Identification of cGnRH-II in the median eminence of Japanese quail (Coturnix coturnix japonica)

In a previous paper, we described the presence of cGnRH-II in the quail (Coturnix coturnix japonica) and chicken (Gallus gallus) median eminence using highly specific antibodies directed against a polypeptide corresponding to the C-terminal portion of cGnRH-II (van Gils et al., 1993). This finding remained very controversial, since no other study, with any other antibody, had ever reported the presence of cGnRH-II immunoreactive fibers in the median eminence of birds. In this study, the cGnRH-II immunoreactive substances in quail median eminence were isolated by RP-HPLC and identified by RIA. To eliminate the possibility that the cGnRH-II-like immunoreactivity in the median eminence was due to a cross-reaction of our anti-cGnRH-II antiserum with an unknown peptide, the cGnRH-II immunoreactive substances, present in a quail median eminence extract, were isolated by immunoaffinity chromatography using immunoaffinity-purified antibodies. In the eluate of the immunoaffinity column only one peptide could be detected by mass spectrometry. This peptide had a mass of 1235.56 Da, which is the same as synthetic cGnRH-II. In addition, MS/MS fragmentation generated an amino acid sequence corresponding to the sequence of cGnRH-II. The present study therefore identified indisputably cGnRH-II in the median eminence of the quail.