Abstract
In mammals, sperm adhesion to the epithelial cells lining the oviductal isthmus plays a key role in the maintenance of motility and in the selection of superior quality subpopulations. In the bovine species, heparin and other sulfated glycoconjugates powerfully induce the synchronous release of sperm adhering to tubal epithelium in vitro and may represent the signal which triggers release at ovulation in vivo. Sperm detachment may be due either to surface remodeling or to hyperactivation brought about by capacitation. In this paper, the dynamics of intracellular free Ca2+concentration ([Ca2+]i) and protein tyrosine phosphorylation in sperm during and after heparin-induced release from in vitro cultured oviductal monolayers were assessed to determine whether this event is due to capacitation. Moreover, Ca2+-ionophore A23187, thapsigargin, thimerosal and caffeine were used to determine whether [Ca2+]i increase and/or hyperactivation can induce sperm release. Results showed that: 1. heparin-released sperm have significantly higher [Ca2+]i than adhering sperm; 2. heparin induces a [Ca2+]i elevation in the sperm head followed by detachment from the monolayers; 3. external Ca2+is not required for heparin-induced release; 4. [Ca2+]i increase and/or hyperactivation are unable to release sperm; and 5. heparin-released sperm have an increased level of tyrosine phosphorylated proteins compared with adhering sperm. In conclusion, although heparin is considered a long-lasting capacitation agent, it quickly modulates the capacitation of bovine sperm adhering to the fallopian epithelium, probably leading to surface remodeling and therefore to the release of sperm selected and stored within the oviduct through adhesion.
Introduction
The mammalian oviduct acts as a functional sperm reservoir, providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation (Harper 1994). In several species, after mating, sperm are sequestered in the lower oviduct through adhesion to the epithelial cells lining its lumen (Wilmut & Hunter 1984, Smith & Yanagimachi 1990). This event is thought to prolong sperm life by delaying capacitation until unknown ovulation-associated signals induce the release of adhering sperm, allowing their progression toward the upper oviduct for fertilization (Smith & Yanagimachi 1991, Harper 1994). Adhesion to the oviduct has been shown to play a key role also in the selection of pre-existing sperm subpopulations characterized by intact acrosomes (cow: Gualtieri & Talevi 2000), an uncapacitated status (horse: Thomas et al. 1995, cow: Lefebvre & Suarez 1996, pig: Fazeli et al. 1999), low internal free calcium content and reduced membrane protein tyrosine phosphorylation (pig: Petrunkina et al. 2001), superior morphology (horse: Thomas et al. 1994), and normal chromatin structure (human: Ellington et al. 1998). Several lines of evidence indicate that the ability of sperm to bind to and be released from oviductal epithelium is modulated by capacitation. In fact, only uncapacitated sperm adhere to oviductal cells (Smith & Yanagimachi 1991, Thomas et al. 1995, Lefebvre & Suarez 1996), whereas the detachment of sperm in the periovulatory period in vivo (Smith & Yanagimachi 1991), as well as the spontaneous release during in vitro co-culture (Gualtieri & Talevi 2000), is thought to be triggered by a remodeling of the sperm plasma membrane and/or by hyperactivation brought about by capacitation. In cattle, heparin-like glycosaminoglycans, normally present in the oviductal fluid, have been regarded as potential in vivo capacitating agents (Parrish et al. 1988, 1989a). In a recent study, heparin, a glycosaminoglycan routinely used to capacitate sperm in bovine in vitro fertilization (IVF), and other sulfated glycoconjugates were shown to induce the release of sperm adhering to the fallopian tube epithelium in vitro (Talevi & Gualtieri 2001). This represented the first demonstration of molecules able to cause the release of sperm adhering to the oviductal epithelium among all species examined so far. Since heparin-like glycosaminoglycans are present in the bovine oviductal fluid, and change in their concentrations and capacitating activities is maximal at estrus (Parrish et al. 1989a), sulfated glycosaminoglycans have been suggested to modulate progressively sperm capacitation, first inducing sperm release from the oviductal reservoir and then enhancing sperm fertilization ability (Talevi & Gualtieri 2001). Recently, the powerful sperm-releasing activity of sulfated glycosaminoglycans allowed us to demonstrate that the sperm subpopulation selected by adhesion to in vitro cultured fallopian tube epithelium has an enhanced zona pellucida binding and fertilization competence compared with the sperm subpopulation unable to adhere within the same ejaculate (Gualtieri & Talevi 2003, Talevi & Gualtieri 2004). Therefore, sperm adhesion and release can represent a crucial selective event during capacitation. Capacitation consists of a complex series of finely tuned events occurring during the sperm transit through the female reproductive tract, and that are essential for sperm to develop the ability to bind the zona pellucida, undergo the acrosome reaction and fertilize the oocyte. Two well-recognized events accompanying capacitation of mammalian sperm are an increase of sperm intracellular free Ca2+concentration ([Ca2+]i) and the phosphorylation of protein tyrosine residues (Visconti et al. 2002). The aim of the present paper was to test the hypothesis that heparin-induced release of sperm adhering to the fallopian tube epithelium in vitro represents a very early event of capacitation. For this purpose, experiments were designed to study the following: 1. the modification of sperm [Ca2+]i and protein tyrosine phosphorylation levels during and/or after heparin-induced sperm release; 2. the role played by extracellular Ca2+in heparin-induced sperm release; and 3. the effect of the pharmacologic agents Ca2+-ionophore A23187, thapsigargin, thimerosal and caffeine that induce [Ca2+]i increase and/or hyperactivation (Ho & Suarez 2001) on the release of sperm adhering to the fallopian tube in vitro. The main findings demonstrate that heparin-induced release is accompanied by increase of both sperm [Ca2+]i and tyrosine phosphorylation, supporting the hypothesis that heparin is able to modulate quickly the capacitation of adhering sperm.
