Hum. Reprod. Advance Access originally published online on April 16, 2007
Human Reproduction 2007 22(6):1617-1625; doi:10.1093/humrep/dem069
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A role for tachykinins in the regulation of human sperm motility
1 Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, 49 Americo Vespucio Ave, 41092 Sevilla, Spain 2 Instituto Valenciano de Infertilidad, 58 Republica Argentina Ave, 41011 Sevilla, Spain 3 Centro de Producción y Experimentación Animal, Universidad de Sevilla, Espartinas, 41807 Sevilla, Spain
4 Correspondence address. Tel: +34-95-4489565; Fax: +34-95-4460565; E-mail: luzcandenas{at}iiq.csic.es
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Abstract |
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BACKGROUND: Tachykinins and tachykinin receptors are widely distributed in the male reproductive tract and appear to be involved in reproduction. However, the function and expression of tachykinins and their receptors in human spermatozoa remain poorly studied. We analysed the effects of tachykinins on sperm motility and characterized the population of tachykinin receptors in human spermatozoa.
METHODS AND RESULTS: Motility analysis was performed following World Health Organization guidelines and we found that substance P (SP), human hemokinin-1 (hHK-1), neurokinin A (NKA) and neurokinin B (NKB) produced concentration-dependent increases in sperm progressive motility. The effects of tachykinins were antagonized by the NK1 receptor-selective antagonist SR 140333, the NK2 receptor-selective antagonist, SR 48968 and, to a lesser extent, also by the NK3 receptor-selective antagonist SR 142801. Immunocytochemistry studies showed expression of the NK1, NK2 and NK3 tachykinin receptor proteins in spermatozoa with different major sites of localization for each receptor. Western blot analysis confirmed the presence of tachykinin receptors in sperm cell homogenates. RT–PCR demonstrated expression of the genes that encode SP/NKA (TAC1), NKB (TAC3) and hHK-1 (TAC4) but not the genes TACR1, TACR2 and TACR3 encoding NK1, NK2 and NK3 receptors, respectively.
CONCLUSIONS: These results show for the first time that the NK1, NK2 and NK3 tachykinin receptor proteins are present in human spermatozoa. Our findings suggest that tachykinins, probably acting through these three tachykinin receptors, play a role in the regulation of human sperm motility.
Key words: human sperm/mRNA expression/tachykinins/tachykinin receptors
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Introduction |
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The coordinated events that regulate sperm transport and development of fertilizing ability between ejaculation and oocyte fertilization are not completely understood. During the time period that sperm reside in the female genital tract, these cells undergo a series of morphological and functional modifications leading to capacitation, changes in motility, binding to the oocyte zona pellucida and the acrosome reaction (Flesch and Gadella, 2000
; Henkel and Schill, 2003; Darszon et al., 2005). It is accepted that the production of functionally competent spermatozoa is associated with several molecular changes that begin at the cell surface. In spite of recent exciting advances, further studies are needed to establish the precise nature of sequentially activated primary receptors and channels in the sperm plasma membrane (Albrizio et al., 2005; Acevedo et al., 2006; Agirregoitia et al., 2006).
Tachykinins comprise a peptide family whose best known members in mammals are substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and hemokinin-1 (HK-1). In humans, SP and NKA are encoded by the TAC1 gene, NKB is encoded by the TAC3 gene and human hemokinin-1 (hHK-1) is encoded by the TAC4 gene (Patacchini et al., 2004; Page, 2004; Pennefather et al., 2004a). The effects of tachykinins are mediated by receptors belonging to the family of G protein-coupled receptors (Gerard et al., 1993; Almeida et al., 2004; Pennefather et al., 2004a). The presently known tachykinin receptors are named NK1, NK2 and NK3, and in humans, are encoded by the TACR1, TACR2 and TACR3 genes, respectively (Page, 2004; Pennefather et al., 2004a). The NK1 receptor is activated preferentially by SP and HK-1, the NK2 receptor by NKA and the NK3 receptor by NKB. However, the naturally occurring tachykinins are not highly selective and can act as full agonists on the three receptors (Almeida et al., 2004; Patacchini et al., 2004).
