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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