Materials and Methods
Chemicals
Bovine serum albumin (BSA) (fraction V), heparin (sodium salt, purified from porcine intestinal mucosa, H3393), dextran sulfate, fucoidan, calcium ionophore A23187, caffeine, thimerosal, Percoll, gelatin and Hoechst 33342 were obtained from Sigma; M199, fetal calf serum (FCS), gentamycin, Fungizone, HEPES and sodium bicarbonate, from Gibco (Milan, Italy); Fluo 3 AM, from Molecular Probe (Milan, Italy); thapsigargin, from Alomone Labs (Milan, Italy); and antiphosphotyrosine monoclonal antibody (clone 1G2) and TRITC-conjugated goat antimouse antibody, from Chemicon (Milan, Italy). Reagents and water for preparation of salines and culture media were all cell culture tested.
Oviduct monolayers
Oviducts were collected at the time of slaughter and transported to the laboratory in Dulbecco’s PBS (D’PBS) supplemented with 50 μg/ml gentamycin at 4 °C. Laminae of epithelial cells were recovered from oviducts of single animals by squeezing and cultured in M199 supplemented with 50 μg/ml gentamycin, 1 μg/ml Fungizone and 10% FCS, as previously described (Gualtieri & Talevi 2000). Bovine oviductal epithelial cells (BOEC) were cultured in 10 cm Petri dishes (Falcon; Becton Dickinson Milan, Italy) for 24–48 h and then transferred into four-well tissue culture dishes (Nunclon, Roskilde, Denmark) with 12 mm, gelatin-coated, German glass, round cover slips on the well bottom. Fresh media changes were performed every 48 h. Cell confluence was attained in about 7–10 days. Monolayers were used within 24–48 h after attainment of cell confluence. Within each experiment, BOEC monolayers from a single individual were washed three times in modified Tyrode’s albumin lactate pyruvate medium (sperm-TALP: Parrish et al. 1988, modified as described in Paula-Lopes et al. 1998), and left in this medium until sperm addition (1–3 h).
Sperm preparation
Frozen bovine semen from three bulls (0.5 ml straws; approximately 40 × 106 sperm per straw), obtained from Semen Italy (San Giuliano Saliceta, Modena, Italy), was used in all experiments. Straws were thawed in a water bath at 38 °C for 30 s and the semen was laid upon a discontinuous (90/40) Percoll gradient with or without 10 μg/ml Hoechst 33342, and centrifuged for 30 min at 180 g. The supernatant was removed and the pellet, resuspended in 2 ml BSA-free sperm-TALP, was centrifuged at 180 g for 10 min, and resuspended in 200 μl BSA-free sperm-TALP. Aliquots of recovered sperm were assessed for concentration and percent motility at the hemocytometer on a microscope stage heated to 38.5 °C.