Many experimental data suggest that tachykinins could play a role in the regulation of male and female reproductive function in Chordata and Prochordata, acting at both central and peripheral levels (Brown et al., 1990; Debeljuk and Lasaga, 1999; Satake et al., 2004; Candenas et al., 2005). In the reproductive tract, these peptides are present within sensory nerves and in non-neuronal cells within the placenta, the ovary and the uterus (Traurig et al., 1984; Lowry, 2003; Pennefather et al., 2004b) and in the testes and the prostate (Chiwakata et al., 1991; Debeljuk et al., 2003; Pinto et al., 2004). They are also expressed in different types of reproductive cells including corpora lutea, oocytes and blastocyst-stage embryos (Reibiger et al., 2001; Pintado et al., 2003).
Sastry et al. (1991) reported that SP caused inhibitory and excitatory effects on human sperm motility. However, little is known about the source and function of tachykinins and there are no studies on the molecular identity of the tachykinin receptors present in sperm cells. The aim of the present study was to investigate the function and expression of tachykinins and the receptors mediating their effects in mature human spermatozoa. We also analysed the expression of the tachykinin-metabolizing enzyme neprilysin in human sperm.
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Chemicals
SP, NKA, NKB, hHK-1 and [MePhe7]-NKB were from Bachem (Bubendorf, Switzerland). SR 140333, SR 48968 and SR 142801 (NK receptor-selective antagonists) were a generous gift from Sanofi Recherche (Montpellier, France). Drugs were dissolved in dilute (0.01 M) hydrochloric acid (SP, NKA, hHK-1 and [MePhe7]-NKB), dimethylsulphoxide (NKB) or absolute ethanol (tachykinin receptor antagonists) and diluted into sperm washing medium to appropriate concentrations.
Semen samples and sperm preparation
This study was approved by the Ethics Committees of CSIC and Instituto Valenciano de Infertilidad, Sevilla, and all donors gave written informed consent.
Freshly ejaculated semen was collected from 30 donors (18–35 years old) with normal sperm parameters and proven fertility. Samples were obtained by masturbation after 3–4 days sexual abstinence and processed immediately upon liquefaction. Quantitative, manual semen analyses were performed on undiluted semen (5 µl) with a Makler Counting Chamber (Sefi Medical Instruments, Haifa, Israel). Samples were examined for concentration and motility according to the World Health Organization (WHO, 1999) guidelines. A minimum of 200 cells were counted per 5 µl drop, and at least two drops were studied per sample.
For capacitation, liquefied semen samples were washed with modified human tubal fluid (mHTF, Irvine Scientific, Santa Ana, CA, USA) supplemented with 0.5% human serum albumin at 37°C. The diluted sperm were concentrated by centrifugation (400 g for 10 min) and the pellets gently reconstituted in 1 ml of fresh mHTF. Sperm suspensions were centrifuged at 400 g for 20 min through a discontinuous Percoll density gradient (Spermgrad-125, Vitrolife, Kungsbacka, Sweden). The samples were then centrifuged (400 g for 15 min), and the pellets collected and washed (400 g for 5 min) in 2 ml of mHTF. Samples were allowed to swim-up for 1 h at 37°C as previously described (Henkel and Schill, 2003; Caballero-Campo et al., 2006) and the supernatant carefully aspirated. Semen motility and concentration were re-examined and the sperm concentration adjusted to 50 million per millilitre for subsequent experiments.
Human sperm motility studies
As indicated above, the motility patterns of sperm samples were examined following WHO procedures. Analyses were carried out by two trained researchers. These researchers performed a monthly intralaboratory control, and the between-workers variation in assessment of sperm concentration and motility was <10% throughout the study. The movement of every encountered sperm was graded as a, b, c or d and defined as follows: a: rapid progressive motility; b: slow progressive motility; c: non-progressive motility and d: immotility (WHO, 1999; Irazusta et al., 2004; Caballero-Campo et al., 2006). Progressive motility (a + b), non-progressive motility (c) and immotility (d) were measured as a percentage of the total (a + b + c + d) that was considered as 100%. According to motility values, samples were divided into three subpopulations: Samples A, with 60–70% spermatozoa of grades a + b; samples B, with 45–60% spermatozoa of grades a + b and samples C, with 30–45% spermatozoa of grade a + b. All samples used in this study had values of immotile, grade d spermatozoa lower than 20% of the total.
