Mol. Hum. Reprod. Advance Access originally published online on January 21, 2005
Molecular Human Reproduction 2005 11(2):141-150; doi:10.1093/molehr/gah142
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Induction of human sperm capacitation and protein tyrosine phosphorylation by endometrial cells and interleukin-6*
1Endocrinologie de la Reproduction and Centre de recherche du CHUQ, Département d'obstétrique et gynécologie, Université Laval, et 2Centre de recherche en Biologie de la reproduction, Canada
3 To whom correspondence should be addressed at: Endocrinologie de la Reproduction, D0-708, Pavillon Saint-François d'Assise, 10, de l'Espinay, Québec, QC, Canada G1L 3L5. Email: pierre.leclerc{at}crsfa.ulaval.ca
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Abstract |
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In order to become fully competent at fertilizing the oocyte, spermatozoa must undergo the maturational process of capacitation during their journey in the female reproductive tract. Endometrial cells secrete an array of growth factors that can affect spermatozoa. Among these factors, it has been previously demonstrated that interleukin-6 (IL-6) affects the fertilizing capacity of human spermatozoa. As the expression of this cytokine varies throughout the menstrual cycle and increases during the periovulatory period, the involvement of IL-6 in human sperm capacitation was investigated, with emphasis on the signal transduction cascade triggered by this agent in sperm cells. Spermatozoa were treated with recombinant human IL-6. Protein phosphotyrosine content and localization of the phosphotyrosine containing proteins were evaluated by western blot and indirect immunofluorescence, respectively, using a monoclonal anti-phosphotyrosine antibody. The acrosomal status was evaluated on IL-6 treated spermatozoa before or after challenge with the ionophore A23187 according to the fluorescent pattern observed upon binding to the Pisum sativum agglutinin conjugated to fluorescein isothiocyanate. In the present study, it is shown that, as for endometrial cell-conditioned media, IL-6 induces human sperm capacitation. The IL-6 effects most likely occur through binding to its receptor, IL-6R
, whose presence in the sperm is also reported in this study. As for the IL-6 receptor, this is the first report on the presence of the tyrosine kinase JAK1 in the spermatozoa. Moreover, this kinase becomes phosphorylated on tyrosine residues upon sperm treatment with recombinant IL-6, which suggests its activation by the cytokine. Taken together, our results demonstrate that the IL-6 intracellular signalling machinery is present in human spermatozoa and might be involved in the acquisition of sperm fertilizing ability, also known as the capacitation process. Key words: capacitation/cytokines/endometrial cells/phosphotyrosine/tyrosine kinases
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Introduction |
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Mammalian sperm must undergo a series of biochemical and physical changes collectively called capacitation in order to fertilize the oocyte (Yanagimachi, 1994
). This includes both the surface and intracellular modifications such as the removal of decapacitation factors from the seminal plasma, and the increase in membrane fluidity and protein tyrosine phosphorylation (Yanagimachi, 1994; de Lamirande et al., 1997; Visconti et al., 2002). Protein tyrosine phosphorylation is a phenomenon tightly controlled by calcium and reactive oxygen species (Leclerc et al., 1998; Herrero et al., 1999; Dorval et al., 2002), which is among the earliest events increasing during the process of capacitation (Baldi et al., 1991). An increase in cAMP concentration also occurs during sperm capacitation, which is another factor involved in the increase in protein tyrosine phosphorylation (Leclerc et al., 1996; Osheroff et al., 1999). Capacitation takes place in the female reproductive tract where the spermatozoa are in contact with different cell types and secretions. Some authors have previously shown that endometrial cells support human sperm capacitation (Fusi et al., 1994; Lai et al., 1996). However, few studies have been conducted to identify the intrauterine effectors that signal the spermatozoa to acquire their functional competence during their transit toward the oocyte (Banerjee and Chowdhury, 1994, 1995). In bovine, it has been demonstrated that the maximal capacitating activity occurs during oestrus (Parrish et al., 1989). Similarly, in a bovine in vitro fertilization system, spermatozoa incubated in culture supernatants from oviductal cells taken in the periovulatory period showed the highest penetration rates (Chian et al., 1995).