Effect of extracellular Ca2+on sperm release
Ca-free sperm-TALP was prepared omitting CaCl2 and adding EGTA at 2 mM final concentration. Sperm suspension recovered after Percoll centrifugation was added to BOEC monolayers cultured on gelatin-coated, 12 mm, round cover slips in NUNC four-well plates in 750 μl sperm-TALP at a concentration of 2–3 × 106/well, and incubated at 39 °C, 5% CO2 in air, 95% humidity, for 60 min. After unbound sperm removal, all wells with adhering sperm were extensively washed with sperm-TALP or with Ca-free sperm-TALP. Control wells were left in these media for 10 min, whereas experimental wells were treated with heparin 100 μg/ml for 10 min. At the end of treatment, sperm-oviductal co-cultures were fixed and analyzed to quantify number of bound sperm, as previously described (Talevi & Gualtieri 2001). Briefly, monolayers grown on cover slips, inseminated with Hoechst-labeled sperm, were fixed in glutaraldehyde 2.5% in PBS, for 1 h at 20–25 °C, extensively washed and mounted with the same buffer on a glass slide with cells facing up. For each well, fields of 0.286 mm2 were acquired at a Zeiss Axioplan microscope equipped with phase-contrast, fluorescence and Nomarsky optics, by means of an Optronix camera and KS 300 software (Zeiss, Milan, Italy). The number of bound sperm was determined by analyzing ten fields of 0.286 mm2 for each well.
[Ca2+]i determinations
Sperm suspensions (2–5 × 106/ml) in BSA-free sperm-TALP were added with 2.5 μM cell-permeable fluo-3FF/AM, 0.02% Pluronic F-127, and coincubated in the dark for 30 min with BOEC monolayers previously washed with BSA-free sperm-TALP at 39 °C, 5% CO2 in air, 95% humidity. At the end of coincubation, the unbound sperm fraction was removed, and after extensive washings with sperm-TALP to eliminate excess dye, the BOEC monolayers with bound spermatozoa were incubated for an additional 20 min at 39 °C to allow de-esterification of the fluo-3FF/AM to its Ca2+-sensitive form, fluo-3FF. In experiments on sperm Ca2+dynamics in response to heparin, Ca2+-ionophore A23187, thimerosal, caffeine or thapsigargin, cover slips with co-cultures were removed from the incubator, transferred in 0.5 ml sperm-TALP in a circular cover-slip chamber, and placed on a thermal plate at 38.5 °C on the microscope stage. In experiments designed to analyze the sperm [Ca2+]i, dye-loaded heparin-released sperm were allowed to attach to the glass by their heads in BSA-free sperm-TALP in the cover slip-chamber, whereas parallel dye-loaded sperm adhering to monolayers cultured on glass were directly mounted in the cover-slip chamber. Intracellular Ca2+changes detected by means of fluorescence intensity of fluo3-FF were analyzed with a computer-controlled photo-multiplier system. Briefly, a digital video microscopy system was based on a Zeiss Axiovert 135 microscope. Stroboscopic illumination was provided by a 100 W xenon arc flash lamp. Light passed through an excitation filter (D500/20), a dichroic mirror (515DCLP), and emission filter (D535/30) to an ORCA-100 Hamamatsu 12-bit digital camera, controlled by a Macintosh G3 workstation. The computer was used to control the microscopy system and to perform all the image acquisitions/elaborations by the Openlab software (Improvision, Coventry, UK).
In experiments performed to analyze the sperm [Ca2+]i dynamics, relative fluorescence intensity (RFI) was calculated by normalizing the fluorescence intensity of the sperm head and midpiece after addition of heparin, or Ca2+-ionophore A23187, thimerosal, caffeine or thapsigargin (F1), against sperm basal fluorescence levels at time 0 (F0) to obtain reliable information regarding transient [Ca2+]i changes from baseline values (RFI = (F1 − F0)/F0).Relative fluorescence intensity was averaged for all recorded cells. In experiments designed to analyze adhering sperm [Ca2+]i changes during heparin-induced release, heparin at 100 μg/ml final concentration was added at 10–20 s after the beginning of acquisitions. Images were typically captured at intervals of 2–10 s for 2–5 min. In experiments designed to analyze the [Ca2+]i in different sperm samples, the fluorescence intensity of sperm was expressed in arbitrary units calculated by subtracting the intensity of background fluorescence measured on areas free of spermatozoa in each record field. In all experiments, single-cell analysis was performed by drawing an ellipse around the head and midpiece of each sperm, and the mean intensity was obtained for all images in the time series. The image series was then analyzed, and if the defined area no longer contained the entire sperm head and midpiece, the data were excluded from analysis.