To investigate the effects of drugs, individual sperm samples were divided into several aliquots and each aliquot was treated with a peptide (SP, NKA, NKB, hHK-1 or [MePhe7]-NKB, final concentration 0.01 nM–10 µM) or the corresponding solvent (time-matched paired controls). Sperm motility was measured 5 min before agent addition (initial value) and after a contact time of 1 and 15 min. In some experiments, the sample was treated with SP (1 µM), hHK-1 (1 µM), NKA (1 µM), NKB (1 µM) or the same volume of solvent and sperm motility measured 5 min before addition (initial value) and after 1, 15, 30, 45, 60, 120 and 240 min contact time periods. In additional experiments, the effect of a tachykinin peptide or its solvent was investigated in aliquots pretreated for 45 min with the NK1 receptor-selective antagonist SR140333 (10 nM), the NK2 receptor-selective antagonist SR48968 (10 nM), the NK3 receptor-selective antagonist SR142801 (10 nM) or its solvent. We observed in preliminary experiments that sample dilutions >10% caused a decrease in sperm motility. Therefore, a maximum of two drug concentrations, or the corresponding solvent volume, were tested on each aliquot.
One concentration–response curve to SP, NKA, NKB, hHK-1 or [MePhe7]-NKB was constructed on each individual sperm sample by applying to each aliquot one concentration of an agonist (0.01 nM–10 µM, in logarithmic increments) in the absence or presence of an antagonist or its solvent. Concentration–response curves were constructed with data obtained after a 15 min contact of the agonist with the sperm aliquot.
Values of sperm progressive motility, non-progressive motility and immotility were calculated as: (values after x min incubation with a drug or the equivalent solvent volume) – (initial values measured 5 min before drug or solvent addition). Responses were expressed as the percentage change (increase or decrease) with respect to the initial value in each aliquot or as a percentage of the maximal response (Emax, maximal change in progressive or non-progressive motility, or immotility) induced by the agonist tested in each sperm sample, considered as 100%.
Immunofluorescence
Sperm immunolocalization of NK1, NK2 and NK3 receptors was assessed by fluorescence microscopy. Spermatozoa was fixed in 1% paraformaldehyde in phosphate-buffered saline (PBS) and then attached to poly-L-lysine-coated slides. Cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min and blocked with 2% bovine serum albumin in PBS. Test slides were incubated overnight at 4°C with primary polyclonal antibodies (Sigma, St Louis, MO, USA) diluted 1:100. The specificity of antibodies had been assessed by the supplier. Negative control slides were not exposed to the primary antibody and were incubated in PBS in the same conditions as the test slides. Samples were next incubated for 2 h with Cy2-conjugated mouse, anti-rabbit secondary antibody (Immuno Research Labs, Baltimore, PA, USA) diluted 1:200 in PBS. Samples were observed using a Leica HCX PLAN-APO 63X 1.4 numerical aperture oil immersion objective attached to a Leica TCS SP2 confocal laser-scanning microscope (Leica, Heidelberg, Germany).
Western blot analysis
For extraction of total proteins, the semen samples were subjected to sonication in 200 µl of urea extraction buffer [1% w/v sodium dodecyl sulphate (SDS), 9 M Urea, 1 mM EDTA, 0.7 M mercapto-ethanol, in 25 mM Tris-HCl, pH 6.8], boiled for 2 min and processed by the PAGEprep Advance kit (Pierce, Rockford, IL, USA). Proteins were also extracted from non-pregnant human uteri, used as positive control. Samples were separated by SDS-polyacrylamide gel electrophoresis on 10% gels and transferred to polyvinyldifluoride membranes for 2 h. Membranes were washed with Tris-buffered saline containing 0.5% Tween-20 (TTBS, pH 7.5) and incubated in blocking solution (2% enhanced chemiluminescence Advance blocking agent, Amersham, Buckinghamshire, UK) for 1 h. The membranes were incubated overnight in 1:10 000 dilutions of primary antibody in TTBS. Blots were washed three times and incubated with 1:100 000 dilutions of horse-radish peroxidase-conjugated secondary antibody (goat anti-rabbit immunoglobulin G, Bio-Rad Laboratories, Hercules, CA, USA). Following three washes in TTBS, proteins were detected using an enhanced chemiluminescence kit (ECL Advance Western Blotting detection kit, Amersham).
RNA extraction and RT–PCR
Sperm samples from 5 to 10 different donors were pooled immediately after capacitation and swim-up, following the procedures described above. Total RNA was extracted using TriReagent (Sigma) and complementary DNA (cDNA) synthesized using the Quantitect Reverse Transcription kit (Qiagen, Venlo, The Netherlands). Specific oligonucleotide primer pairs used to amplify TAC1, TAC3, TAC4, TACR1, TACR2, TACR3, MME (gene encoding neprilysin), 
-actin (ACTB) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) were synthesized and purified by Sigma Genosys (Cambridge, UK). The sequences of the specific primers are shown in Table 1. ACTB and GAPDH were used as housekeeping genes to control RT–PCR reactions among samples. A pool of cDNAs from 20 different human tissues (human total RNA master panel, Clontech, Palo Alto, CA, USA) was used as a positive control of amplification.
