Research note: tissue- and stage-specific expression of ovocalyxin-32 during eggshell formation in laying hens

The avian eggshell is a biomineralized structure that protects the developing embryo from physical damage and microbial contamination. Ovocalyxin-32 (OCX32) is an eggshell matrix protein implicated in shell formation and antimicrobial defense. However, its tissue distribution and stage-dependent expression in the reproductive tract throughout the egg-laying cycle remain poorly characterized. This study investigated the tissue distribution of chicken OCX32 and its expression in the eggshell gland during the egg-laying cycle. A rabbit polyclonal antibody was generated against a C-terminal peptide of chicken OCX32 and validated using DF-1 cells expressing full-length OCX32. Laying hens were classified into seven physiological stages based on the egg location in reproductive tract and the progression of shell formation: no egg (S1); an egg in the infundibulum, magnum, or isthmus (S2-S4); an egg in the eggshell gland with a soft, hard unpigmented, or hard pigmented shell (S5-S7). OCX32 expression in the eggshell gland was examined by quantitative real-time PCR and Western blotting. Two immunoreactive bands of approximately 27 and 29 kDa were detected in both DF-1 cells overexpressing OCX32 and eggshell gland tissue. Tissue distribution analysis revealed predominant OCX32 protein expression in the eggshell gland, weaker expression in the isthmus, and no detectable expression in other tissues examined. In the eggshell gland, OCX32 mRNA expression was significantly higher at S5 and S6 than at S1-S3, while S4 and S7 showed intermediate expression levels (P < 0.05). OCX32 protein was barely detectable at S1-S4 but markedly increased during the eggshell gland stages (S5-S7), beginning with soft-shell formation and remaining abundant through shell hardening and pigmentation. These findings demonstrate that OCX32 is predominantly expressed in the eggshell gland and exhibits stage-dependent changes in both mRNA and protein abundance, with increased expression coinciding with active shell formation. This spatiotemporal expression pattern supports a specialized role for OCX32 in eggshell formation and potentially in antimicrobial defense of the chicken egg.