Evidence that luteinizing hormone-releasing hormone statin from ovine retetestis fluid is immunologically related to alpha C inhibin

LHRH Statin is a putative gonadal protein that increases the interval betwe en two consecutive LHRH pulses. The present work was aimed at analyzing the immunological homology between LHRH Statin and the N-terminal region of th e alpha C subunit of inhibin. Thus, rete testis fluid (RTF) proteins were p urified by immunoaffinity chromatography using antibodies against residues 1-7 plus 7-30 (experiment 1, A-fractions) and 14-28 of the alpha C inhibin subunit (experiment 2, B-fractions), and the LHRH Statin activity of the fr actions was examined by intracerebroventricular administration in castrated rams followed by RIA of plasma LH levels in 15-min blood samples. Fraction s that bound to the immunoaffinity column with low affinity were eluted wit h 0.5 M NaCl, pH 7.4 (-F2); then highly bound fractions were eluted sequent ially in acidic (pH 2.5, -F3) followed by basic conditions (pH 11.5, -F4). In experiment 1, RTF (40 mu g, n = 4) and highly bound fractions (A-F3, 30 ng, n = 8, 150 ng, n = 3; A-F4, 120 ng, n = 5) decreased LH mean plasma lev els between 4 and 6 h after injection by 39%, 29%, 43%, and 37%, respective ly (P < 0.001 to 0.01), while the weakly bound fractions (A-F2, 180 ng, n = 4) and albumin control (40 mu g, n = 4) had no activity. In experiment 2, RTF (100 mu g, n = 4) and B-F3 (100 ng, n = 3) decreased plasma LH levels b y 48% and 38%, respectively (P < 0.001 to 0.05), whereas B-F4 (100 ng, n = 4) and albumin control (100 mu g, n = 4) had no effect. A fraction obtained from B-F3 by gel filtration had significant LHRH Statin activity (63%, n = 6, P < 0.001). PAGE with colloidal gold staining revealed 3 high molecular weight bands and 5 low molecular weight bands in B-63. The 3 high molecula r weight bands were shown to belong to the clusterin family and did not app ear to have LHRH Statin activity. The 5 low molecular weight bands were all labeled by anti-alpha C inhibin antibodies. Collectively, these results st rongly suggest that LHRH Statin has some homology with the 14-28 alpha C in hibin sequence.