Chick Oviduct Research Articles

Chicken oviduct-the target tissue for growth hormone action: effect on cell proliferation and apoptosis and on the gene expression of some oviduct-specific proteins.

The aim of this study was to examine the in vivo effect of growth hormone (GH) on cell proliferation and apoptosis and on the gene expression of selected proteins in the chicken oviduct before sexual maturity (first oviposition). Ten-week-old Hy-Line Brown chickens were injected three times a week with 200 μg · kg-1 body weight of recombinant chicken GH (cGH) until 16 weeks of age. Control hens received 0.9 % NaCl with 0.05 % bovine serum albumin as a vehicle. Treatment with cGH increased (P < 0.05) oviduct weight at 16 weeks of age, i.e. 1–2 weeks before onset of egg laying. The highest number of proliferating (determined by proliferating cell nuclear antigen [PCNA] immunocytochemistry) and apoptotic (determined by TUNEL assay) cells in the oviduct was found in the mucosal epithelium, and the lowest in the stroma. Administration of cGH did not increase (P > 0.05) the number of PCNA-positive cells but it decreased (P < 0.01) the number of TUNEL-positive cells, thus increasing the proliferating-to-apoptotic cell ratio in the oviduct. Gene expression (determined by real-time polymerase chain reaction) of apoptosis-related caspase-2 in the magnum and caspase-3 in the magnum and isthmus and their activity (determined by fluorometric assay) in the magnum were attenuated (P < 0.05) in cGH-treated hens. The gene expression of the magnum-specific ovalbumin and the shell-gland-specific ovocalyxins 32 and 36 was increased (P < 0.05) in cGH-treated chickens. In contrast, the expression of Bcl-2 and of caspases 8 and 9 was not affected by cGH in any of the oviductal segments. The results suggest that GH, via the orchestration of apoptosis and expression of some oviduct-specific proteins, participates in the development and activity of the chicken oviduct prior to the onset of egg laying.

Pivotal roles for hormonally regulated expression of the HEP21 gene in the reproductive tract of chickens for oviduct development and in ovarian carcinogenesis

Hen egg protein (HEP21) is a 21-kDa secreted protein and has a single copy of the Ly6/uPAR domain. Although HEP21 is expressed primarily in the chicken oviduct, its biological function(s) in the reproductive system of chickens is not known. Thus, in the present study, we investigated expression patterns of HEP21 with respect to hormonal regulation, oviduct development, changes in expression in laying hens undergoing induced molting, and in the development of ovarian carcinogenesis in laying hens. Results of present study indicated that HEP21 messenger RNA (mRNA) expression increased (P < 0.001) in the chicken oviduct in response to estrogen. In situ hybridization analyses revealed expression of HEP21 mRNA predominantly in glandular (GE) and luminal epithelia of the magnum of the chicken oviduct in response to estrogen. The expression of HEP21 mRNA decreased (P < 0.001) as the oviduct regressed during induced molting and increased (P < 0.001) with recrudescence of the oviduct following molting. HEP21 mRNA was most abundant in GE of the oviduct during recrudescence, but not during oviduct regression following induced molting. Moreover, we found abundant expression of HEP21 in GE of cancerous ovaries, but not in normal ovaries of hens. Collectively, results of present study suggest that HEP21 is an estrogen-responsive gene in the oviduct of hens that likely regulates development of the chicken oviduct, and egg production and formation. Furthermore, there is increased expression of HEP21 in epithelial-derived ovarian cancer suggesting that HEP21 could be used for diagnosis and monitoring carcinogenesis in laying hens and in women.

The study of factors affecting the efficiency of somatic transgenesis in chickens

In this article we studied the impact of the use of retroviral vectors for genetic transformation of chicken oviduct cells in vivo to produce transgenic chickens bioreactors and investigated factors affecting the efficiency of local transgenesis in chickens. By using of retroviral vector system it was shown that with the efficiency of chickens oviduct cells transformation was at least 17,2 ± 3,1%.

