We used Japanese quail ≥ 10 weeks old and laying on a regular clutch. 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. The oviposition times of each quail were automatically recorded (Egg Counter, O’Hara & Co., Ltd. Tokyo, Japan) and estimated oviposition time was calculated from former several days’ oviposition time. Quail laying on a regular clutch were selected and used at a certain time before estimated oviposition or after actual oviposition. All procedures were conducted in accordance with the guidelines for animal experiments, College of Bioresource Science, Nihon University.

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. After centrifugation (1600× g, 5 min, 4˚C), the supernatant was collected; 100 µL was placed in each well of a 96-well culture plate (#3860-096, Iwaki, Tokyo, Japan) and the fluorescence was determined on a Synergy 2 Multi-Mode Microplate Reader (BioTek Instrument Inc., Winooski, VT, USA) using a 400-nm, 30-nm-bandwidth excitation filter and a 620-nm, 40-nm bandwidth emission filter [12] . The fluorescence intensity was used as the value of PpIX.

The shell gland epithelial cells were collected from the same quails as in 2.2. The cell layer was isolated from the shell gland epithelium as described [12] . In brief, the tissues were placed in Ham’s F12 medium (Nissui Pharmaceutical, Tokyo, Japan) with 5 mM CaCl₂ (Nacalai Tesque, Tokyo, Japan) and 0.001% collagenase (Wako, Tokyo, Japan) or in the same volume of Accumax cell dissociation solution (Innovative Cell Technologies, Inc., San Diego, CA, USA). The mixture was cultured in a shaking water bath for 10 min (collagenase) or 15 min (Accumax) at 37˚C. The cells were centrifuged (30× g, 5 min, 4˚C) to wash out the enzyme. The precipitated cells were resuspended in Ham’s F12 medium with 5 mM CaCl₂ and stood for several minutes to allow unseparated tissues to settle out. This process was repeated until the supernatant was clear. Then the supernatant was centrifuged (30× g, 5 min, 4˚C) once more. The sediment was used as shell gland epithelial cells in experiments. For some experiments (e.g. Figure 2), unincubated cells were resuspended in Ham’s F12 + 20% HCl (final, 4%), stirred vigorously, and centrifuged (2000× g, 5 min, 4˚C), and PpIX in the supernatant was measured promptly as in 2.2.

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. Cell viability was assessed by the trypan blue method. Cells suspension (2 × 10⁵ cells/well) were placed in each of 3 wells per quail in a 96-well culture plate with 10 µL pooled plasma (final, 4%) taken from normal quail collected at any time (or the sampled quail) or with the same volume of medium as a control (total, 250 µL) and cultured in an incubator (5% CO₂, 37˚C) for 4 h. After incubation, 200 µL of culture supernatant was collected and mixed with Ham’s F12 + 20% HCl (final, 4%), stirred vigorously, and measured as described in 2.2; the mean value of 3 wells was defined as PpIX secreted from the cells. The remainder in each well was then mixed with 200 µL medium (Ham’s F12) and centrifuged (30× g, 5 min, 4˚C), and the sediment was resuspended in 200 µL medium (Ham’s F12 + 20% HCl) as cell samples. PpIX was measured as described in 2.2 and the values were defined as PpIX in the cells.

To test the effect of hormones, we individually dissolved Prostaglandin F2α Tris salt (PGF2α; Sigma, Tokyo, Japan), Progesterone-water soluble (P4; Sigma), and β-Estradiol-water soluble (E2; Sigma) in 0.22-µm-filter-sterilized PBS. Five µL of hormones were added at either 200 or 2 µM (PGF2α, P4) or at either 200 or 2 nM (E2) to cell suspension (total, 250 µL in Ham’s F12 medium with 5 mM CaCl₂). To exclude steroid hormones from plasma, we mixed half of the quail plasma with activated charcoal powder (Wako) at 0.02 g/mL overnight with stirring at 4˚C. The sample was centrifuged (1600× g, 20 min, 4˚C), then the supernatant was centrifuged again (5000× g, 40 min, 4˚C). The supernatant was filtered at 0.45 µm and used as charcoal-treated plasma. The other half of the plasma was left untreated. Cells collected at 5 h before estimated oviposition were cultured as described in 2.4. After incubation, 200 µL of culture supernatant was collected and mixed with Ham’s F12 + 20% HCl (final, 4%), stirred vigorously, and measured as described in 2.2; the values were defined as PpIX secreted from the cells.

We prepared 1 M CaCl₂ solution in Ham’s F12 medium, diluted in the medium, and added 15 µL of diluted solution to the cell suspension (final, 250 µL) to a final concentration of 2, 3, 5, or 7 mM. Cells collected at 5 h before estimated oviposition were prepared and cultured as described in 2.4. After incubation, 200 µL of culture supernatant was collected and measured as described in 2.5; the values were defined as PpIX secreted from the cells.

We added 25 µL of 10% BSA (Wako) dissolved in sterilized PBS (final, 1%) or 10 µL of quail plasma to cell suspension collected at 5 h before estimated oviposition (total, 250 µL in Ham’s F12 with 7 mM CaCl₂). After 1 h incubation, we removed 200 µL of culture medium (1st culture) and added 200 µL of fresh medium (Ham’s F12 with 0, 3, or 7 mM CaCl₂) and cultured it for a further 2 h (2nd culture) in an incubator (5% CO₂, 37˚C). After incubation, 200 µL of culture supernatant was collected and measured as described in 2.5; the values were defined as PpIX secreted from the cells.

All data obtained from 3 wells per quail were averaged. Data from all experiments were analyzed by Tukey-Kramer’s HSD multiple range test or Student’s t-test. Correlations were tested with Pearson’s coefficient. Values were determined to be significant at P < 0.05.

Figure 1 shows the appearance of eggs and shell gland epithelium at an estimated 5 and 3 h before oviposition and 1 h after oviposition. Eggs collected at −3 h varied in appearance.

The PpIX concentration (indicated by fluorescence intensity) in eggshell increased significantly from −5 h to −3 h to +1 h (Figure 2). 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%).