time PCR analysis indicates that the DRD1 mRNA is widely expressed in all examined tissues. Five single nucleotide polymorphisms (SNPs) (c.189A > T, c.507C > T, c.681C > T, c.765A > T, c.1044A > G) in the CDS of duck DRD1 gene were indentified, c.681C > T and c.765A > T were genotyped and analyzed in a two generations duck population by using of PCR-RFLP. Association analysis demonstrated that the c.681C > T genotypes were significantly associated with body weight at sexual maturity (when laying their first egg) (P < 0.01), egg production within 360 days (P < 0.05) and 420 days (P < 0.01); the c.765A > T genotypes were significantly associated with egg shape index and egg shell strength (P < 0.05). Those results suggest that the DRD1 gene may be a potential genetic marker to improve some reproductive traits in ducks.

WT1 suppresses follicle-stimulating hormone-induced progesterone secretion by regulating ERK1/2 pathway in chicken preovulatory granulosa cells.

Multiple Wilms tumor gene 1 (WT1) splicing variants are expressed in mammals, and these variants regulate tumorigenesis and mediate the development of multiple tissues and organs, including gonads. However, WT1 splicing variants (+KTS or -KTS) are expressed in only two nonmammalian vertebrates, and unexpectedly, their functions in chicken ovaries remain elusive. Progesterone (P4) secreted by chicken granulosa cells (GCs) participates in various physiological processes and plays an important role in maintaining reproductive performance. The purpose of this study was to investigate the effect of WT1(+KTS) and WT1(-KTS) on chicken P4 secretion in preovulatory GCs. First, we detected WT1 mRNA expression in GCs from follicles of different developmental stages by Quantitative real-time PCR (RT-qPCR) and found that WT1 mRNA expression was considerably increased in preovulatory GCs compared with prehierarchical GCs. Primary cells collected from preovulatory follicles were treated with WT1(+KTS) or WT1(-KTS) overexpression vectors and subsequently cultured in the absence or presence of follicle-stimulating hormone (FSH). The mRNA levels of FSH-receptor (FSHR) and steroidogenesis genes were determined by RT-qPCR, and the P4 levels in the cell supernatants were measured by radioimmunoassay (RIA). Both WT1(+KTS) and WT1(-KTS) significantly decreased P4 secretion due to a reduction in FSHR, STAR and CYP11A1 mRNA levels. Western blotting revealed that ERK1/2 and BRAF phosphorylation levels were suppressed after overexpression of WT1(+KTS) or WT1(-KTS), whereas total protein and mRNA levels were not significantly changed. In addition, CREB protein and phosphorylation levels were inhibited after overexpression of WT1(+KTS) or WT1(-KTS). In conclusion, WT1(+KTS) and WT1(-KTS) inhibited CREB protein activity and significantly reduced FSHR, STAR and CYP11A1 mRNA levels, which subsequently suppressed FSH-induced P4 secretion in preovulatory GCs by modulating ERK1/2 signaling.

Assessment of the Endophytic Fungal Composition of Lactobacillus plantarum and Enterococcus faecalis-Fermented Astragalus membranaceus Using Single-Molecule, Real-Time Sequencing Technology.

Endophytic fungus represents microorganisms existing within the healthy plant organs, which can significantly influence metabolic product production in plants, a process with great research value and broad prospects for development. To investigate the effect of fermentation with probiotic cultures on the endophytic fungal diversity and composition of Astragalus membranaceus, we used single-molecular, real-time sequencing (Pacific Biosciences) for 18S ribosomal RNA (rRNA) sequencing. The results showed that the endophytic fungi of A. membranaceus mainly belonged to Aspergillus, Penicillium, Cystofilobasidium, Candida, Guehomyces, and Wallemia. Furthermore, the endophytic fungal diversity and abundance of A. membranaceus were more variable after fermentation with Enterococcus faecium and/or Lactobacillus plantarum. Our data lays a solid and comprehensive foundation for further exploration of endophytic fungi from A. membranaceus as potential sources of functional compounds.

Cloning and expression profiling of the VLDLR gene associated with egg performance in duck (Anas platyrhynchos).

BACKGROUND: The very low density lipoprotein receptor gene (VLDLR), a member of the low density lipoprotein receptor (LDLR) gene family, plays a crucial role in the synthesis of yolk protein precursors in oviparous species. Differential splicing of this gene has been reported in human, rabbit and rat. In chicken, studies showed that the VLDLR protein on the oocyte surface mediates the uptake of yolk protein precursors into growing oocytes. However, information on the VLDLR gene in duck is still scarce. METHODS: Full-length duck VLDLR cDNA was obtained by comparative cloning and rapid amplification of cDNA ends (RACE). Tissue expression patterns were analysed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). Association between the different genotypes and egg performance traits was investigated with the general linear model (GLM) procedure of the SAS® software package. RESULTS: In duck, two VLDLR transcripts were identified, one transcript (variant-a) containing an O-linked sugar domain and the other (variant-b) not containing this sugar domain. These transcripts share ~70 to 90% identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences showed that duck VLDLR proteins were closely related with those of chicken and zebra finch. The two duck VLDLR transcripts are differentially expressed i.e. VLDLR-a is mainly expressed in muscle tissue and VLDLR-b in reproductive organs. We have localized the duck VLDLR gene on chromosome Z. An association analysis using two completely linked SNP sites (T/C at position 2025 bp of the ORF and G/A in intron 13) and records from two generations demonstrated that the duck VLDLR gene was significantly associated with egg production (P < 0.01), age of first egg (P < 0.01) and body weight of first egg (P < 0.05). CONCLUSIONS: Duck and chicken VLDLR genes probably perform similar function in the development of growing oocytes and deposition of yolk lipoprotein. Therefore, VLDLR could be a candidate gene for duck egg performance and be used as a genetic marker to improve egg performance in ducks.