Endometrial cells secrete a variety of cytokines, including interleukin-6 (IL-6) (Kishimoto, 1989; Vandermolen and Gu, 1996), a mediator of the inflammatory response (Kishimoto, 1989). IL-6 has been shown to affect the fertilizing capacity of human sperm as it induces acrosomal exocytosis and, at higher concentrations, reduces sperm viability and motility (Naz and Kaplan, 1994a,b). In the endometrium, IL-6 is produced primarily by epithelial cells (Tabibzadeh and Sun, 1992) and its expression increases upon ovulation (Tabibzadeh et al., 1995; Vandermolen and Gu, 1996; von Wolff et al., 2002). IL-6 plasma levels increase during the proliferative phase of the menstrual cycle and rapidly decrease after ovulation (Angstwurm et al., 1997). IL-6 affects cell activity through a signalling pathway triggered by binding to its specific receptor, gp80 (IL-6R), which then forms a high affinity complex with gp130 (IL-6Rß) (Kishimoto, 1989). The latter is the signal transducing subunit of the receptor that mediates the signal transduction cascade of IL-6, as well as other cytokines through interaction with their specific receptor (Taga et al., 1992). None of the subunits possess an intrinsic tyrosine kinase activity although following ligand binding to the receptor, there is an activation of members of the janus kinase (JAK) family of cytosolic tyrosine kinases (Taga, 1996). These kinases are known to phosphorylate transcription factors identified as signal transducers and activators of transcription (STAT), which transduce signal to the cytoplasm or nucleus. The IL-6 receptor can be released from the plasma membrane by a proteolytic cleavage, also called shedding, regulated by protein kinase C. The soluble IL-6 receptor (sIL-6R), a 55 kDa protein, can even bind IL-6 with the same affinity as the membrane receptor and may associate with the membranous gp130 to transduce the signal (Mullberg et al., 1993).
Given that endometrial cells secrete IL-6 and that this cytokine can affect the fertilizing capacity of the human spermatozoa, we were interested in studying the effect of IL-6 in human sperm capacitation and to determine whether it activates a signal transduction pathway similar to what is known in somatic cells. Here we demonstrate that (1) endometrial cells support capacitation and secrete IL-6, (2) IL-6 induces sperm capacitation and protein tyrosine phosphorylation and (3) the effects of IL-6 might be mediated by the JAK1-mediated signalling pathway.
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Materials
IL-6 concentration was measured by enzyme linked immunosorbent assay (ELISA) using the Pelikine Compact Kit from Research Diagnostics (Flanders, NJ). The peroxidase substrate for ELISA (TMB peroxidase EIA substrate solution A and B) was from Bio-Rad laboratories (Hercules, CA). Ham's-F10 medium, Dulbecco's modified Eagle's medium-F12 (DMEMF12) and Hank's medium were purchased from Life Technologies (Grand Island, NY). The antibiotics–antimycotics, trypsine and calf serum used for cell culture were from Invitrogen (Burlington, ON, Canada). Streptavidine conjugated to fluorescein isothiocyanate (FITC) was obtained from Gibco BRL. Tween 20, bovine serum albumin (BSA), leupeptine, aprotinine, pepstatine A, phenylmethylsulphonyl fluoride, FITC-conjugated Pisum sativum agglutinin, (PSA-FITC) collagenase IA, the calcium ionophore A23187, DMSO and the anti-bleaching agent 1,4-diazobicyclo-(2,2,2)-octane were all purchased from Sigma Chemical Co. (St Louis, MO). Recombinant human IL-6 was bought from R&D (Minneapolis, MN). Biotinylated goat anti-rabbit immunoglobulin G (IgG), horse-radish peroxidase conjugated goat anti-mouse IgG and goat anti-rabbit IgG were from Jackson Immunoresearch Laboratories (West Grove, PA). The anti-phosphotyrosine monoclonal antibody (clone 4G10) was bought from Upstate Biotechnology (Lake Placid, NY). The anti-phosphotyrosine antibody PY-20 clone and the JAK1 monoclonal antibody were from Transduction Laboratories (Lexington, KY). The polyclonal antibody directed against the IL-6 receptor (gp80) and its blocking peptides were purchased from Santa Cruz biotechnology (Santa Cruz, CA). The Enhanced Chemiluminescence detection (ECL) kit, protein G-coupled sepharose, Percoll and Ficoll-Paque solutions were bought from Amersham Bioscience Corp. (Baie d'Urfé, PQ, Canada). Nitrocellulose (0.22 µm pore size) was purchased from Micron Separations Inc. (Westboro, MA) and X-ray films were from Fuji (Tokyo, Japan).
Endometrial cell culture
Women who were undergoing tubal ligation were recruited into this study at the gynaecology clinic of the Saint-François d'Assise hospital (Québec, QC). All women gave their informed consent and ethical approval was obtained from the ethical committees for research on human subjects of the Saint-François d'Assise hospital (CHUQ) and the Laval University. The following inclusion criteria were used to select normal fertile subjects: (1) no history of endometriosis or other related pathology, pelvic inflammatory disease or abnormal bleeding; (2) no hormonal or intrauterine contraceptive device for at least 3 months prior to the study; and (3) normal hormonal profile and regular menstrual cycles. All women were between 24 and 44 years of age. Endometrial biopsies were performed during laparoscopy for tubal ligation using a pipelle and dated according to menstruation history and histological evaluation when enough biological material was available.