Treatment of sperm with pharmacologic agents
Sperm adhering to BOEC monolayers were treated with Ca2+-increasing and/or sperm hyperactivating agents, that is, Ca2+ionophore A23187, thapsigargin, caffeine and thimerosal, to determine their effectiveness at inducing sperm release. Ca2+-ionophore A23187 was tested at 1–10 μM, thapsigargin at 5 μM, caffeine at 10 mM and thimerosal at 50 μM. Ca2+- ionophore A23187 and thapsigargin were dissolved in DMSO, and aliquots were stored frozen at − 30 °C. Their working solutions were prepared immediately before addition to the sperm suspension by diluting the stock solution in Ca2+-free sperm-TALP. Thimerosal was prepared in Ca2+-free sperm-TALP just before use. Caffeine was dissolved in Ca2+-free sperm-TALP by heating at 70 °C (Patel et al. 1997).
Immunocytochemistry of sperm tyrosine phosphorylated proteins
The immunocytochemical localization of tyrosine phosphorylated proteins was performed on the following: 1. the initial sperm suspension recovered after Percoll centrifugation; 2. unbound sperm collected after 1 h co-culture with monolayers; 3. bound sperm adhering to monolayers cultured on gelatin-coated, round cover slips at 1 h co-culture; and 4) bound sperm collected at 5 min after induction of release by addition of heparin at 100 μg/ml. Sperm suspensions were spotted on glass slides and air-dried, whereas sperm adhering to monolayers were directly air-dried. Samples were fixed in methanol for 10 min; washed for 3 × 5 min in D’PBS; blocked in 50% goat serum, Triton-X100 0.1% in D’PBS, overnight at 4 °C; washed for 2 × 5 min in Triton-X100 0.1% in D’PBS; incubated with mouse antiphosphotyrosine monoclonal antibody 10 μg/ml in 1% goat serum, Triton-X100 0.1% in D’PBS, for 1 h at 37 °C; washed for 3 × 10 min in Triton-X100 0.1% in D’PBS; incubated with TRITC-conjugated goat antimouse immunoglobulin (Ig) G 10 μg/ml in 1% goat serum, Triton-X100 0.1% in D’PBS, for 1 h at 37 °C; washed for 3 × 10 min in Triton-X100 0.1% in D’PBS; and mounted in glycerin 90% in D’PBS. Images were acquired at a Zeiss Axioplan microscope equipped with phase-contrast, fluorescence and Nomarsky optics, by means of an Optronix camera and KS 300 software (Kontron, Zeiss, Milan, Italy). For each sample, at least 200 sperm were counted in three different experiments. Specificity controls were performed by omission of the primary antibody or by saturation of the primary antibody with 20 mM o-phosphotyrosine.
Statistical analysis
Fluorescence intensity data of adhering and heparin-released sperm were compared by GLM procedure of ANOVA (SAS/STAT Users Guide 1988). Raw data from the immunolocalization experiment were modified by arcsine transformation to normalize data. Then, the GLM procedures were used for all analyses of variance. In the experiments for testing the influence of extracellular Ca2+on sperm release, the model included the presence/absence of external Ca and heparin. For immunolocalization data, the model included incubation time (0 and 1 h), heparin treatment and sperm condition (adhering and released). Pairwise comparisons of means were made with Tukey’s honestly significant difference.
Results
Sperm [Ca2+]i and heparin-induced release
Experiments to analyze [Ca2+]i during heparin-induced release of sperm adhering to oviductal monolayers were designed as described in the Materials and Methods section.
Cells of the oviductal monolayers did not show any detectable fluorescence under the dye-loading conditions used, whereas adhering sperm showed a slight fluorescence at the level of the sperm head. When 100 μg/ml heparin were added to dye-loaded co-cultures, all adhering sperm were released within 10 min under incubator conditions. This demonstrates that dye loading did not interfere with the sperm ability to respond to heparin. In a first series of experiments, basal fluorescence of adhering sperm (control) was registered in co-cultures in BSA-free sperm-TALP. Single-cell analysis showed a stable level of fluorescence over the time series with a progressive slight decrease of fluorescence probably due to photobleaching. After heparin treatment, released sperm were recovered and allowed to attach to a glass slide in BSA-free sperm-TALP. The slides were then immediately registered for fluorescence. Under these conditions, 80–90% of adhering sperm detached from the monolayer. After subtraction of the background, the mean fluorescence intensity of released sperm was increased about 1.4-fold compared with the basal levels registered at the same time in control sperm (mean± s.d.: 1018 ± 335 vs 756 ± 189; P < 0.0001; n = 3; sperm analyzed = 150).