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Amplification was performed in 25-µl of PCR buffer containing 3 µl of cDNA reaction mixture, 2.5 mM MgCl2, 0.2 µM primers, 200 µM dNTPs and 1.5 U of heat-activated thermostable DNA polymerase (Immolase, Bioline, London, UK). PCR was performed for 35 cycles with cycling parameters of 15 s at 94°C, 20 s at 60°C and 20 s at 72°C. The PCR products were separated by agarose gel electrophoresis, purified and sequenced at Newbiotechnic SA (Sevilla, Spain).
Statistical analysis
Values (mean ± SEM) were obtained by pooling individual data. Unless otherwise indicated, n represents the number of experiments in sperm samples from n different donors. Multiple means were compared by one-way analysis of variance and Dunnett's post-test. These procedures were undertaken using GRAPHPAD PRISM (version 4.0) program. A value of P < 0.05 was considered significant.
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Effects of tachykinins on human sperm motility
All tachykinins tested caused concentration-dependent increases in the proportion of progressively motile sperm whereas causing a parallel decrease in the percentage of non-progressively motile sperm (Figs 1 and 2). The proportion of immotile sperm was unaffected (see Fig. 1 for SP). Tachykinin effects were dependent on the initial motility and were more pronounced when the initial sperm progressive motility was lower. Sperm samples were thus divided in three different subpopulations according to the initial percentage of progressive and non-progressive motile sperm (samples A, B and C, see Methods), and we found that the functional effects of exogenously applied tachykinin peptides were highly reproducible within each type of sample (see Fig. 1 for effects of SP after 15 min incubation in sperm samples A and C). In samples B (with 45–60% spermatozoa of grades a + b), sperm progressive motility measured 5 min before (initial values) and 15 min after tachykinin exposure were 51.4 ± 3.4 and 65.6 ± 4.0 (n = 7, P < 0.001) for 1 µM SP, 54.7 ± 2.7 and 66.8 ± 1.6 (n = 6, P < 0.001) for 1 µM NKA, 51.4 ± 3.4 and 60.8 ± 3.9 (n = 5, P < 0.01) for 1 µM NKB, and 56.5 ± 1.6 and 68.3 ± 3.3 (n = 6, P < 0.001) for 1 µM hHK-1.
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Figure 2 shows the log concentration–response curves for SP, hHK-1, NKA, NKB and [MePhe7]-NKB after a 15 min incubation period in sample C (with a 30–45% of progressive motile spermatozoa). SP appeared as the most active tachykinin assayed, in terms of potency and maximal effect (Fig. 2). This tachykinin caused significant increases in sperm progressive motility and concomitant decreases in non-progressive motility even at a concentration of 0.1 nM (P < 0.05, n = 5–8, Figs 1 and 2). For NKA, NKB and hHK-1, a 10-fold higher concentration (1 nM) was needed to observe an appreciable effect on motility (Fig. 2). The NK3 receptor-selective agonist [MePhe7]-NKB caused smaller increases in progressive motility, which were observed at higher concentrations (
10 nM, Fig. 2). On the basis of positions of the concentration–response curves, the relative order of potency was SP > NKA = hHK-1 NKB = [MePhe7]-NKB (Fig. 2B). The effects of SP, hHK-1, NKA and NKB (each at 1 µM) on sperm motility were maintained during prolonged incubation and remain similar after 4 h of incubation (Table 2). In addition to the effects on progressive motility, it was also observed for all tachykinins that sperm swam straighter, with a more symmetrical movement of the flagellum.