Tissue processing from biopsies
Endometrial cells were prepared and cultured according to a well established and routinely used protocol (Akoum et al., 1995). Briefly, the endometrial tissue was minced into small pieces and dissociated with collagenase to separate the epithelial glands from the fibroblast-like cells. These two cell populations were further purified using Percoll density gradients. The purity of the primary endometrial cell cultures was verified morphologically by light microscopy and immunocytochemically on parallel coverslip cultures as previously described (Akoum et al., 1995). No CD-45 positive leukocytes were detected in culture and less than 1% contamination by endothelial factor VIII-positive cells was generally observed. Endometrial cells were cultured at 37 °C in DMEMF12 containing 10% fetal bovine serum (FBS) and a mix of antibiotics–antimycotics. At confluence, the cells were washed twice with Hank's, serum-depleted Ham's-F10 medium was added and the cells were cultured for another 24 h after which the medium was collected (conditioned medium, CM). The CM was centrifuged to remove particulate debris, aliquoted and kept at –80 °C until use. The stromal and epithelial cells were cultured either together (mixed cells) or isolated and cultured separately. A total of 12 biopsies were processed.
On four occasions, the cells were cultured after a single freeze/thaw cycle. For freezing, the cells were freshly dissociated as described above, put in 1 ml of cryopreservative medium (50% FBS, 40% culture medium and 10% DMSO) before being immediately frozen at –80 °C for 24 h and placed thereafter in liquid nitrogen. At the time of culture, the cells were thawed at 37 °C and washed from cryopreservative medium in 25 ml of Hank's medium (110 g for 8 min at room temperature). The cells were then cultured in DMEMF12 containing FBS and antibiotics–antimycotics and CM was produced as described above. No major differences were observed between frozen–thawed and freshly prepared cells as determined with the densitometric analysis of the anti-phosphotyrosine western blots performed following incubation of the fresh spermatozoa with culture media from these endometrial cells.
Measurement of IL-6 concentration
The concentrations of IL-6 protein were determined in the CM using a commercially available ELISA Kit. The assays were performed according to the manufacturer's instructions and the sensitivity was 0.5 pg/ml.
Preparation of sperm
Fresh semen samples were obtained by masturbation from healthy volunteers after a minimum of 2 days of sexual abstinence. After liquefaction, the semen was layered on top of a gradient composed of 2 ml fractions each of 20%, 40% and 65% and 0.1 ml of 95% percoll. Percoll was made isoosmotic in HEPES-buffered saline (HBS: 25 mM HEPES, 130 mM NaCl, 4 mM KCl, 0.5 mM MgCl2, 14 mM fructose, pH 7.6) and centrifuged (30 min, 900 g) to wash the spermatozoa from the seminal plasma. Sperm cells at the 65–95% interface and within the 95% percoll fraction, which represents the highly motile population, were pooled and used for incubations.
Sperm capacitation
The Percoll-washed spermatozoa were incubated at 20x106 cells/ml for 4 h at 37 °C in a 5% CO2 atmosphere with the medium conditioned by different endometrial cell populations. At the end of incubation, the spermatozoa were divided into three aliquots: the first one was washed in HBS (150 mM NaCl, 10 mM HEPES, pH 7.2), sperm proteins were solubilized in sample buffer [2% sodium dodecyl sulphate (SDS), 10% glycerol, 5% ß-mercaptoethanol, 62.5 mM Tris–HCl, pH 6.8] and heated at 100 °C for 5 min. This sample was next processed for the evaluation of phosphotyrosine-containing proteins by western blot. The other two aliquots were washed in phosphate-buffered saline (PBS: 1.5 mM KH2PO4, 8.1 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) and resuspended in Ham's-F10 medium containing 3 mg/ml of BSA. One aliquot was added with Ca2+ ionophore A23187 (5 µM final) and was incubated for 1 h in the same conditions as mentioned earlier to determine the induced acrosome reaction while the other served as a control for spontaneous acrosome reaction. The spermatozoa were next washed in PBS, fixed-permeabilized in methanol for 30 min on ice, smeared on slides, air-dried, and the acrosomal status was evaluated by PSA-FITC (75 µg/ml) (Cross et al., 1986). The binding pattern of PSA-FITC was determined under UV illumination and more than 200 sperm were scored in each treatment.
On several occasions, the spermatozoa were incubated in the presence of IL-6. The effects of IL-6 on sperm capacitation and protein phosphotyrosine content were evaluated as for spermatozoa incubated with endometrial cell CM.