Poultry infected with Mycoplasma gallisepticum or Mycoplasma synoviae produce antibodies to their cysteine protease CysP

Major poultry pathogens, Mycoplasma gallisepticum and Mycoplasma synoviae possess cysteine protease CysP, which in vitro cleaves chicken immunoglobulin G (IgG) into Fab (antigen-binding fragment) and Fc (crystallisable region fragment). We used recombinant CysP of M. synoviae (rCysP) in enzyme immunoassays to detect antibodies to CysP in sera, synovial fluids, and in washings of respiratory tract and oviducts of chickens and turkeys experimentally or naturally infected with M. gallisepticum or M. synoviae. In poultry infected with M. synoviae, 70.4 % of samples contained antibodies reacting with rCysP. In birds infected with M. gallisepticum we detected CysP antibodies in 63.1 % of samples. Our data demonstrate that CysP is immunogenic for chickens and turkeys and indicate that M. gallisepticum and M. synoviae infecting chickens and turkeys synthesise CysP in vivo. This is the first study demonstrating that proteases of any Mycoplasma species can induce production of specific antibodies in the natural host.

Hormonal regulation of beta-catenin during development of the avian oviduct and its expression in epithelial cell-derived ovarian carcinogenesis

Beta-catenin (CTNNB1) is a dual function molecule that acts as a key component of the cadherin complex and WNT signaling pathway. It has a crucial role in embryogenesis, tumorigenesis, angiogenesis and progression of metastasis. Recently, it has been suggested that the CTNNB1 complex is a major regulator of development of the mouse oviduct and uterus. However, little is known about the CTNNB1 gene in chickens. Therefore, in this study, we focused on the CTNNB1 gene in the chicken reproductive tract and hormonal control of its expression in the chicken oviduct. CTNNB1 was localized specifically to the luminal and glandular epithelium of the four segments of chicken oviduct and DES (diethylstilbestrol, a synthetic non-steroidal estrogen) increased its expression primarily in LE of the magnum. In addition, CTNNB1 mRNA and protein were expressed abundantly in glandular epithelium of endometrioid-type ovarian carcinoma, but not in normal ovaries. Moreover, CTNNB1 expression was post-transcriptionally regulated via its 3′-UTR by binding with target miRNAs including miR-217, miR-1467, miR-1623 and miR-1697. Collectively, these results indicate that CTNNB1 is a novel gene regulated by estrogen in epithelial cells of the chicken oviduct and that it is also abundantly expressed in epithelial cells of endometrioid-type ovarian carcinoma suggesting that it could be used as a marker for diagnosis of ovarian cancer in laying hens and women.

In vitro Effects of TCDD, PCB126 and PCB153 on Estrogen Receptors, Caspases and Metalloproteinase-2 mRNA Expression in the Chicken Shell Gland

Among the environmental chemicals which disturb endocrine functions, dioxins and polychlorinated biphenyls (PCBs) are known as the most toxic. Numerous studies in mammals revealed that dioxins and PCBs disrupt functions of the uterus, delay implantation and increase embryo loss. The direct effect of these chemicals on the avian oviduct is not known. Therefore, in the study chicken shell gland tissues were used to examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), coplanar PCB126 and non-coplanar PCB153 on estrogen receptors (ERs), initiator caspase-1, executioner caspase-3 and metalloproteinase-2 (MMP-2) mRNA expression. Fragments of shell gland tissue isolated from the laying chicken were incubated for 24h with TCDD (100nM), PCB126 (100nM) or PCB153 (100 microM). Quantitative PCR analysis showed that: (1) TCDD increased ER beta (ERbeta) mRNA expression, (2) PCB126 increased ER alpha (ERalpha), ERbeta and caspase-1, and decreased MMP-2 mRNA expression, (3) PCB153 elevated the ERbeta and caspase-1 expression levels and (4) expression of caspase-3 was not altered by any investigated xenobiotics. The results obtained using the shell gland explants model indicate that dioxins and PCBs have a direct effect on the chicken oviduct, especially the shell gland, by affecting the expression of genes involved in the function of this oviductal segment. It is suggested that coplanar PCBs such as PCB126, by changing cellular and extracellular regulators gene expression, may lead to disruption of shell gland activity and impair egg components formed in this organ.

Effect of Plant Derived Antimicrobials on Salmonella Enteritidis Adhesion to and Invasion of Primary Chicken Oviduct Epithelial Cells in vitro and Virulence Gene Expression