For study of the [Ca2+]i dynamics during sperm detachment from monolayers, heparin addition to co-cultures was performed directly in the imaging chamber during the acquisitions. The graph and corresponding micrographs in Fig. 1 show the changes of the fluorescent signal of a sperm adhering to an oviductal monolayer in a representative experiment. Heparin at 100 μg/ml was added at about 20 s after starting the acquisition. Fluorescence started to increase 10–20 s after heparin addition at the level of the sperm head and continued until detachment from the monolayer, which occurred 120–130 s later. Sperm release was complete in the areas of co-cultures unexposed to the excitation light, but it was far less efficient in the imaged areas probably due to the phototoxic effects of the excitation light. Single-cell analysis of adhering sperm that were released after heparin addition revealed peak values of 0.8 ± 0.22 RFI (mean± s.d.; n = 6; sperm analyzed = 87).
Effect of external Ca2+on sperm release
Preliminary experiments showed that incubation of oviductal monolayers in Ca2+-free sperm-TALP for more than 15 min causes the dissociation of cells from the monolayer. Therefore, to investigate the involvement of external Ca2+in heparin-induced release, sperm were first co-cultured with oviductal monolayers in sperm-TALP; after removal of unbound sperm, the following co-cultures were treated: 1) sperm-TALP; 2) Ca2+-free sperm-TALP; 3) heparin 100 μg/ml in sperm-TALP; and 4) heparin 100 μg/ml in Ca2+-free sperm-TALP. All co-cultures were fixed for quantitative analysis 10 min after heparin addition. As shown in Fig. 2, treatment with Ca2+-free sperm-TALP did not induce sperm release; addition of heparin in Ca2+-free sperm-TALP caused sperm release at an extent comparable to that recorded in sperm-TALP.
Effect of pharmacologic agents on sperm [Ca2+]i and release
Experiments were designed to test the ability to evoke sperm release from oviductal monolayers by increasing either sperm [Ca2+]i or flagellar beat frequency by means of pharmacologic agents. Hence, sperm oviductal co-cultures were treated with Ca2+-ionophore A23187, thapsigargin, thimerosal or caffeine, as described in the Materials and Methods section. All pharmacologic agents tested were able to increase sperm [Ca2+]i but failed to induce sperm release. Parallel samples not exposed to the dye were treated with pharmacologic agents in the cover-slip chamber and observed at intervals of 10 min for 1 h under phase-contrast microscopy to ascertain: 1. that dye loading did not interfere with the ability of sperm to be released by the agent; and 2. that an eventual releasing activity of the pharmacologic agents needed a longer incubation time to occur. Even under these conditions, all agents failed to induce the release of adhering sperm. Figure 3 shows the increase of fluorescence caused by 10 μM ionophore in five adhering sperm during a 40 s acquisition (one frame/5 s) in a representative experiment. Ca2+-ionophore caused a sharp fluorescence increase (peak, mean± s.d.: 1.5 ± 0.41 RFI; n = 5; sperm analyzed = 65) in both the sperm head and midpiece that started about 5 s after addition of the reagent. However, this treatment caused immobilization of all adhering sperm. Repeated resuspensions of the medium in the cover-slip chamber after ionophore treatment did not displace sperm, showing that they were still firmly bound to monolayers. In the next series of experiments, treatment with 1 μM ionophore maintained sperm motility, causing sperm [Ca2+]i elevations without inducing sperm release from the monolayer (data not shown).
Thapsigargin, a Ca2+-ATPase inhibitor that increases [Ca2+]i through release from internal stores (Thastrup 1990), has been reported to increase sperm [Ca2+]i in several species and to induce hyperactivation in bovine sperm (Ho & Suarez 2001). Addition of 5 μM thapsigargin to sperm bound to oviductal monolayers caused a sudden increase of sperm [Ca2+]i (peak, mean± s.d.: 2.1 ± 0.8 RFI), which recovered to basal values in about 120 s (Fig. 4A) (n = 4; sperm analyzed = 72).
Thimerosal, a drug able to release [Ca2+]i from both inositol trisphosphate (IP3)- and ryanodine-operated stores, has been reported to induce elevations of [Ca2+]i and hyperactivation in bovine sperm (Ho & Suarez 2001). Treatment of sperm adhering to monolayers with thimerosal 50 μM caused a rapid [Ca2+]i increase that reached maximum value at about 60 s (peak, mean± s.d.: 0.8 ± 0.4 RFI) (Fig. 4B) (n = 4; sperm analyzed = 67).
Treatment of co-cultures with caffeine, an agent that releases [Ca2+]i from ryanodine-operated stores and hyperactivates bovine sperm (Ho & Suarez 2001), induced [Ca2+]i elevations (peak, mean± s.d.: 1.6 ± 1.0 RFI) (Fig. 4C) (n = 4; sperm analyzed = 93). Although caffeine was the hyperactivating agent that more effectively promoted the augmentation of adhering sperm flagellar beat frequencies, it failed to induce the release of adhering sperm under our experimental conditions.
