Avian WNT4 in the female reproductive tracts: potential role of oviduct development and ovarian carcinogenesis

Abstract

The wingless-type MMTV integration site family of proteins (WNTs) is highly conserved secreted lipid-modified signaling molecules that play a variety of pivotal roles in developmental events such as embryogenesis, tissue homeostasis and cell polarity. Although, of these proteins, WNT4 is known to be involved in genital development in fetuses of mammalian species, its role is unknown in avian species. Therefore, in this study, we investigated expression profiles, as well as hormonal and post-transcriptional regulation of WNT4 expression in the reproductive tract of female chickens. Results of this study demonstrated that WNT4 is most abundant in the stromal and luminal epithelial cells of the isthmus and shell gland of the oviduct, respectively. WNT4 is also most abundant in the glandular epithelium of the shell gland of the oviduct of laying hens at 3 h post-ovulation during the laying cycle. In addition, treatment of young chicks with diethylstilbestrol (DES, a synthetic estrogen agonist) stimulated WNT4 only in the glandular epithelial cells of the isthmus and shell gland of the oviduct. Moreover, results of our study demonstrated that miR-1786 influences WNT4 expression via specific binding sites in its 3'-UTR. On the other hand, our results also indicate that WNT4 is expressed predominantly in the glandular epithelium of cancerous ovaries, but not in normal ovaries of hens. Collectively, these results indicate cell-specific expression of WNT4 in the reproductive tract of chickens and that it likely has crucial roles in development and function of oviduct as well as initiation of ovarian carcinogenesis in laying hens.

Figure 1. Expression and localization of WNT4 in the chicken oviduct.

Both RT-PCR [A] and quantitative PCR [B] analyses were performed using cDNA templates from each segment of the chicken oviduct. These experiments were conducted in triplicate and normalized to control ACTB expression. [C] In situ hybridization analysis for cell-specific changes in expression of WNT4 in the each segment of the chicken oviduct. Both RT-PCR [D] and quantitative PCR [E] analyses were performed using cDNA templates from the magnum and the shell gland segment at 3 h and 20 h after ovulation. [F] In situ hybridization analysis for cell-specific changes in expression of WNT4 in the magnum and the shell gland at 3 h and 20 h after ovulation. Legend: ST, stromal cells; GE, glandular epithelium; LE, luminal epithelium. Scale bar represents 50 μm (the first and the third horizontal panels) and 20 μm (the second horizontal panel). The tissue samples were from oviducts of 1- to 2- year-old female laying hens (n = 5). The asterisks denote statistically significant differences (***P<0.001 and **P<0.01).

Figure 2. Effect of DES on tissue-specific expression of chicken WNT4.

Both RT-PCR [A and B] and quantitative PCR [C and D] analyses were performed using cDNA templates from DES-treated and untreated oviducts. These experiments were conducted in triplicate and normalized to control ACTB expression. [E] In situ hybridization analyses revealed cell-specific expression of WNT4 mRNA in oviducts of DES-treated and untreated chicks. Cross-sections of the four segments of chicken oviduct (infundibulum, magnum, isthmus, and shell gland) from chicks treated with DES or vehicle were hybridized with antisense or sense chicken WNT4 cRNA probes. Legend: GE, glandular epithelium LE, luminal epithelium. Scale bar represents 50 μm (the first and the third horizontal panel) and 20 μm (the second horizontal panel). The tissue samples were used from 37-day-old chick oviducts (n = 5). The asterisks denote statistically significant differences (***P<0.001 and *P<0.05).

Figure 3. In vitro target assay of miR-1786 on WNT4 transcript.

[A] Diagram of miR-1786 binding site in the 3′-UTR of the WNT4 gene. [B] Expression vector maps for eGFP with the 3′-UTR of WNT4 and Ds-Red with miR-1786. The 3′-UTR of the WNT4 transcript was subcloned between the eGFP gene and the polyA tail to generate the fusion construct of the GFP transcript following the miRNA target 3′-UTR (pcDNA-eGFP-3′UTR) (upper panel) and miRNA expression vector was designed to co-express DsRed and miRNA (pcDNA-DsRed-miRNA) (lower panel). [C and D] After co-transfection of pcDNA-eGFP-3′UTR for the WNT4 transcript and pcDNA-DsRed-miRNA for the miR-1786, the fluorescence signals of GFP and DsRed were detected using FACS [C] and fluorescent microscopy [D]. [E] Quantitative expression of miR-1786 between untreated and DES-treated oviducts of chicks was analyzed by qRT-PCR. This experiment was normalized to control U6 snRNA expression. The asterisk denotes statistically significant differences (**P<0.01).

Figure 4. Expression and localization of WNT4 between normal and cancerous ovaries.

[A] Quantitative PCR analysis was performed using cDNA templates from normal and cancerous ovaries. [B] In situ hybridization analyses revealed cell-specific expression of WNT4 mRNA between normal and cancerous ovary. Legend: GE, glandular epithelium. Scale bar represents 50 μm (the first and the third horizontal panel) and 20 μm (the second horizontal panel). The tissue samples were from normal and cancerous ovaires of 2- to 3- year-old laying hens (n = 5). The asterisks denote statistically significant differences (*P<0.05).