Salmonella Enteritidis (SE) is a major foodborne pathogen in the United States and one of the most frequently reported Salmonella serotypes globally. Eggs are the most common food product associated with SE infections in humans. The pathogen colonizes the intestinal tract in layers, and migrates to reproductive organs systemically. Since adhesion to and invasion of chicken oviduct epithelial cells (COEC) is critical for SE colonization in reproductive tract, reducing these virulence factors could potentially decrease egg yolk contamination. This study investigated the efficacy of sub-inhibitory concentrations of three plant-derived antimicrobials (PDAs), namely carvacrol, thymol and eugenol in reducing SE adhesion to and invasion of COEC, and survival in chicken macrophages. In addition, the effect of PDAs on SE genes critical for oviduct colonization and macrophage survival was determined using real-time quantitative PCR (RT-qPCR). All PDAs significantly reduced SE adhesion to and invasion of COEC (p < 0.001). The PDAs, except thymol consistently decreased SE survival in macrophages (p < 0.001). RT-qPCR results revealed down-regulation in the expression of genes involved in SE colonization and macrophage survival (p < 0.001). The results indicate that PDAs could potentially be used to control SE colonization in chicken reproductive tract; however, in vivo studies validating these results are warranted.

An EAV-HP Insertion in 5′ Flanking Region of SLCO1B3 Causes Blue Eggshell in the Chicken

The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an F2 chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5′ flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level.

Developmental changes in survivin mRNA expression in the chicken oviduct

Developmental changes in survivin mRNA expression in the chicken oviduct

Apoptosis and proliferation as the processes concomitant with the chicken oviduct development during sexual maturation

The Notch signaling pathway plays vital roles in vascular development and homeostasis. However, the functional role of HRT1, a primary downstream effector of Notch signaling in VSMC, is poorly characterized. In the present study, we postulated that HRT1 plays fundamental roles in modulating VSMC fate. To test the hypothesis that HRT1 is coupled to growth regulation, we generated VSMC lines constitutively overexpressing HRT1 (HRT1SMC) and demonstrated an exaggerated growth behavior compared to its control cell line. The lack of cell cycle arrest at confluence in HRT1SMC was associated with an attenuated up-regulation of the cell cycle inhibitor, p21WAF1/CIP1. We further established that both transient and constitutive HRT1 signaling promoted VSMC survival in response to serum deprivation and pro-apoptotic Fas ligand. Resistance to apoptosis was associated with the induction of Akt expression/activity, a well-described anti-apoptotic mediator. Overall, these findings provide initial evidence that HRT1 functions as a critical determinant of VSMC proliferation and survival.

Differential Expression of Select Members of the SLC Family of Genes and Regulation of Expression by MicroRNAs in the Chicken Oviduct1

The yolk and white of eggs from chickens contain proteins and other molecules either secreted or transported by cells of the reproductive tract, or secreted by the liver and transported to the ovarian follicles of laying hens. Nutrients transported by solute carriers (SLCs) include glucose, electrolytes, and amino acids. Although SLC genes have been investigated in mammals, there are few studies of expression of SLC genes in the chicken oviduct. Therefore, we investigated temporal and cell-specific expression of selected SLC genes at 3 h and 20 h postovulation and regulation of their expression by microRNAs (miRs). Expression of SLC1A4 (glutamate and neutral amino acid transporter), SLC13A2 (dicarboxylate transporter), and SLC35B4 (UDP-xylose: UDP-N-acetylglucosamine transporter) mRNAs was limited to glandular epithelium (GE), while SLC4A5 (sodium bicarbonate cotransporter) and SLC7A3 (cationic amino acid transporter) mRNAs were expressed predominantly in the luminal epithelium of the magnum. Interestingly, SLC1A4, SLC4A5, SLC13A2 and SLC35B4 mRNAs were abundant only in GE of the shell gland, whereas SLC7A3 was not detected in the shell gland. In the magnum, SLC7A3 and SLC4A5 were expressed, but SLC1A4, SLC35B4, and SLC13A2 were not expressed at 20 h postovulation. In the shell gland, all SLC mRNAs were expressed at both time points, except for SLC7A3. The miRNA target validation assay revealed that miR-1764 and miR-1700 bind directly to SLC13A2 and SLC35B4 transcripts, respectively, to regulate expression. Results of this study demonstrate cell-specific and temporal changes in expression of selected SLC genes and regulation of SLC13A2 and SLC35B4 expression by miRs in the oviduct of laying hens.