Pairwise comparisons of mean peak values recorded after application of pharmacologic agents demonstrated that thimerosal was the least effective [Ca2+]i increasing agent (P < 0.01).
Immunocytochemistry of sperm tyrosine phosphorylated proteins
Bovine sperm processed for the immunocytochemical localization of tyrosine phosphorylated proteins exhibited the following three different labeling patterns (Fig. 5): 1. equatorial segment (pattern E); 2. acrosome (pattern A); and 3. equatorial segment and acrosome (pattern EA). Specificity controls performed either by omission of the primary antibody or by saturation of the primary antibody with 20 mM o-phosphotyrosine were both negative. For study of the dynamics of tyrosine phosphorylation during capacitation, the following samples were analyzed: 1. initial sperm suspension recovered after Percoll centrifugation at times 0 and 1 h; 2. sperm adhering to monolayers at 1 h of co-culture; and 3. initial suspension and released sperm at 1 h of culture in sperm-TALP plus 5min treatment with heparin 100 μg/ml. Preliminary findings showed that labeling of the equatorial segment was the first pattern observed during capacitation, whereas labeling of the acrosome alone or of both the equatorial segment and acrosome was observed only at longer capacitation times. Therefore, since A and EA may represent more advanced stages of capacitation than E, these patterns were grouped together as shown in Table 1. Data on control sperm showed a general progression both in the number of labeled sperm and in the labeling pattern. In fact, at 0 h, 4% of sperm were labeled and showed pattern E, whereas, at 1 h, labeled sperm increased at 41%, and both patterns E and A plus EA were observed (Table 1). Moreover, treatment of this sample for 5 min with heparin increased the percentage of labeled sperm to 75%. By contrast, only 2% of adhering sperm were labeled at 1 h of co-culture and showed exclusively the E pattern. Treatment for 5 min with heparin caused the release of adhering sperm and the increase of labeled sperm to 15% (Table 1).
Discussion
The epithelial cells of the isthmic portion of mammalian fallopian tube can select a fraction of uncapacitated sperm and maintain their fertile life by delaying capacitation until unknown ovulation-associated signals, inducing sperm hyperactivation and/or surface remodeling, ultimately release selected and stored sperm for fertilization (DeMott & Suarez 1992, Smith 1998). Since sperm adhesion to oviductal epithelial cells is one of the first events occurring during capacitation, it is likely that some mechanisms of capacitation are modulated through such cell–cell interaction.
In the bovine species, an acrosome-intact sperm sub-population endowed with a superior zona pellucida binding and fertilization competence can be selected by adhesion to in vitro cultured oviductal monolayers, and readily and synchronously released by nanomolar to micromolar amounts of the sulfated glycoconjugates heparin, fucoidan and dextran sulfate (Gualtieri & Talevi 2000, 2003, Talevi & Gualtieri 2001, 2004). On this basis, heparin-like glycosaminoglycans have been suggested to represent the physiologic signal for the release of sperm stored in the oviductal reservoir in the bovine species.
The aim of the present paper was to determine whether heparin-induced release of selected sperm represents a very early event of sperm capacitation within the female reproductive tract. To this end, experiments were designed to study sperm [Ca2+]i and protein tyrosine phosphorylation, two well-recognized markers of sperm capacitation, during heparin-induced release of bovine sperm adhering to the fallopian tube epithelium in vitro.
Main results indicate the following: 1. [Ca2+]i increase accompanies heparin-induced sperm release; 2. heparin-induced sperm release does not require extracellular Ca2+; 3. triggering of sperm hyperactivation and/or [Ca2+]i elevations by means of pharmacologic agents fails to elicit release of sperm adhering to the fallopian tube in vitro; and 4. adhesion to the oviduct in vitro maintains a low level of protein tyrosine phosphorylation in sperm, whereas heparin-released sperm have increased protein tyrosine phosphorylation.
