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.

Central effect of rete testis fluid, inhibin 32K, and follicular fluid on plasma gonadotropin concentrations in sheep: inhibin is not the rete testis fluid protein able to suppress luteinizing hormone pulses.

We have previously shown that peripheral administration of rete testis fluid (RTF) proteins was able to suppress LH pulses through the suppression of LHRH pulses. This activity was named "LHRH Statin." The aims of the present work were to analyze LH inhibition after an intracerebroventricular injection of RTF and to determine whether inhibin is the factor responsible for this inhibition. Castrated rams (experiment 1) or ewes (experiment 2) received an intracerebroventricular injection of RTF, purified bovine inhibin 32K, bovine follicular fluid, or human serum albumin as control. Animals were bled every 15 min for 5 h before injection and for 7 h after injection. LH mean levels were significantly lowered (p < 0.01) only in the RFT-treated groups. FSH levels were not affected irrespective of group, source, or dose of inhibin. These experiments show first, that protein(s) present in ovine RTF can suppress LH secretion in sheep; second, that bovine follicular fluid or purified bovine inhibin 32K have no effect on LH secretion. Furthermore, the results suggest that centrally administered inhibin has no effect on FSH secretion under our experimental conditions. Together, these experiments clearly demonstrate that inhibin 32K does not exert any "LHRH statin" activity.