Expression and regulation of beta-defensin 11 in the oviduct in response to estrogen and in ovarian tumors of chickens

Avian beta-defensins (AvBDs), also known as gallinacins, are small cationic peptides having three cysteine disulfide bonds between their cysteine residues. They play essential roles in the innate immune system as well as stimulate proliferation of epithelial cells and fibroblasts. Although we found the avian homolog of human beta-defensin 11 to be highly expressed in chicks treated with the diethylstilbestrol (DES, a synthetic estrogen agonist), little is known about the hormonal and transcriptional regulation of AvBD-11 in the chicken oviduct and its expression in cancerous ovaries of chickens. Results of this study of young chicks revealed that DES induced AvBD-11 mRNA and protein in the oviduct, specifically luminal and glandular epithelial cells. In addition, microRNA-1615 was discovered to influence AvBD-11 expression via its 3′-UTR which suggests post-transcriptional regulation of AvBD-11 expression in chickens. Furthermore, we compared the expression patterns of the AvBD-11 gene in normal and cancerous ovaries from laying hens which are models for human epithelial ovarian cancer. Our results demonstrated that AvBD-11 is most abundant in the glandular epithelium of endometrioid-type ovarian tumors, but not normal ovaries of laying hens. Collectively, these results suggest that AvBD-11 is an estrogen-induced gene during oviduct development and that it may be used as a biomarker for diagnosis of ovarian cancer and for monitoring effects of therapeutics on progression of ovarian carcinogenesis.

AHCYL1 Is Mediated by Estrogen-Induced ERK1/2 MAPK Cell Signaling and MicroRNA Regulation to Effect Functional Aspects of the Avian Oviduct

S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP3 receptor-binding protein released with IP3 (IRBIT), regulates IP3-induced Ca2+ release into the cytoplasm of cells. AHCYL1 is a critical regulator of early developmental stages in zebrafish, but little is known about the function of AHCYL1 or hormonal regulation of expression of the AHCYL1 gene in avian species. Therefore, we investigated differential expression profiles of the AHCYL1 gene in various adult organs and in oviducts from estrogen-treated chickens. Chicken AHCYL1 encodes for a protein of 540 amino acids that is highly conserved and has considerable homology to mammalian AHCYL1 proteins (>94% identity). AHCYL1 mRNA was expressed abundantly in various organs of chickens. Further, the synthetic estrogen agonist induced AHCYL1 mRNA and protein predominantly in luminal and glandular epithelial cells of the chick oviduct. In addition, estrogen activated AHCYL1 through the ERK1/2 signal transduction cascade and that activated expression of AHCYL1 regulated genes affecting oviduct development in chicks as well as calcium release in epithelial cells of the oviduct. Also, microRNAs, miR-124a, miR-1669, miR-1710 and miR-1782 influenced AHCYL1 expression in vitro via its 3′-UTR which suggests that post-transcriptional events are involved in the regulation of AHCYL1 expression in the chick oviduct. In conclusion, these results indicate that AHCYL1 is a novel estrogen-stimulated gene expressed in epithelial cells of the chicken oviduct that likely affects growth, development and calcium metabolism of the mature oviduct of hens via an estrogen-mediated ERK1/2 MAPK cell signaling pathway.

Identification and Characterization of Avian Pleiotrophin as Estrogen-Stimulated Gene in the Chicken Oviduct and Its Differential Expression Patterns in Cancerous Ovaries.

Pleiotrophin (PTN), also known as heparin-binding growth factor 8 (HBGF-8), is a developmentally-regulated growth factor expressed in normal tissues and in carcinomas. Although PTN is involved in regulation of cellular development and differentiation and the etiology of carcinogenesis, little is known about its expression and regulation in either the oviduct or normal and cancerous ovaries of chickens. This novel study compared chicken and mammalian PTN genes with respect to structure, phylogenetic evolution, tissue specific expression of PTN mRNA and protein, and regulation of expression by estrogen in the chicken oviduct. We performed RT-PCR, quantitative RT-PCR, in situ hybridization and immunohistochemical analyses to detect PTN mRNA expression and protein localization in organs of adult hens, young chicks treated with diethylstilbestrol (DES) and laying hens with ovarian carcinoma. Chicken PTN is highly conserved with respect to mammalian PTN genes (91-92.6%) and its mRNA expression was most abundant in brain, heart and oviduct. This study focused on the avian PTN gene in the female chicken reproductive organs. Treatment of young chicks with diethylstilbestrol induced PTN mRNA and protein only in luminal and glandular epithelia of the oviduct. Further, PTN gene expression is influenced by several micro RNAs, specifically miR-499 and miR-1709 through its 3'-UTR, which suggests that post-transcriptional regulation influences PTN expression in chickens. We also compared CpG methylation status of the PTN gene in normal and cancerous ovaries from laying hens to discover high expression of the PTN gene in glandular epithelia of ovarian carcinoma in laying hens with 30- and 40% of -1311 and -1339 CpG sites being demethylated in ovarian cancer cells, but not in normal ovarian epithelial cells. In conclusion, chicken PTN is a novel estrogen-stimulated gene expressed abundantly in oviductal epithelia implicating roles in oviduct development and egg formation. PTN is also a biomarker for epithelial ovarian carcinoma that could be used for diagnosis and monitoring effects of cancer therapies. Research supported by the World Class University (WCU) program (R31-10056) and by Basic Science Research Program (2010-0013078) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology.