Heparin, a well-known capacitating molecule routinely used in bovine IVF (Parrish et al. 1988), is similar to endogenous glycosaminoglycans of the oviductal fluid, whose concentration peaks around the time of ovulation (Parrish et al. 1989a). Several studies have shown that incubation with heparin for at least 4 h is required to induce capacitation and to increase sperm [Ca2+]i, cAMP, intracellular pH and protein tyrosine phosphorylation (Parrish et al. 1988, 1989b, 1994, Galantino-Homer et al. 1997, Visconti et al. 1998). On the other hand, heparin has an immediate effect on release of sperm adhering to the oviductal epithelium in vitro (Talevi & Gualtieri 2001). If release is due to the capacitating activity of heparin, this means that some events of capacitation may occur very rapidly. Present results on sperm [Ca2+]i determinations agree with this hypothesis. In fact, heparin increases the [Ca2+]i in the head of adhering sperm until they detach from the monolayer; moreover, heparin-released sperm have significantly higher [Ca2+]i than adhering sperm. Previous studies in horses demonstrated that adhesion to oviductal cells or to apical plasma membrane preparations is responsible for the maintenance of low sperm [Ca2+]i and that spontaneously released sperm have [Ca2+]i about three times higher than adhering sperm (Dobrinski et al. 1996, 1997). The maintenance of low [Ca2+]i in adhering sperm may account for the reported ability of oviduct adhesion to prolong sperm fertile life and delay capacitation. On the other hand, the observation that released sperm have higher [Ca2+]i than bound sperm might be explained by the removal of the inhibitory influence of oviduct adhesion on sperm [Ca2+]i. Results of the present study, in which the [Ca2+]i dynamics of single adhering sperm was continuously monitored during heparin-induced release, demonstrate for the first time that [Ca2+]i increase precedes the release of adhering sperm. Quantitative experiments on heparin-induced sperm release in Ca2+-free sperm-TALP were designed to determine whether sperm [Ca2+]i increase is caused by the entry of extracellular Ca2+from the external medium or by release from intracellular stores. Data indicate that such an early capacitation event does not require extracellular Ca2+but may be due to release from internal stores. This finding agrees with recent evidence of the existence of sperm internal Ca2+stores and their involvement in the regulation of sperm functions (Walensky & Snyder 1995, Ho & Suarez 2001, Rossato et al. 2001). Interestingly, in the bovine species, an IP3-gated Ca2+store has been shown to be involved in the initiation of hyperactivated motility (Ho & Suarez 2001). Moreover, heparin has been reported to cause a sudden increase of adhering sperm flagellar beat followed by detachment of sperm from the oviductal monolayers (Talevi & Gualtieri 2001), and to induce hyperactivation of free-swimming sperm (Chamberland et al. 2001). Therefore, it can be hypothesized that addition of heparin to sperm adhering to oviductal monolayers induces first the release of Ca2+from internal stores accompanied by the augmentation of flagellar beat frequencies and then sperm detachment.
Experiments with pharmacologic agents to induce sperm [Ca2+]i elevations artificially and/or hyperactivation demonstrated that neither [Ca2+]i elevation nor hyperactivation can cause sperm release. Capacitation is supposed to induce the release of sperm adhering to the fallopian tube epithelium, either by a rapid remodeling of the sperm plasma membrane, which involves the inactivation of adhesion molecules directed toward the oviductal epithelium, or merely by inducing sperm hyperactivation, which provides an additional force sufficient to release sperm from their cellular contacts (Smith & Yanagimachi 1991, DeMott & Suarez 1992, Lefebvre & Suarez 1996). In the present paper, several agents were selected for their reported ability to increase sperm [Ca2+]i (Ca2+ionophore A23187) or both [Ca2+]i and hyperactivation in free-swimming sperm (thapsigargin, thimerosal and caffeine: Ho & Suarez 2001). However, under our experimental conditions, pharmacologic agents failed to release sperm, although they were able to increase sperm [Ca2+]i, and, at least as regards caffeine, to induce a clear increase of sperm flagellar beat frequency. Overall, present findings support the hypothesis that heparin-induced sperm release is due to a rapid remodeling of the plasma membrane that leads to a loss of affinity for the molecules involved in such cell–cell interaction.