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Tachykinin receptors mediating tachykinin responses
We analysed first the tachykinin receptors that mediated the actions of SP using sperm samples C. Incubation of the sample for 45 min with the NK1 receptor-selective antagonist SR140333 (10 nM, n = 7) or the NK2 receptor-selective antagonist SR48968 (10 nM, n = 6) caused a great inhibition of SP-induced increases in progressive motility and a shift to the right of the concentration–response curves (Fig. 3A). SR140333 and SR48968 inhibited concomitantly the SP-induced decreases in non-progressive motility (Fig. 3B). The NK3 receptor-selective antagonist SR142801 (10 nM, n = 5) caused a smaller but significant inhibition of SP responses (Fig. 3A and B). The three tachykinin receptor antagonists had similar inhibitory effects on the responses to NKA, hHK-1 and NKB (not shown). The responses to [MePhe7]-NKB were inhibited by SR142801 (10 nM, n = 3) but were unaffected by SR140333 (10 nM, n = 3) and SR48968 (10 nM, n = 3) (Fig. 3C). In time-matched paired aliquots, the tachykinin receptor antagonists (10 nM) or the solvent did not modify sperm motility (not shown) and the antagonist solvent did not modify tachykinin responses (see Fig. 3 for SP and [MePhe7]-NKB).
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Immunodetection of tachykinin receptors in human sperm
Immunocytochemistry demonstrates a positive immunostaining for tachykinin NK1, NK2 and NK3 receptors, which was seen in all samples assayed (n = 6 different human sperm for each receptor, Fig. 4). Tachykinin NK1 receptor immunostaining was intense and distributed throughout the acrosomal region of the head, the midpiece and the flagellum. The NK2 receptor staining was mainly found on the acrosomal region, the middle region of the sperm head and the end of the flagellum. The NK3 receptor was almost exclusively located in the midpiece, and a diffuse labelling was observed in the proximal region of the flagellum (Fig. 4). The specificity of labelling was confirmed by the complete absence of immunofluorescence in the absence of the primary antibody (not shown). Furthermore, in the case of the tachykinin NK1 receptor, we tested a second antibody (sc-5218, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and found results similar to those using the antibody from Sigma.
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Western blot analysis of sperm homogenates confirmed the presence of tachykinin receptors in human sperm (Fig. 5). The polyclonal antibodies against human NK1 and NK3 receptors labelled single bands of about 59 and 55 kDa, respectively. The anti-human NK2 receptor polyclonal antibody recognized two bands, one of the expected size (52 kDa) and a second one of about 105 kDa, which could result from post-translational modifications, such as receptor glycosylation (Wheatley and Hawtin, 1999
). The immunoreactive bands for tachykinin receptors were also observed in homogenates obtained from human uterus, used as a positive control, and were absent when primary antibodies were omitted (data not shown).
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mRNA expression of tachykinins and neprilysin in human sperm
The PCR products expected for TACR1, TACR2 and TACR3 were not detected in human sperm cDNAs (Fig. 6). Conversely, the mRNAs of TAC1, TAC3, TAC4, MME and the housekeeping genes ACTB and GAPDH were all detected (Fig. 6). The identity of the fragments was established by sequencing the RT–PCR products.
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The mRNAs of all target and housekeeping genes were detected in the cDNA pool used as positive control (Fig. 6). Conversely, no PCR product was detectable in the two negative controls (no reverse transcriptase and no template) showing the absence of genomic DNA contamination and that reagents were free of target sequence contamination (Fig. 6).
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Discussion |
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This study shows for the first time that the three tachykinin receptors, NK1, NK2 and NK3, are present in human sperm. Tachykinins appear to increase sperm progressive motility (with a concomitant decrease in non-progressive motility) by acting at tachykinin NK1, NK2 and NK3 receptors. Moreover, the mRNAs of all presently known human genes encoding tachykinins are expressed in mature sperm cells.
Tachykinins affect reproductive functions by acting at both central and peripheral levels. These peptides modulate the secretion of different hormones by the hypothalamus and the anterior pituitary and modify hypothalamic–pituitary–gonadal axis function in both males and females (Debeljuk and Lasaga, 1999; Candenas et al., 2005). At the peripheral level, tachykinins, classically considered as neuropeptides, are present in capsaicin-sensitive neurons supplying the male and female genital tract (Traurig et al., 1984; Debeljuk et al., 2003; Pennefather et al., 2004b). In addition, they are expressed in many different types of non-neuronal reproductive cells (Chiwakata et al., 1991; Lowry, 2003; Page et al., 2003; Patak et al., 2003; Pintado et al., 2003). In a previous study, Sastry et al. (1991) showed the presence of the SP protein in human sperm. Our data extend this observation and show that the mRNAs of TAC1, TAC3 and TAC4, encoding SP/NKA, NKB and hHK-1, respectively, are expressed in spermatozoa. These data support the notion that tachykinins could act as intercellular signalling molecules and be involved in reproduction and fertilization processes.