HISTOLOGICAL EVIDENCE FOR BLOOD SUPPLY TO THE SPERM-HOST GLANDS (SHG) IN THE OVIDUCT OF NATIVE CHICKEN (GALLUS DOMESTICUS)

Blood supply to the uterova.-inal sperm-host glands of native chicken was investigated in the present study using light microscopy (X40) during the period from January to June 2002. Samples were collected from the oviduct of deshi chicken and stained with H and E stain in Histology Laboratory of the Department of Anatomy and Histology, BAU, Mymensingh, Bangladesh. Results of the present study revealed that the arterial and venous capillaries were present mainly in the submucosa, also in the core of the villi of the oviduct. However, the evidence for the blood supply of the sperm-host glands in the chicken's oviduct suggesting nutlitional supplies to these glands as well as spermatozoa.

Cell-Specific and Temporal Aspects of Gene Expression in the Chicken Oviduct at Different Stages of the Laying Cycle1

Egg formation and embryonic development occur as the yolk passes through the magnum, isthmus, and shell gland of the oviduct before oviposition in hens. The present study identified candidate genes associated with secretory function of the chicken oviduct after ovulation and contributing to egg formation and oviposition. Hens (n = 5 per time point) were euthanized to recover the reproductive tract when the egg was in the magnum (3 h after ovulation) and the shell gland (20 h after ovulation). Total RNA was extracted from each segment of the oviducts and subjected to Affymetrix chicken GeneChip analysis. Quantitative PCR and in situ hybridization analyses of selected genes confirmed the validity of the gene expression patterns detected using microarray analysis. In particular, ACP1, CALB1, CYP26A1, PENK, RCAN1 and SPP1 expression increased significantly in the shell gland between 3 h and 20 h postovulation, whereas only RCNA1 expression increased significantly in the magnum between 3 h and 20 h postovulation. Results of the high-throughput analysis revealed cell-specific and temporal changes in gene expression in the oviduct at 3 h and 20 h postovulation in laying hens provide novel insight into changes at the molecular and cellular levels of candidate genes related to formation of the egg and oviposition.

Differential expression of secreted phosphoprotein 1 in response to estradiol-17β and in ovarian tumors in chickens

Secreted phosphoprotein 1 (SPP1), a highly phosphorylated protein containing a polyaspartic acid sequence and a conserved RGD motif, plays important roles in physiological processes such as inflammatory responses, calcification, organ development, immune cell function and carcinogenesis. Results of the present study indicate expression of SPP1 mRNA in various organs such as oviduct, small intestine and kidney from chickens, particularly in the glandular epithelium (GE) of the shell gland and, to a lesser extent, in luminal epithelium (LE) of the infundibulum and magnum, and GE of the isthmus of the oviduct. We determined that DES (diethylstilbestrol, a synthetic nonsteroidal estrogen) decreases SPP1 expression in the oviduct and that SPP1 mRNA and protein are significantly more abundant in GE of ovarian endometrioid carcinoma, but not the other cancerous and normal ovaries of hens. Further, microRNA-140 was discovered to influence SPP1 expression via its 3′-UTR which suggests that post-transcriptional regulation influences SPP1 expression in chickens. Collectively, results of this study indicate that SPP1 is novel in that its expression is down-regulated by estrogen in epithelial cells of the chicken oviduct and that it is up-regulated in chicken ovarian endometrioid tumor that could be used for monitoring effects of therapies for this disease in laying hens.

