Extragonadal alpha-inhibin precursor proteins circulate in human male serum.
The role of inhibin as a negative feedback regulator of pituitary FSH secretion in men remains controversial inasmuch as serum inhibin and FSH levels are often not correlated, in part because of the known alpha-inhibin subunit cross-reactivity of the most widely used inhibin antiserum (Monash #1989). The objective of this study was to identify the nature of these alpha-inhibin proteins in male serum using antisera specific for the alpha-inhibin precursor. Three polyclonal antisera were raised against synthetic peptide fragments from the proregion (amino acids 21-35 = precursor alpha inhibin (PIN) 1 and 42-56 = PIN 2) and alpha-N segment (113-127 = PIN 3) of the human alpha-inhibin precursor protein. These antisera were then used in individual RIAs with the homologous peptide as both standard and radioligand. Because pure human alpha-inhibin subunit proteins are not available, recombinant alpha-inhibin medium and porcine follicular fluid were used as reference preparations in the PIN assays. All assays were specific for alpha-inhibin proteins, i.e. 1) they showed no significant cross-reactivity with other alpha-inhibin peptide fragments, dimeric 32-kDa inhibin, recombinant activin, FSH, human albumin, or the inhibin binding proteins alpha-2 macroglobulin and follistatin, and 2) they recognized native protein in alpha-inhibin precursor-containing porcine follicular fluid and in medium from a recombinant alpha-inhibin-only secreting cell line. Furthermore, immunoreactivity of all serum proteins seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with the PIN antisera was abolished or significantly reduced (40-100%) by preincubation of each PIN antiserum with the homologous peptide. Serial dilutions of serum from normal, GnRH-deficient, and castrate males exhibited equivalent displacement curves in each precursor (PIN) assay, and there were no significant group differences in PIN 2 immunoreactivity between normal (n = 14), GnRH-deficient (n = 8), and castrate men (n = 3). Western blotting of serum samples from a normal and a GnRH-deficient male revealed immunoreactive proteins of approximately 57 and 29 kDa under reducing conditions with all three PIN antisera. An additional 40-kDa protein was observed with the pro-alpha-inhibin antisera, PIN 1 and 2, and a protein of more than 97 kDa was seen with the PIN 2 antiserum.(ABSTRACT TRUNCATED AT 400 WORDS)
Lambert-Messerlian
G M
GM
Reproductive Endocrine Unit, Massachusetts General Hospital, Boston 02114, USA.
Crowley
W F
WF
Schneyer
A L
AL
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D003429:Cross Reactions; D005246:Feedback; D005640:Follicle Stimulating Hormone / Q000097:blood / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000172:deficiency; D006801:Humans; D007106:Immune Sera; D007265:Inhibins / Q000097:blood* / Q000302:isolation & purification; D008297:Male; D010446:Peptide Fragments / Q000097:blood / Q000276:immunology; D011498:Protein Precursors / Q000097:blood* / Q000302:isolation & purification; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000032:analysis; D012016:Reference Values; D013552:Swine
D007106:Immune Sera; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; C095971:inhibin-alpha subunit precursor; D007987:Gonadotropin-Releasing Hormone; D007265:Inhibins; D005640:Follicle Stimulating Hormone