Calcium Participates in Secretion of Porphyrin from Shell Gland Epithelial Cells of Japanese Quail (Coturnix coturnix japonica)

Bird eggshell coloring depends on superficial pigments. Japanese quail eggshells are pigmented with protoporphyrin IX (PpIX). The PpIX is accumulated by the shell gland epithelial cells and secreted onto the shell surface 3.5 - 2 h before oviposition.

In this paper, to investigate factors involved in the secretion of protoporphyrin IX (PpIX), a superficial eggshell pigment, from shell gland epithelial cells of Japanese quail, we cultured cells in Ham’s F12 medium with calcium chloride and quail plasma.

Japanese quail ≥ 10 weeks old were used and laid on a regular clutch in the experiment. The quail were individually caged in a windowless environment-controlled room under a cycle of 14 h light/10 h dark. Food and water were available ad libitum. Eggs were collected at 5, 3, or 0.5 h before estimated oviposition or 1 h after oviposition. Shells removed of shell membrane were extracted in 6 mL hydrogen chloride-methanol solution (1:1, 20% HCl and 100% methanol) for 24 h in the dark at room temperature. The shell gland epithelial cells were also collected from the same quails. The isolated epithelial cells collected at 5 h before estimated oviposition were counted and adjusted to 2 × 10⁶ cells/mL in Ham’s F12 medium with 5 mM CaCl₂. All data obtained from 3 wells per quail were averaged.

The results showed that the PpIX concentration (indicated by fluorescence intensity) in eggshell increased significantly from −5 h to −3 h to +1 h. The concentration in the epithelial cells also increased marginally before oviposition, but decreased greatly at −0.5 h. At −3 h, the correlation between eggshells and cells was significantly negative (n = 7, Pearson’s r = −0.927, P < 0.01%). The addition of hormones (prostaglandin F2α, progesterone, estradiol-17β) to the medium did not change the PpIX concentration in the culture supernatant, but change the calcium chloride (CaCl₂) concentration: a lower concentration of CaCl₂ led to a higher PpIX concentration; 0 mM CaCl₂ enhanced the secretion of PpIX from epithelial cells prepared at 5 or 7 mM CaCl₂.

In conclusion, the PpIX concentration in culture medium containing quail plasma depended on the concentration of CaCl2 but not on the presence or concentration of hormones. The rapid drop of CaCl2 concentration in the culture medium might mimic the environment of the cells and the timing of the end of shell calcification in vivo, and thus might be an important factor in triggering PpIX secretion.

Article by Osamu Kai, from Nihon University, Fujisawa, Japan.

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