Western blots
Following incubations with CM and IL-6, sperm proteins solubilized in sample buffer were separated by SDS-PAGE (Laemmli, 1970) and were electrotransferred onto nitrocellulose membrane (Towbin et al., 1979). Non-specific sites on the membrane were blocked with 5% (w/v) skimmed milk in Tris buffered saline (150 mM NaCl, 20 mM Tris–HCl, pH 7.4, 0.1% Tween 20). The nitrocellulose membrane was incubated for 1 h with the monoclonal anti-phosphotyrosine antibody. After several washes in Tris buffered saline, the membrane was next incubated with goat anti-mouse IgG conjugated to horse-radish peroxidase for 1 h. Again, the membrane was extensively washed and positive immunoreactive bands were detected by chemiluminescence using ECL according to the manufacturer's instructions.
The same western blot protocol was used for the investigation of the IL-6 receptor and the tyrosine kinase JAK1 using anti-rabbit IgG and anti-mouse IgG, respectively, as secondary antibodies conjugated to horse-radish peroxidase.
Subcellular localization of JAK1
Localization of JAK1 was investigated in the subcellular fractions of Percoll-washed spermatozoa. Sperm cells were resuspended in 5 ml HBS and were subjected to nitrogen cavitation for 10 min at 800 psi at 4 °C in order to separate the membrane from the cytosol or the skeletal elements (Noland et al., 1983). The cells were next centrifuged at 4 °C, for 15 min, 1000 g, the supernatant was centrifuged at 4000 g (30 min, 4 °C) to remove particulate materials, then subjected to an ultra-centrifugation (100 000 g, 4 °C, 60 min). The resulting supernatant (cytosol) was concentrated using a Microcon device (10 kDa cut off; YM10, Amicon, Bedford, MA) and the pellet (membranes) was resuspended in HBS. Aliquots were taken at each step of the procedure, the proteins solubilized in sample buffer and heated at 100 °C for 5 min. Sperm proteins were separated by SDS-PAGE, electrotransferred on nitrocellulose and the presence of JAK1 was determined by western blot as described earlier.
Indirect immunofluorescence
The immunolocalization of the IL-6 receptor (IL-6R subunit; gp80) in the spermatozoa was investigated by indirect immunofluorescence. Percoll-washed spermatozoa were put on a poly-L-Lysine-coated coverslip, fixed for 15 min in 3.7% formaldehyde in PBS, washed with PBS, permeabilized for 10 min in 0.2% Triton X-100 in PBS and washed again with PBS. Non-specific sites were blocked with porcine serum (10%) in PBS. Coverslips were incubated for 1 h at 37 °C with the IL-6R polyclonal antibody, washed with PBS and incubated at 37 °C for a further hour with an FITC-conjugated goat anti-rabbit IgG secondary antibody. After extensive washes in PBS, the coverslips were mounted on slides with 90% glycerol containing 1,4-diazobicyclo-(2,2,2)-octane (1.5% w/v) as an anti-bleaching agent. Positive signal was detected by epifluorescence microscopy under UV illumination. The Sperm were also incubated with IL-6 at 6 pg/ml for 4 h at 37 °C prior to being fixed, permeabilized and processed as described above to verify whether there was shedding of IL-6 receptor during incubation (Vermes et al., 2002). The specificity of the signal was confirmed by using, in parallel, the IL-6R antibody in the presence of the blocking peptide provided by the company. In some other experiments, we used the phosphotyrosine antibody 4G10 to verify whether there were some differences in the amount and localization of phosphotrytosine-containing proteins in sperm incubated with or without IL-6.
Immunoprecipitation of sperm proteins
The tyrosine phosphorylation status of sperm JAK1 upon IL-6 treatment was examined by immunoprecipitation of phosphotyrosine-containing proteins. The spermatozoa were incubated for 4 h (37 °C, 5% CO2 in air) with or without IL-6. Sperm were then centrifuged (5 min, 500 g), resuspended in 100 x 106 spz/ml and lysed for 30 min at 4 C in RIPA buffer (final concentrations: 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, 1 mM EDTA, 0.2 mM vanadate) containing protease inhibitors (leupeptine, pepstatine and aprotinine, all at a final concentration of 10 µg/ml, and 50 µM of phenylmethylsulphonyl fluoride). The lysate was centrifuged (20 min, 13 000 g, 4 °C) to remove cellular debris and the supernatant was added with 1 µg of the anti-phosphotyrosine antibody (PY-20). The tubes were incubated for 2 h at 4 °C with agitation, then 35 µl of sepharose beads (washed and resuspended in RIPA) was added and the incubation prolonged for a further 2 h at 4 °C. The tubes were centrifuged (3 min, 4 °C, 4600 g) and the beads washed three times with the RIPA buffer. At the end, the beads and the associated immune complex were resuspended in SDS-PAGE sample buffer, heated at 100 °C for 5 min, subjected to SDS-PAGE and electrotransfered onto nitrocellulose. The presence and the phosphorylation of the tyrosine kinase JAK1 was investigated by western blot using the JAK1 monoclonal antibody.
