Molecular signatures of epithelial oviduct cells of a laying hen (Gallus gallus domesticus) and quail (Coturnix japonica)

Abstract

Background: In this work we have determined molecular signatures of oviduct epithelial and progenitor cells. We have proposed a panel of selected marker genes, which correspond with the phenotype of oviduct cells of a laying hen (Gallus gallus domesticus) and quail (Coturnix japonica). We demonstrated differences in characteristics of those cells, in tissue and in vitro, with respect to different anatomical and functional parts of the oviduct (infundibulum (INF), distal magnum (DM, and proximal magnum (PM)). The following gene expression signatures were studied: (1) oviduct markers (estrogen receptor 1, ovalbumin, and SPINK7 - ovomucoid), (2) epithelial markers (keratin 5, keratin 14, and occludin) and (3) stem-like/progenitor markers (CD44 glycoprotein, LGR5, Musashi-1, and sex determining region Y-box 9, Nanog homebox, OCT4/cPOUV gene encoding transcription factor POU5F3).

Results: In chicken, the expression of oviduct markers increased toward the proximal oviduct. Epithelial markers keratin14 and occludin were high in distal oviduct and decreased toward the proximal magnum. In quail oviduct tissue, the gene expression pattern of oviduct/epithelial markers was similar to chicken. The markers of progenitors/stemness in hen oviduct (Musashi-1 and CD44 glycoprotein) had the highest relative expression in the infundibulum and decreased toward the proximal magnum. In quail, we found significant expression of four progenitor markers (LGR5 gene, SRY sex determining region Y-box 9, OCT4/cPOUV gene, and CD44 glycoprotein) that were largely present in the distal oviduct. After in vitro culture of oviduct cells, the gene expression pattern has changed. High secretive potential of magnum-derived cells diminished by using decreased abundance of mRNA. On the other hand, chicken oviduct cells originating from the infundibulum gained ability to express OVM and OVAL. Epithelial character of the cells was maintained in vitro. Among progenitor markers, both hen and quail cells expressed high level of SOX9, LGR5 and Musashi-1.

Conclusion: Analysis of tissue material revealed gradual increase/decrease pattern in majority of the oviduct markers in both species. This pattern changed after the oviductal cells have been cultured in vitro. The results can provide molecular tools to validate the phenotype of in vitro biological models from reproductive tissue.

Keywords: Epithelial cells; Laying hen; Laying quail; Molecular signatures; Oviduct; Progenitor cells.

Fig. 1

A graphical representation of selected panel of epithelial genetic markers associated with oviduct cells. Three panels of epithelial genetic markers were proposed to provide a pattern of molecular signatures in the oviduct of hen and quail in 3 compartments: INF – infundibulum, DM – distal magnum, PM – proximal magnum. The first panel shown in the picture refers to stem-like markers: Nanog homebox (NANOG), octamer-binding protein 4 (OCT4/cPOUV) and sex determining region Y-box 9 (SOX9); and epithelial progenitor cells: cell surface glycoprotein CD44, leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), and Musashi-1 (MSI-1). The second panel refers to epithelial cells: keratins KRT 5 and 14 and occludin (OCLN). The third panel refers to functional avian oviduct cells: estrogen receptor-1 (ESR1), ovalbumin (OVAL) and ovomucoid (OVM)

Fig. 2

Phenotypes displayed by hen and quail oviduct cell colonies in vitro. a–b: confluent monolayers and visible spheres of colony-initiating cells isolated from the region of infundibulum neck (INF); magnification: × 100. c–d: confluent monolayers of epithelial cells isolated from distal magnum (DM), showing typical cobble-like morphology; magnification × 100. e–f: confluent epithelial monolayer, typically observed in cultivated cells that are originating from the oviduct magnum, showing mostly fibroblast-like morphology; magnification × 100. In each case, the cells were seeded at a density of 4 × 10⁴ cells/cm²

Fig. 3

Expression of oviduct, epithelial, and progenitor markers in different fragments of hen (a) and quail (b) oviduct tissue. Relative gene expression analysis was conducted with RT-qPCR method in three oviduct fragments: infundibulum (INF), distal magnum (DM) and proximal magnum (PM). Pairwise t-test was conducted to determine the significant modulation of the gene expression in the oviduct as compared to the external calibrator (breast muscle) (P < 0.05). An asterisk (*) indicates that the gene is differentially expressed, compared to the calibrator. Letters A, B, and C in brackets indicate results of one-way ANOVA multiple comparisons between different fragments of the oviduct (P < 0.05)

Fig. 4

Expression of oviduct, epithelial, and progenitor markers in chicken (a) and quail (b) oviduct epithelial cells. Relative gene expression analysis was performed with RT-qPCR method in three oviduct fragments: infundibulum (INF), distal magnum (DM) and proximal magnum (PM). Pairwise t-test was conducted to determine the significant modulation of the gene expression in the oviduct as compared to the external calibrator (breast muscle) (P < 0.05). An asterisk (*) indicates that the gene is differentially expressed, compared to the calibrator. Letters A, B, and C in brackets indicate results of one-way ANOVA multiple comparisons between different fragments of the oviduct (P < 0.05)