Evidence that luteinizing hormone-releasing hormone statin from ovine rete testis fluid is immunologically related to alphaC inhibin.
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 alphaC 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 alphaC 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 microg, 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 microg, n = 4) had no activity. In experiment 2, RTF (100 microg, 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 microg, 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-alphaC inhibin antibodies. Collectively, these results strongly suggest that LHRH Statin has some homology with the 14-28 alphaC inhibin sequence.
Cariou-Guennoc
C
C
URA CNRS 1291, Laboratoire de Physiologie de la Reproduction des Mammifères Domestiques, 37380 Nouzilly, France.
Poirier
J C
JC
Calas
B
B
Locatelli
A
A
Dacheux
J L
JL
Blanc
M R
MR
D000818:Animals; D000918:Antibody Specificity; D001826:Body Fluids / Q000737:chemistry*; D002846:Chromatography, Affinity; D000939:Epitopes / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D007987:Gonadotropin-Releasing Hormone / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D007158:Immunologic Techniques; D007265:Inhibins; D007986:Luteinizing Hormone / Q000097:blood; D008297:Male; D010455:Peptides / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D012152:Rete Testis / Q000378:metabolism*; D017385:Sequence Homology; D012756:Sheep
D000939:Epitopes; D010455:Peptides; C094377:inhibin-alpha subunit; D007987:Gonadotropin-Releasing Hormone; D007265:Inhibins; D007986:Luteinizing Hormone