Statistical analysis
Statistical analyses on the percentages of acrosome-reacted spermatozoa and protein phosphotyrosine content upon different treatments were performed using Tukey's multiple comparisons tests after ANOVA.
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Results |
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The culture medium conditioned by endometrial cells induce human sperm capacitation
In human as in other mammalian species, an increase in protein phosphotyrosine content is generally observed during sperm capacitation. Previous studies from our and other laboratories have shown that sperm protein tyrosine phosphorylation increases as human spermatozoa are incubated under conditions conducive to capacitation for more than 4 h (Osheroff et al., 1999
; Dorval et al., 2002). Therefore, to determine whether endometrial cells secrete a factor that induces sperm capacitation, anti-phosphotyrosine western blots were performed following a 4-h incubation of spermatozoa with endometrial cell CM. As shown by the phosphotyrosine content of p105 and 81, the two major human sperm tyrosine phosphorylated proteins (Figure 1), the culture medium conditioned by stromal, epithelial and mixed endometrial cells induced an increase in sperm protein tyrosine phosphorylation. Unfortunately, the effect of CM could not be assessed at the same time with the same ejaculates to clearly evaluate the differences among the cell types as sperm capacitation inducers. However, although not statistically significant, the values (Figure 1B and C) were slightly lower than those achieved when the spermatozoa were incubated with BSA, the total amount of proteins in the CM were 
14 times lower than in the BSA-containing medium (215 µg/ml versus 3 mg/ml).
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The capacitation inducing ability of the different endometrial cell populations was also evaluated by measuring the spontaneous and the Ca2+ ionophore (A23187)-induced acrosome reaction in the spermatozoa incubated with the respective CM. As shown in Figure 2, no treatment caused a spontaneous acrosomal exocytosis. However, when the spermatozoa were incubated for 4 h in culture medium conditioned by mixed populations of endometrial cells, the Ca2+ ionophore A23187 significantly induced an increase in the sperm acrosome reaction as observed in the spermatozoa incubated with BSA or fetal calf serum (FCS; Figure 2).
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Effect of IL-6 on human sperm capacitation
IL-6 is an inflammatory cytokine produced by endometrial cells that has been shown to affect sperm motility and promote the acrosomal exocytosis in human sperm (Naz and Kaplan, 1994b
). However, its effect as a capacitation inducer was not determined. Washed sperm were incubated with increasing concentrations of IL-6 in order to determine the optimal concentration of this cytokine, which affects the phosphotyrosine content of sperm proteins. IL-6 stimulated the tyrosine phosphorylation of human sperm proteins in a concentration-dependent manner with the maximal effect at 6 pg/ml (Figure 3). Although a similar effect was observed using 10 pg/ml IL-6, when the concentration was further increased to 60 pg/ml in the incubation medium, deleterious effects on the phosphotyrosine-content of human sperm proteins were observed (Figure 3). In addition, sperm motility (as qualitatively evaluated by light microscopy) was affected as well (not shown). The positive effect of IL-6 on sperm protein phosphotyrosine content was already observed after 1 h of incubation (Figure 4).
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Although IL-6 clearly caused an increase in the phosphotyrosine content of human sperm proteins, the localization of tyrosine-phosphorylated proteins were next investigated by indirect immunofluorescence. Five different phosphotyrosine expression patterns were classified according to the localization and the intensity of the signal (Figure 5). In non-incubated freshly washed spermatozoa (Figure 6A), 58% of the cells exhibited pattern 4, a moderate expression of phosphotyrosine-containing proteins in the principal piece of the flagellum. However, only 15% of the sperm expressed a strong signal in the principal piece (pattern 1). Twenty per cent of the sperm cells expressed a moderate signal both in the principal piece and the equatorial segment (pattern 2), whereas 6% of the sperm expressed a moderate signal both in the principal piece and the whole acrosome (pattern 3). The rarest sperm expression pattern of phosphotyrosine containing proteins was both a strong signal in the principal piece and a moderate signal in the equatorial segment (2%; pattern 5). After a 4 h incubation, spermatozoa exhibiting only a strong tyrosine phosphorylation of the principal piece proteins (pattern 1) increased up to 31% (Figure 6B), and the presence of IL-6 (6 pg/ml) in the incubation medium did not affect this expression pattern. Conversely, this increase was associated with a decrease in the percentage of spermatozoa exhibiting a moderate phosphorylation in the principal piece (pattern 4; Figure 6B). The number of spermatozoa expressing protein tyrosine phosphorylation in both the acrosomal and the principal piece areas (pattern 3) increased up to 16% and 17.4%, respectively after a 4 h incubation in the absence or presence of IL-6. However, differences in the expression patterns (2 and 5) of tyrosine phosphorylated proteins were observed when the spermatozoa were incubated with IL-6. The percentage of sperm cells expressing a moderate phosphorylation of the principal piece and the equatorial segment (pattern 2) was significantly higher than the control (19.3% versus 7.4%; Figure 6B). Similarly, a higher number of sperm showed strong tyrosine phosphorylation labelling in the principal piece and a moderate expression in the equatorial segment (pattern 5) when they were incubated in the presence of IL-6 (3.7% versus 0.9%).
