Pekary A E, Reeve J R, Smith V P, Friedman S
Endocrinology Research Laboratory, Veterans Administration Wadsworth Medical Center, Los Angeles, CA 90073.
Int J Androl. 1990 Jun;13(3):169-79. doi: 10.1111/j.1365-2605.1990.tb00974.x.
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.
促甲状腺激素释放激素（TRH）及相关肽在人类精液中浓度很高。TRH来源于一种242个氨基酸的前体蛋白，即前促甲状腺激素释放激素原（prepro - TRH），它有六个重复序列（-Lys - Arg - Gln - His - Pro - Gly - Lys/Arg）-Arg-，由疏水连接序列相连。针对TRH - Gly（pGlu - His - Pro - Gly）（TRH形成的最终前体）的抗体，被用于检测这种四肽以及其他与这些抗体发生交叉反应的前促甲状腺激素释放激素原片段。输精管切除术后，总TRH - Gly免疫反应性显著降低。精液中的TRH - Gly免疫反应性在0℃或37℃体外孵育期间显著增加，在5小时达到峰值，随后呈指数下降，37℃时半衰期为11小时。然而，在60℃时，TRH - Gly免疫反应性持续上升，20小时后达到孵育开始时水平的2.2倍（P＜0.001）。反相高效液相色谱（HPLC）显示精液中存在疏水和亲水的TRH - Gly免疫反应性肽，两类肽在加热至60℃时均显著增加。对在60℃孵育的混合人精液进行阳离子交换色谱分析，结果显示与合成TRH - Gly共洗脱的一个TRH - Gly免疫反应性峰增加了4.3倍，另一个鉴定为Glu - His - Pro - Gly的TRH - Gly免疫反应性峰增加了30%。一个较小的TRH - Gly免疫反应性峰在60℃下20小时内增加了50倍（P＜0.001）。该物质与Arg - Gln - His - Pro - Gly共洗脱，后者是由前促甲状腺激素释放激素原中该序列两侧的成对碱性残基经酶切形成的。当合成的Arg - Gln - His - Pro - Gly与新鲜精液在60℃孵育时，大部分该肽在30分钟内迅速转化为Glu - His - Pro - Gly、Gln - His - Pro - Gly和TRH - Gly。这些数据与60℃时酰胺化和降解酶的热失活一致，而在成对碱性残基处切割前体肽的类胰蛋白酶则相对未受影响。因为其他研究者发现C末端酰胺化酶与分泌小泡相关，并与小泡内容物一起共同分泌，所以我们认为男性生殖系统的分泌上皮细胞分泌TRH和TRH相关前体肽以及α - 酰胺化酶，这些酶在射精后的精液中继续对前促甲状腺激素释放激素原进行加工。