In vitro optimization of the Gallus domesticus oviduct epithelial cells culture

The aim of this experiment was to establish an efficient method for isolation and further culture in vitro of the normal chicken oviduct epithelial cells (COEC) for cell-based research models. Different factors were tested to optimize COEC primary culture for repeatable results: the origin of isolated cells (oviduct Infundibulum or Magnum section); the oviduct tissue dissociation procedure (mechanical scrapping or mincing), tissue digestion times (15, 30 and 45 min), the culture plates coating (colagene I, polystyrene surface or 3T3 feeder layer), the growth media (classic DMEM/Ham's F12 and defined serum-free medium, Lonza Switzerland), incubation temperature (37 °C vs 41°C) and different cell seeding numbers: 0.2M, 0.5M and 1.0M cells/well. The COEC isolated by mincing the Infundibular neck and digestion of tissue for 30 min formed cell aggregates of bright colour and gave proliferating colonies of epithelial-like character which was the best result obtained from all applied procedures in our studies. The fibroblast-like cells considered as contaminants occurred only sporadically up to day 7 of culture. Seeding about 1M cells in 1 mL of serum-free medium onto 12-well dishes gave the optimal growth of colonies resulting in 5 to 7 confluent culture wells from a single oviduct sample. Feeder layer and collagen I did not improve adhesion of the COEC to the culture vessel. Adoption of 37 °C and 41 °C did not reveal apparent differences to the condition of cultured COEC. Cell differentiation and proliferation potential depends on number and replicative capacity of isolated progenitors. The progenitors are responsible for holoclones formation and good culture growth. The percentage of colonies developed from the cells isolated from Infundibulum was greater than that of other samples in our studies. We conclude that the model of COEC primary cultures from different segments of oviduct, in particular infundibulum, should be incorporated to the range of avian cells research as this work generates questions about undocumented sources of oviduct progenitor cells.

ERBB receptor feedback inhibitor 1: Identification and regulation by estrogen in chickens

The ERBB receptor feedback inhibitor 1 (ERRFI1) is a scaffolding adaptor protein, that plays a pivotal role in the epidermal growth factor receptor (EGFR) cell signaling cascade as a negative regulator affecting many important physiological processes. It was recently reported that ERRFI1 is a critical regulator of the response of the endometrium to estrogen regulation of tissue homeostasis in mice. But, very little is known about ERRF11 and hormonal regulation of the ERRFI1 gene in chickens. Therefore, in the present study, ERRFI1 gene was cloned and its differential expression profile analyzed at different embryonic stages, in various adult organs, and in oviducts from estrogen-treated chickens. Chicken ERRFI1 has an open-reading frame of 2848 nucleotides that encode for a protein of 465 amino acids that has considerable homology to mammalian ERRFI1 proteins (>62% identity). Importantly, ERRFI1 mRNA is abundantly distributed in various organs from chickens. We then determined that DES (diethylstilbestrol, a synthetic nonsteroidal estrogen) induced ERRFI1 mRNA and protein predominantly in luminal and glandular epithelial cells of the oviduct. Further, we determined whether microRNAs, specifically miR-200b, miR-429 and miR-1639, influence ERRFI1 expression via its 3′UTR and found that it does not directly target the 3′UTR of ERRFI1 mRNA. Therefore, it is unlikely that post-transcriptional regulation influences ERRFI1 expression in the chicken oviduct. In conclusion, our results indicate that ERRFI1 is a novel estrogen-stimulated gene expressed in epithelial cells of the chicken oviduct that likely plays an important role in oviduct growth and differentiation during early development of the chicken.

Tissue specific expression and estrogen regulation of SERPINB3 in the chicken oviduct

Serine protease inhibitors (SERPINs) comprise the largest superfamily of protease inhibitors and appear to be ubiquitously expressed in a variety of species. Of these, squamous cell carcinoma antigen 1 (SCCA1), also known as a SERPINB3, was first identified in squamous cell carcinoma tissue from the cervix of women. However, there is little known about the expression and hormonal regulation of SERPINB3 in chickens. Therefore, the avian SERPINB3 gene was compared with those of other species with respect to structure, phylogenetic evolution and tissue- and cell-specific expression in hens. Chicken SERPINB3 has moderate homology to mammalian SERPINB3 proteins (36–47%). Of particular note, SERPINB3 mRNA was most abundant in the chicken oviduct and cell-specific expression was in glandular (GE) and luminal (LE) epithelial cells of the oviduct of laying hens. Treatment of young chicks with DES (diethylstilbestrol, a synthetic nonsteroidal estrogen) induced SERPINB3 mRNA and protein in GE and LE, but not in other cell types of the oviduct. Western blot analyses determined that immunoreactive SERPINB3 protein was also increased by DES in LE and GE of the oviduct of chicks. Collectively, these results indicate that SERPINB3 is an estrogen-induced gene expressed only in LE and GE of the chicken oviduct and implicate SERPINB3 in regulation of oviduct development and egg formation.