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The effects of IL-6 were next investigated on the acrosomal exocytosis of human spermatozoa occuring either spontaneously or induced by the Ca2+ ionophore A23187. When used at 60 pg/ml, IL-6 significantly induced acrosome reaction compared to sperm cells incubated in the presence of IL-6 at concentration ranging from 0 to 10 pg/ml (Figure 7). However, for the A23187-induced acrosome reaction, it was only at 10 pg/ml that IL-6 had a significant effect (14.4% versus 5.4%; Figure 6).
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To determine whether or not the effect observed with IL-6 could relate to those observed with endometrial cell CM, the concentrations of IL-6 in the latter were assayed by ELISA. Production of IL-6 the by stromal cells was detected to the same extent whether these cells were cultured from biopsies obtained in the proliferative (14.0 ± 7.2 pg/ml; n=3) or secretory (9.0 ± 2.6 pg/ml; n=3) phase of the menstrual cycle. The concentration of IL-6 in epithelial cell CM could not be evaluated in the two samples available: one had undetectable IL-6 values and the other had IL-6 values higher than the upper limit of the dosage kit. The production of IL-6 by mixed cell populations (n=4) was variable among the samples, two specimens having IL-6 values within detection limit of the test (two secretory endometria 39.0 pg/ml and 32.6 pg/ml) and the others having an IL-6 concentrations either higher or lower than the detection limit of the kit. Therefore, the IL-6 concentrations measured in most of the endometrial cell CM were within the range of those that induced an increase in sperm protein tyrosine phosphorylation and prepared spermatozoa to undergo acrosomal exocytosis upon an ionophore challenge.
Expression of IL-6 receptor
As our preceding results demonstrated a capacitating effect of IL-6 on human spermatozoa, experiments were designed to investigate the mechanisms or signalling pathways involved in the induction of sperm capacitation and protein tyrosine phosphorylation by IL-6. The expression of the IL-6 receptor was first investigated. As shown in Figure 8, an 80 kDa protein was detected in the protein extract from human spermatozoa, which is in agreement with the reported molecular mass of the transmembranous form of the receptor.
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The localization of the IL-6 receptor in human spermatozoa was next assessed by indirect immunofluorescence. In human spermatozoa, the IL-6 receptor is detected on the entire length of the flagellum (Figure 9A). This signal was completely abolished when the antibody was pre-adsorbed on the blocking peptide, which suggests that this sperm localization of the IL-6 receptor is specific (Figure 9B). No major difference in the presence or localization of this receptor was observed when the spermatozoa were incubated for 4 h in the presence or absence of IL-6 (data not shown) suggesting that no shedding of the receptor occurred.
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Role of the tyrosine kinase JAK1 in human sperm capacitation
In somatic cells, binding of IL-6 to its receptor induces tyrosine phosphorylation and activation of the tyrosine kinase JAK1. Therefore, the involvement of JAK1 in the effects mediated by IL-6 on sperm capacitation and protein tyrosine phosphorylation was next investigated. As shown in Figure 10, JAK1 is present in human spermatozoa, mostly in the membrane fraction. Unfortunately, all the indirect immunofluorescence attempts to determine the localization of JAK1 in spermatozoa using the commercial monoclonal antibody that we had, were unsuccessful. The next set of experiments was to determine whether the tyrosine kinase JAK1 is activated upon IL-6 treatment of human spermatozoa. This was achieved by the evaluation of the phosphotyrosine content of JAK1, which increases upon activation. When spermatozoa were treated for 4 h with IL-6, a stronger JAK1 signal was detected among the proteins immunoprecipitated by an anti-phosphotyrosine antibody as compared to the spermatozoa incubated in the absence of the cytokine (Figure 11). This result clearly shows that JAK1 is phosphorylated on its tyrosine residues, which strongly suggests that it is activated, when spermatozoa are treated with IL-6.
