Autocrine regulation of human sperm motility by tachykinins

Abstract

Background: We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa.

Methods: Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA).

Results: The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective).

Conclusion: These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins.

Figure 1

Gene expression of tachykinin precursors, neprilysin (MME) and neprilysin-2 (MMEL1) in human sperm (SPZ) and testis (TES). In human sperm, the specific bands corresponding to TAC1 γ and δ isoforms, TAC4 and MME were only detected after cDNA reamplification. Acrosin (ACR) was present in sperm and testis cDNA while CD4 was only detected in testis. (+), positive control showing the expression of all genes in a pool of cDNAs from twenty different human tissues. (-), negative control with no RNA in the reverse transcriptase reaction; M, molecular size standards. The figure is representative of results in 6 pools of sperm samples from 8 different donors.

Figure 2

Immunofluorescent localization of tachykinins in human sperm. (A) Immunofluorescence and corresponding differential interference contrast images of sperm cells stained with primary antibodies against substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) showing specific localizations for each peptide. (B) Immunofluorescence and corresponding phase contrast images of sperm cells stained with a primary antibody against hemokinin-1 (HK-1) in the absence (a) and in the presence (b) of immunogenic peptide. For each tachykinin, experiments were performed at least six times with similar results. Scale bar, 10 μM.

Figure 3

Localization of neprilysins in human sperm. (A) Immunofluorescence and corresponding differential interference contrast images of sperm cells stained with primary antibodies against neprilysin (NEP) and neprilysin-2 (NEP2). Experiments were performed at least six times with similar results. Scale bar, 10 μM. (B) Western Blot analysis of NEP and NEP2 in human spermatozoa (SPZ) and seminal plasma (SP) homogenates. For NEP2, SP and SPZ₁ represent results obtained with the NEP2 antibody sc-104450 and SPZ₂ represents results obtained with the NEP2 antibody HPA 007876 (see text for further details). Results are representative of at least five separate protein preparations, each from eight different donors.

Figure 4

Time- and concentration-dependent effects of phosphoramidon on human sperm motility. Motility analysis was performed manually (A, B) or using a computer-assisted sperm analysis (CASA) system (C). (A) Effects of phosphoramidon (1 nM-1 μM) or its solvent on progressive motility (grade a+b sperm) at different times of incubation (B) Effects of phosphoramidon (1 μM) on progressive motility (grade a+b sperm), non-progressive motility (grade c sperm) and immotility (grade d sperm) at different times of incubation. (C) Effects of phosphoramidon (1 μM) on grade a and grade b sperm at different times of incubation. Bars are means with SEM of 6-13 different experiments and represent percentage changes in motility in samples treated with phosphoramidon relative to the value observed at the same time in untreated (A) or solvent-treated (B, C) paired controls. *P < 0.05, significant difference vs. control responses.

Figure 5

Tachykinin receptor-selective antagonists inhibit the effect of phosphoramidon on sperm motility. The effects of phosphoramidon (1 μM, 60 min incubation) on human sperm progressive motility (grade a+b sperm) were analyzed in the presence of SR140333 (NK₁ antagonist, 10 nM), SR48968 (NK₂ antagonist, 10 nM), SR142801 (NK₃ antagonist, 10 nM), a combination of the three antagonists, or the antagonist solvent. Motility analysis was performed manually following WHO guidelines. Bars are means with SEM of 6-8 different experiments and represent percentage changes in motility in samples treated with phosphoramidon relative to the value observed at the same time in phosphoramidon solvent-treated paired controls. *P < 0.05, significant difference vs. response to phosphoramidon.