All tachykinins assayed, including the endogenous ligands SP, NKA, NKB and hHK-1, influence sperm motility patterns without affecting immotility. The tachykinin effect appeared very quickly and its magnitude was dependent on tachykinin concentration and on the initial proportion of cells with progressive and non-progressive motility in the sperm sample. Sastry et al. (1991) found variable, inhibitory and stimulatory effects of SP on human sperm motility. The difference with our results is probably due to the use of different experimental conditions, as we found in preliminary experiments that, whatever the solvent used, sample dilutions higher than 10% had a negative effect on motility.
The relative order of potency of tachykinins in modulating sperm motility was SP > NKA = hHK-1 NKB = [MePhe7]-NKB. Studies with the tachykinin receptor-selective antagonists SR140333, (NK1-selective) (Emonds-Alt et al., 1993), SR48968 (NK2-selective) (Emonds-Alt et al., 1992) and SR142801 (NK3-selective) (Emonds-Alt et al., 1995) suggested that tachykinin effects were mostly mediated by tachykinin NK1 and NK2 receptors and, to a lesser extend, also by NK3 receptors. The participation of the NK3 receptor was further supported by the following observations: (i) the NK3 receptor-selective agonist [MePHe7]-NKB modified sperm motility and (ii) The effect of [MePHe7]-NKB was unaffected by SR140333 and SR48968 but was inhibited by SR142801. The presence of the three tachykinin receptors in human sperm was further confirmed by immunocytochemistry and western blot studies.
The effects of tachykinins on human sperm motility were well maintained during several hours. This contrast with results obtained in most types of cells, where tachykinin receptors, particularly the NK1 and NK3 receptor types, are rapidly desensitized after exposure to micromolar concentrations of tachykinins (Schmidlin et al., 2003). This suggests that the mechanisms responsible for desensitization of tachykinin receptors are not functional in mature sperm cells.
Immunofluorescence studies showed a differential localization for each tachykinin receptor type, suggesting that, in addition to effects on motility, the NK1, NK2 and/or NK3 receptors could play additional roles in human sperm function. This is particularly evident in the case of the NK3 receptor which, compared with the NK1 and NK2 receptors, had a less important effect on motility. Tachykinin NK3 receptor immunostaining was concentrated in the midpiece, the region of sperm rich in mitochondria and related with cell energy production and metabolism (Solakidi et al., 2005). The possibility that the NK3 receptor could participate in energy consumption and/or sperm metabolism should be explored.
In spite of immunolocalization of the three tachykinin receptor proteins, we failed to detect the mRNAs of TACR1, TACR2 and TACR3 in human sperm. A similar result has been recently reported for the 
-opioid receptor by Agirregoitia et al. (2006). As suggested by these authors, the presence of the receptor protein and the absence of its mRNA could reflect that mature cells have a need for the protein but not for the corresponding transcript (Agirregoitia et al., 2006). In this context, it is widely accepted that RNA synthesis terminates during midspermiogenesis, long before spermatozoa differentiation (Miller et al., 2005; Iguchi et al., 2006) and the mRNAs of the three tachykinin receptors may have been lost in previous maturation stages.
We found, in contrast, that sperm cells expressed the mRNAs of TAC1, TAC3 and TAC4, coding for the human tachykinin peptides. Human sperm also expressed MME, the gene encoding neprilysin, the major peptidase that degrades tachykinins in most human tissues, including the uterus (Patak et al., 2003; Irazusta et al., 2004; Pennefather et al., 2004b). These data support recent important observations showing that mature spermatozoa contain its own repertoire of mRNAs (Ostermeier et al., 2002; Miller et al., 2005; Albrizio et al., 2005, Acevedo et al., 2006; Agirregoitia et al., 2006) and could be able to translate these mRNAs into proteins (Gur and Breitbart, 2006; Miller and Ostermeier, 2006). Thus, sperm cells could selectively retain a minor range of mRNAs that may be necessary for fertilization or for initial development of the embryo (Miller et al., 2005; Miller and Ostermeier, 2006; Iguchi et al., 2006). Tachykinins form part of this range of mRNAs and this would suggest that mature sperm cells have a need for tachykinin and neprilysin transcripts. Further studies are needed to determine the precise role of sperm mRNAs, and particularly of tachykinin mRNAs, in sperm cell function and their contribution to mammalian fertilization.
