In-vitro production of precursor peptides for thyrotropin-releasing hormone by human semen.

Thyrotrophin-releasing hormone (TRH) and related peptides occur in high concentrations in human semen. TRH derives from a 242-amino acid precursor protein, prepro-TRH, with six repetitive sequences of -Lys-Arg-Gln-His-Pro-Gly-Lys/Arg)-Arg- connected by hydrophobic linking sequences. Antibodies to TRH-Gly (pGlu-His-Pro-Gly), a final precursor for TRH formation, were used to detect this tetrapeptide as well as other prepro-TRH fragments which cross-react with these antibodies. The total TRH-Gly immunoreactivity decreased significantly after vasectomy. The TRH-Gly immunoreactivity in semen increased significantly during in-vitro incubation at 0 or 37 degrees C, to a peak value at 5 h, followed by an exponential decline, with t 1/2 equal to 11 h at 37 degrees C. At 60 degrees C, however, the TRH-Gly immunoreactivity rose continuously, attaining, after 20 h, a level 2.2 times that at the start of the incubation (P less than 0.001). Reversed-phase high pressure liquid chromatography (HPLC) revealed both hydrophobic and hydrophilic TRH-Gly immunoreactive peptides in semen with both classes of peptides increasing significantly with heating to 60 degrees C. Cation exchange chromatography of pooled human semen incubated at 60 degrees C revealed a 4.3-fold increase in a TRH-Gly immunoreactive peak which co-eluted with synthetic TRH-Gly, and a 30% increase in another TRH-Gly immunoreactive peak identified as Glu-His-Pro-Gly. A minor, TRH-Gly immunoreactive peak increased 50-fold (P less than 0.001) during 20 h at 60 degrees C. This material co-eluted with Arg-Gln-His-Pro-Gly which is formed by enzymic cleavage of the paired basic residues flanking this sequence in prepro-TRH. When synthetic Arg-Gln-His-Pro-Gly was incubated with fresh semen at 60 degrees C a rapid conversion of most of this peptide to Glu-His-Pro-Gly, Gln-His-Pro-Gly and TRH-Gly occurred within 30 min. These data are consistent with thermal inactivation of the amidation and degrading enzymes at 60 degrees C while the trypsin-like enzymes which cleave the precursor peptide at the paired basic residues remain relatively unaffected. Because other investigators have found the C-terminal amidating enzymes to be associated with secretory vesicles and to be co-secreted with the vesicular contents, we suggest that secretory epithelia of the male reproductive system secrete TRH and TRH-related precursor peptides along with the alpha-amidating enzymes which continue processing of prepro-TRH in the post-ejaculatory seminal fluid.