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Discussion |
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Sperm capacitation is a process which renders sperm cells fertilization-competent and which normally occurs within the female reproductive tract. Nonetheless, only few studies have been conducted on the involvement of endometrial cells in human sperm capacitation. The aim of the present study was to evaluate the effects of the endometrial secretions on the acquisition of the fertilizing potential of human spermatozoa. During this process, spermatozoa go through a large number of biochemical modifications, one of which being an increase in the protein phosphotyrosine content. Human spermatozoa were incubated with the culture media conditioned by stromal, epithelial and a mix of stromal and epithelial cells and their capacitation status were investigated as an assessment of the effects of endometrial secretions on sperm cell functions. Both epithelial and stromal cells, cultured either together or separately, induced an increase in the phosphotyrosine content of sperm proteins. However, because of the small number of biopsies leading to the preparation of stromal, epithelial or the mixed populations of endometrial cells, no comparison between the cell types could be done to determine which cell type is the best sperm capacitation inducer. In addition, because of the small number of biopsies, no correlation could be established between the ability to induce sperm capacitation and the hormonal status (proliferative of secretory) of the women providing the biopsies from whom endometrial cells were isolated and cultured. It has been shown that secretions or cells collected in the periovulatory period are better sperm capacitation inducers (Parrish et al., 1989
; Chian et al., 1995).
In the present study, it is demonstrated that during the culture period, the endometrial cells secrete IL-6. Moreover, after a 24 h culture in the absence of serum, the IL-6 concentration is within the range of concentrations effective to promote capacitation as evaluated by the increase in the phosphotyrosine content of sperm proteins and the percentage of acrosome reaction. Endometrial IL-6 protein production has already been investigated by others (Laird et al., 1993; Tabibzadeh et al., 1995; Vandermolen and Gu, 1996; von Wolff et al., 2002). An increase in endometrial IL-6 production occurs as the uterus goes from a proliferative to a secretory stage (Tabibzadeh et al., 1995; Vandermolen and Gu, 1996; von Wolff et al., 2002). Epithelial cells appear to be the main cell type producing IL-6 during the proliferative phase (Tabibzadeh and Sun, 1992; von Wolff et al., 2002). The increase in IL-6 immunoreactivity during the secretory phase was attributed not only to epithelial cells but also to stromal cells and leukocytes (von Wolff et al., 2002). The fluctuations of endometrial IL-6 production throughout the menstrual cycle could be explained, at least in part, by the sex steroids: a previous study has demonstrated that the production of IL-6 by stromal cells can be inhibited by estradiol (Tabibzadeh et al., 1989). On the other hand, the IL-6 concentrations present in the circulating blood differ from those measured in the endometrium (Angstwurm et al., 1997). In ovulating women, blood IL-6 concentrations were the highest during the pre-ovulatory period and the lowest in the luteal phase.
The sperm signalling system is very sensitive to IL-6, since a concentration as low as 6 pg/ml can induce a positive response, which contrasts to what is found in most of the IL-6 affected cells where this cytokine acts at a concentration ranging from 1 to 100 ng/ml. In the present study, a significant increase in spontaneous acrosome reaction was observed when sperm were incubated with 60 pg/ml of IL-6. However, this high percentage is rather indicative of a toxic effect, as a decrease in phosphotyrosine-containing proteins was also noted in sperm incubated with this IL-6 concentration (Figure 3). Similar induction of human sperm acrosome reaction by IL-6 has been reported before, although at concentrations 10–100-fold higher (Naz and Kaplan, 1994b). In somatic cells, IL-6 acts through binding to its receptor (Heinrich et al., 2003). This latter is composed of an 80 kDa ligand binding subunit, the -subunit gp80, which, upon ligand binding, associates with a homodimer of the signal transducing 130 kDa subunit, gp130. This signal transducer is also known as the ß-subunit of the IL-6 receptor and also acts as a subunit of the receptor for other cytokines such as LIF, oncostatin, IL-11 and CNTF (Lutticken et al., 1994; Stahl et al., 1994; Heinrich et al., 2003). To our knowledge, the present study is the first report on the expression of the IL-6 receptor, gp80, in mature human spermatozoa. The presence of this receptor was evidenced by the mean of two different techniques, western blot and indirect immunofluorescence. Although others have investigated the involvement of IL-6 in mature sperm functions and fertilizing capacity (Camejo et al., 2001; Eggert-Kruse et al., 2001; Naz and Kaplan, 1994a,b), none verified whether the effect of this cytokine was mediated by its receptor.
The effect of IL-6 on protein tyrosine phosphorylation has been demonstrated about 10 years ago (Kumar et al., 1994; Lutticken et al., 1994; Stahl et al., 1994) in a signalling pathway involving the tyrosine kinase JAK1. Different studies have demonstrated that cytoplasmic tyrosine kinases of the JAK family associates with the transmembranous gp130 (Lutticken et al., 1994; Stahl et al., 1994). The presence of gp130, the signal transducing subunit of the IL-6 receptor, has recently been reported in the human spermatozoa (Yoshida et al., 2004). In addition, IL-6-induced protein tyrosine phosphorylation in somatic cells has also been shown to involve Fyn, Hck and Lyn, all members of the src family of tyrosine kinases (Hallek et al., 1997). In human spermatozoa inhibitors of src-related tyrosine kinases, herbimycin A or PP2, prevent protein tyrosine phosphorylation (Leclerc et al., 1997; Dorval et al., 2002). In the present study, the tyrosine kinase inhibitor PP2 inhibited, only weakly, IL-6-induced sperm protein tyrosine phosphorylation and had no effect on sperm acrosome reaction percentages (data not shown). With the exception of c-yes (Leclerc and Goupil, 2002), the presence of tyrosine kinases of the src family has not been demonstrated in mature spermatozoa. It would be therefore important to determine which kinases are responsible for the increase in sperm protein phosphotyrosine-content that occurs during the capacitation process.