In conclusion, the NK1, NK2 and NK3 tachykinin receptors are present in human sperm and are functionally active. Tachykinins are able to improve sperm motility; their effects are maintained during several hours and varied in magnitude depending on the initial motility of the sample. This study also shows for the first time that the mRNAs of the known human genes encoding tachykinins are expressed in human sperm. These data strongly argue for a role of tachykinins in the regulation of human sperm function.
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We are very grateful to Dr Emonds-Alt for generous gifts of SR 140333, SR 48968 and SR 142801. This work was supported by a grant from Ministerio de Educación y Ciencia (BFU2005-04495-C02-01/BFI), Spain. M.S. is the recipient of a fellowship from Laboratorios del Dr Esteve (Barcelona, Spain).
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Acevedo JJ, Mendoza-Lujambio I, de la Vega-Beltran JL, et al. (2006) KATP channels in mouse spermatogenic cells and sperm, and their role in capacitation. Dev Biol 289:395–405.[CrossRef][ISI][Medline]
Agirregoitia E, Valdivia A, Carracedo A, et al. (2006) Expression and localization of , 
and µ opioid receptors in human spermatozoa and implications for sperm motility. J Clin Endocrinol Metab 91:4969–4975.
Albrizio M, Guaricci AC, Maritato F, et al. (2005) Expression and subcellular localization of the µ-opioid receptor in equine spermatozoa: evidence for its functional role. Reproduction 129:39–49.
Almeida TA, Rojo J, Nieto PM, et al. (2004) Tachykinins and tachykinin receptors: structure and activity relationships. Curr Med Chem 11:2045–2081.[ISI][Medline]
Brown ER, Harlan RE, Krause JE. (1990) Gonadal steroid regulation of substance P (SP) and SP-encoding messenger ribonucleic acids in the rat anterior pituitary and hypothalamus. Endocrinology 126:330–340.[Abstract]
Caballero-Campo P, Chirinos M, Fan XJ, et al. (2006) Biological effects of recombinant human zona pellucida proteins on sperm function. Biol Reprod 74:760–768.
Candenas L, Lecci A, Pinto FM, et al. (2006) Tachykinins and tachykinin receptors: effects in the genitourinary tract. Life Sci 76:835–862.[CrossRef][ISI]
Chiwakata C, Brackmann B, Hunt N, et al. (1991) Tachykinin (substance-P) gene expression in Leydig cells of the human and mouse testis. Endocrinology 128:2441–2448.[Abstract]
Darszon A, Nishigaki T, Wood C, et al. (2005) Calcium channels and Ca2+ fluctuations in sperm physiology. Int Rev Cytol 243:79–172.[CrossRef][ISI][Medline]
Debeljuk L and Lasaga M. (1999) Modulation of the hypothalamo–pituitary–gonadal axis and the pineal gland by neurokinin A, neuropeptide K and neuropeptide gamma. Peptides 20:285–299.[CrossRef][ISI][Medline]
Debeljuk L, Rao JN, Bartke A. (2003) Tachykinins and their possible modulatory role on testicular function: a review. Int J Androl 26:202–210.[CrossRef][ISI][Medline]
Emonds-Alt X, Vilain P, Goulaouic P, et al. (1992) A potent and selective non-peptide antagonist of the neurokinin A (NK2) receptor. Life Sci 50:L101–L106.
Emonds-Alt X, Doutremepuich JD, Heaulme M, et al. (1993) In vitro and in vivo biological activities of SR 140333, a novel potent non-peptide tachykinin NK1 receptor antagonist. Eur J Pharmacol 250:403–413.[CrossRef][ISI][Medline]
Emonds-Alt X, Bichon D, Ducoux JP, et al. (1995) SR 142801, the first potent non-peptide antagonist of the tachykinin NK3 receptor. Life Sci 56:L27–L32.
Flesch FM and Gadella BM. (2000) Dynamics of the mammalian sperm plasma membrane in the process of fertilization. Biochim Biophys Acta 1469:197–235.[Medline]
Gerard NP, Bao L, Xiao-Ping H, et al. (1993) Molecular aspects of the tachykinin receptors. Regul Pept 43:21–35.[CrossRef][ISI][Medline]
Gur Y and Breitbart H. (2006) Mammalian sperm translate nuclear-encoded proteins by mitochondrial-type ribosomes. Genes Dev 20:411–416.
Henkel RR and Schill WB. Sperm preparation for ART. Reprod Biol Endocrinol 1:108.
