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Biology of Reproduction 62, 1551-1563 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Evidence That Luteinizing Hormone-Releasing Hormone Statin from Ovine Rete Testis Fluid Is Immunologically Related to {alpha}C Inhibin1

C. Cariou-Guennoca, J.C. Poiriera, B. Calasb, A. Locatellia, J.L. Dacheuxa, and M.R. Blanc2,a

a URA CNRS 1291, Laboratoire de Physiologie de la Reproduction des Mammifères Domestiques, 37380 Nouzilly, France b CRBM-CNRS (ERS 155 et INSERM U249), BP 5051 34033 Montpellier, Cedex, France

LHRH Statin is a putative gonadal protein that increases the interval between two consecutive LHRH pulses. The present work was aimed at analyzing the immunological homology between LHRH Statin and the N-terminal region of the {alpha}C subunit of inhibin. Thus, rete testis fluid (RTF) proteins were purified 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 fractions was examined by intracerebroventricular administration in castrated rams followed by RIA of plasma LH levels in 15-min blood samples. Fractions that bound to the immunoaffinity column with low affinity were eluted with 0.5 M NaCl, pH 7.4 (-F2); then highly bound fractions were eluted sequentially in acidic (pH 2.5, -F3) followed by basic conditions (pH 11.5, -F4). In experiment 1, RTF (40 µg, 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 levels between 4 and 6 h after injection by 39%, 29%, 43%, and 37%, respectively (P < 0.001 to 0.01), while the weakly bound fractions (A-F2, 180 ng, n = 4) and albumin control (40 µg, n = 4) had no activity. In experiment 2, RTF (100 µg, n = 4) and B-F3 (100 ng, n = 3) decreased plasma LH levels by 48% and 38%, respectively (P < 0.001 to 0.05), whereas B-F4 (100 ng, n = 4) and albumin control (100 µg, 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-F3. The 3 high molecular weight bands were shown to belong to the clusterin family and did not appear to have LHRH Statin activity. The 5 low molecular weight bands were all labeled by anti-{alpha}C inhibin antibodies. Collectively, these results strongly suggest that LHRH Statin has some homology with the 14–28 {alpha}C inhibin sequence.

First decision: 10 February 1999.

1 C.C.G. was supported by a grant in aid by INRA (50%) and Région Centre (50%).

2 Correspondence. FAX: 33 2 47 42 77 43; michel.blanc{at}tours.inra.fr






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