Expression of enkephalin-degrading enzymes in human semen and implications for sperm motility

To investigate the presence of two enkephalin-degrading enzymes (aminopeptidase N [APN] and neutral endopeptidase 24.11 [NEP]) in different fractions of human semen, their distribution in sperm cells, and their effect on sperm motility.

We performed expression assays for APN and NEP by real-time polymerase chain reaction, Western blot, and immunofluorescence techniques in sperm cells and performed motility analysis after incubation of semen samples with enzyme inhibitors and the opioid receptor antagonist naloxone.

Assisted reproduction unit and academic research laboratory.

Semen from 50 normozoospermic healthy human donors.

Spermatozoa isolated from semen on a discontinuous Percoll gradient (40%-80%), followed by swim-up, were used for all techniques except for sperm motility analysis, for which fresh semen was used.

Immunoblotting blots, indirect immunofluorescence antibody assays, cycle threshold values for real-time polymerase chain reaction, and percentage of motile sperm.

We found APN in the equatorial segment of the upper post-acrosomal region of the sperm head, in the neck and along the tail of spermatozoa, in prostasomes, and in seminal fluid, whereas NEP was present in a very restricted area of a few sperm cells and in prostasomes. Messenger RNA of both enzymes was detected in spermatozoa. The inhibition of enkephalin-degrading enzymes attenuated the time-dependent decrease of sperm motility; this effect was reversed by naloxone.

Enkephalin-degrading enzymes are present in human semen and may be involved in the control of sperm motility, mainly by the regulation of endogenous opioid peptides.