Iguchi N, Tobias JW, Hecht NB. (2006) Expression profiling reveals meiotic male germ cell mRNAs that are translationally up- and down-regulated. Proc Natl Acad Sci USA 103:7712–7717.
Irazusta J, Valdivia A, Fernandez D, et al. (2006) Enkephalin-degrading enzymes in normal and subfertile human semen. J Androl 25:733–739.
Lowry PJ. (2003) Has the mechanism by which the endocrine placenta scavenges the mother whilst sparing the foetus been unmasked? J Mol Endocrinol 31:341–347.[Abstract]
Miller D and Ostermeier GC. (2006) Towards a better understanding of RNA carriage by ejaculate spermatozoa. Hum Reprod Update 12:757–767.
Miller D, Ostermeier GC, Krawetz SA. (2005) The controversy, potential and roles of spermatozoal RNA. Trends Mol Med 11:156–163.[CrossRef][ISI][Medline]
Ostermeier GC, Dix DJ, Miller D, et al. (2002) Spermatozoal RNA profiles of normal fertile men. Lancet 360:772–777.[CrossRef][ISI][Medline]
Page NM. (2004) Hemokinins and endokinins. Cell Mol Life Sci 61:1652–1663.[ISI][Medline]
Page NM, Bell NJ, Gardiner SM, et al. (2003) Characterization of the endokinins: human tachykinins with cardiovascular activity. Proc Natl Acad Sci USA 100:6245–6250.
Patacchini R, Lecci A, Holzer P, et al. (2004) Newly discovered tachykinins raise new questions about their peripheral roles and the tachykinin nomenclature. Trends Pharmacol Sci 25:1–3.[CrossRef][Medline]
Patak E, Candenas ML, Pennefather JN, et al. (2003) Tachykinins and tachykinin receptors in human uterus. Br J Pharmacol 139:523–532.[CrossRef][ISI][Medline]
Pennefather JN, Lecci A, Candenas ML, et al. (2004a) Tachykinins and tachykinin receptors: a growing family. Life Sci 74:1445–1463.[CrossRef][ISI][Medline]
Pennefather JN, Patak E, Pinto FM, et al. (2004b) Mammalian tachykinins and uterine smooth muscle: the challenge escalates. Eur J Pharmacol 500:15–26.[CrossRef][ISI][Medline]
Pintado CO, Pinto FM, Pennefather JN, et al. (2003) A role for tachykinins in female mouse and rat reproductive function. Biol Reprod 69:940–946.
Pinto FM, Almeida TA, Hernandez M, et al. (2004) mRNA expression of tachykinins and tachykinin receptors in different human tissues. Eur J Pharmacol 494:233–239.[CrossRef][ISI][Medline]
Reibiger I, Aust G, Tscheudschilsuren G, et al. (2001) The expression of substance P and its neurokinin-1 receptor mRNA in the bovine corpus luteum of early developmental stage. Neurosci Lett 299:49–52.[CrossRef][ISI][Medline]
Sastry BV, Janson VE, Owens LK. (1991) Significance of substance P- and enkephalin-peptide systems in the male genital tract. Ann NY Acad Sci 632:339–353.[Abstract]
Satake H, Ogasawara M, Kawada T, et al. (2004) Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: evolutionary origin of the vertebrate tachykinin family. J Biol Chem 279:53798–53805.
Schmidlin F, Roosterman D, Bunnett NW. (2003) The third intracellular loop and carboxyl tail of neurokinin 1 and 3 receptors determine interactions with -arrestins. Am J Physiol Cell Physiol 285:C945–C958.
Solakidi S, Psarra AM, Nikolaropoulos S, et al. (2005) Estrogen receptors 
and (ER and ER) and androgen receptor (AR) in human sperm: localization of ER and AR in mitochondria of the midpiece. Hum Reprod 20:3481–3487.
Traurig HH, Saria A, Lembeck F. (1984) Substance P in primary afferent neurons of the female rat reproductive system. Naunyn Schmiedebergs Arch Pharmacol 326:343–346.[CrossRef][ISI][Medline]
World Health Organization. (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm-cervical Mucus Interaction 4th edn. (Cambridge University Press, Cambridge, UK).
Wheatley M and Hawtin SR. (1999) Glycosylation of G-protein-coupled receptors for hormones central to normal reproductive functioning: its occurrence and role. Hum Reprod Update 5:356–364.
Submitted on December 12, 2006; resubmitted on February 15, 2007; accepted on February 22, 2007.
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