The present study, represents also the first report on the presence of the tyrosine kinase JAK1 in the ejaculated human spermatozoa. Moreover, its major expression within the membrane fractions (Figure 9) is in agreement with the reported localization of JAK1 in close proximity to the cytokine receptor (Heinrich et al., 2003). In addition, in the present study, it is demonstrated that tyrosine phosphorylation of JAK1 occurs in the spermatozoa incubated with IL-6, which is an important step in the activation of the tyrosine kinase JAK. The presence of JAK1 in the human sperm is in contrast with a previous study on the JAK/STAT signalling pathway in human spermatozoa where TYK2 but none of JAK1, JAK2 or JAK3 were detected (D'Cruz et al., 2001). In this same study, the presence of STAT1 and its tyrosine phosphorylation upon sperm treatment with interferon- or 
was also reported. Both TYK2 and JAK1 can be activated by IL-6 (Heinrich et al., 1998), which would result in the phosphorylation of STAT1 tyrosine residues. Conversely, interferon- or induced tyrosine phosphorylation of sperm STAT1 (D'Cruz et al., 2001), might also occur through the activation of JAK1 as the activation of this kinase by interferon is a known phenomenon (Kalvakolanu, 2003). Therefore, the presence and activation of either JAK1 and/or TYK2 are not exclusive and both kinases can phosphorylate STAT1 in sperm cells. In the present study, the presence/activation of STAT proteins has not been investigated.
It has been previously shown that the major phosphotyrosine-containing proteins that become phosphorylated during capacitation are located to the fibrous sheath of the sperm flagellum (Carrera et al., 1996; Leclerc et al., 1997; Sakkas et al., 2003). In the present study, we report that increases in tyrosine phosphorylated proteins are also observed in the equatorial segment of IL-6 treated spermatozoa in addition to the principal piece of the flagellum (Figure 5). In addition, the localization of phosphotyrosine-containing proteins in the principal piece of the sperm flagellum is in agreement with the localization of the IL-6 receptor (present study) and the tyrosine kinase TYK2 (D'Cruz et al., 2001). Unfortunately, no sperm localization of the tyrosine kinase JAK1 could be achieved using our commercial antibody in the indirect immunofluorescence experiments. Therefore, these results are in agreement with the positive effect of low concentrations of IL-6 on sperm motility (Naz and Kaplan, 1994b). The involvement of protein tyrosine phosphorylation in the regulation of sperm motility has been shown in different studies (Leclerc et al., 1996; Mahony and Gwathmey, 1999; Si and Okuno, 1999). In the present study, an increase in protein tyrosine phosphorylation was induced by IL-6 (up to 10 pg/ml). On the other hand, a negative correlation is observed between sperm motility and high concentration of this cytokine in the seminal plasma (Naz and Kaplan, 1994a), this is also in agreement with the negative effects of high IL-6 concentration on sperm protein phosphotyrosine content reported in the present study.
In the present study, the involvement of endometrial cells in human sperm capacitation and its associated increase in protein tyrosine phosphorylation was demonstrated. In addition, the involvement of the IL-6 mediated signalling pathway in sperm protein tyrosine phosphorylation is more likely as most of the members are present and active: gp80, the IL-6 receptor (present study); gp130, the signalling subunit of the receptor (Yoshida et al., 2004); the tyrosine kinases JAK1 (present study) and TYK2 (D'Cruz et al., 2001); and the substrate STAT1 (D'Cruz et al., 2001). Since endometrial cells secrete IL-6 and because its concentration in the circulating blood increases during the periovulatory period, this cytokine might be a good candidate as a human sperm capacitation inducer. However, the importance of STAT1 or other specific substrates remains to be established.
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The authors are thankful to Dr L. Turcot-Lemay for her help with the statistical analysis, to Mr S. Goupil for his technical assistance in various protocols and to Mr F. Bigonnesse for his help in the preparation and culture of the endometrial cells. We heartly thank all the volunteers, male and female, who participated in this study.
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*This project was supported by a grant from the Canadian Institutes of Health Research.
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Submitted on October 27, 2004; accepted on December 15, 2004.
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