Several studies have shown the involvement of the cAMP/protein kinase A pathway in the tyrosine phosphorylation of specific subsets of sperm proteins, as occurs during capacitation (Visconti et al. 1995, Galantino-Homer et al. 1997, Gadella & Harrison 2000). In particular, sperm protein tyrosine phosphorylation is involved in different steps of capacitation, such as the development of hyperactivated motility and the induction of acrosome reaction (Flesch & Gadella 2000). Present findings indicate that thawed frozen bull sperm are mostly uncapacitated at time 0, whereas, at longer times, adhesion to the oviductal epithelium maintains low levels of tyrosine phosphorylation compared with those detected in free-swimming sperm. This agrees with recent reports in other species (Petrunkina et al. 2001, 2003) demonstrating that oviduct adhesion selects sperm with suppressed tyrosine phosphorylation and stores them in such an uncapacitated condition, thus prolonging sperm fertility. Moreover, in the bovine species, heparin treatment of both free-swimming and adhering sperm quickly triggers tyrosine phosphorylation. Interestingly, the rate of tyrosine phosphorylation in response to heparin in adhering sperm is faster than in free-swimming sperm, suggesting that the suppression of capacitation caused by adhesion to the oviduct may render the adhering sperm subpopulation more responsive to capacitation signals. On the other hand, it is also possible that oviduct adhesion selects a pre-existing sperm sub-population endowed with higher responsiveness to heparin. Whatever the case, this finding may be related to the superior sperm–zona pellucida binding and fertilization competence of oviduct-selected sperm (Gualtieri & Talevi 2003).
In conclusion, the present data on sperm [Ca2+]i and tyrosine phosphorylation dynamics during heparin-induced release from in vitro cultured tubal epithelium add new information suggesting that adhesion to the oviduct suppresses capacitation, and that release of adhering sperm is due to a very early event of capacitation that remodels the sperm surface, leading to a rapid loss of affinity for the oviductal epithelium.
Labeling patterns of bovine sperm processed for the immunolocalization of tyrosine phosphorylated proteins (n = 3; means± s.d.). Control 0 and 1 h: sperm suspension at 0 and 1 h of incubation in sperm-TALP. 1 h + Heparin: sperm suspension at 1 h of incubation in sperm-TALP followed by treatment with heparin 100 μg/ml for 5 min. Bound 1 h: sperm adhering to oviductal monolayers at 1 h of co-culture in sperm-TALP. Heparin-released: sperm co-cultured for 1 h in sperm-TALP and released by treatment with heparin 100 μg/ml for 5 min.
| Control 0 h | Control 1 h | 1 h + Heparin | Bound 1 h | Heparin-released | |
|---|---|---|---|---|---|
| a,b,c,d,e,f and g,hValues within rows differ significantly (P < 0.01). | |||||
| Labeling patterns: E, equatorial segment; A, acrosome; EA, equatorial segment and acrosome. | |||||
| Labeled sperm (%) | 4 ± 3a | 41 ± 8bce | 76 ± 16d | 2 ± 2fg | 15 ± 4 h |
| Pattern E (%) | 4 ± 3a | 14 ± 3bc | 24 ± 7 | 2 ± 2de | 15 ± 4f |
| Pattern A + EA (%) | 0a | 27 ± 5bce | 51 ± 9d | 0f | 0 |
Effect of heparin (100 μg/ml) on [Ca2+]i of sperm adhering to oviductal monolayers. (A) Graph shows a typical [Ca2+]i increase, expressed in relative fluorescence units, of the sperm depicted in the upper left corner of panel B in a representative experiment. The decline in fluorescence at about 160 s was due to the detachment of the imaged sperm from the oviductal monolayer. (B) Time-lapse imaging of adhering sperm during heparin-induced release. Oviductal cells were not visualized under the fluo-3FF/AM charging and acquisition conditions utilized. Numbers at the lower left corner of figures represent time in seconds. Bar = 10 μm.
Citation: Reproduction 129, 1; 10.1530/rep.1.00374
Effect of Ca-free medium on the release of sperm adhering to oviductal monolayers (n = 3; means± s.d.). In all samples, sperm–oviduct adhesion was performed for 1 h in sperm-TALP, and then unbound sperm were removed. Control: 10 min in sperm-TALP; Ca-free: 10 min in Ca-free sperm-TALP; heparin: 10 min in sperm-TALP plus 100 μg/ml heparin; heparin in Ca-free: 10 min in Ca-free sperm-TALP plus 100 μg/ml heparin (control vs heparin; Ca-free vs heparin in Ca-free: P < 0.0001).
Citation: Reproduction 129, 1; 10.1530/rep.1.00374
Effect of Ca2+ionophore A23187 (10 μM) on [Ca2+]i of sperm adhering to oviductal monolayers. (A) Graph shows the [Ca2+]i increase expressed in relative fluorescence units of five sperm imaged in panel B in one representative experiment (n = 5; number of sperm analyzed was 65). Shaded and unshaded symbols indicate each sperm and relative [Ca2+]i trace. (B) Time-lapse imaging of adhering sperm during ionophore treatment. Sperm remained firmly bound to the oviductal monolayers after treatment. Oviductal cells were not visualized under the fluo-3FF/AM charging and acquisition conditions utilized. Numbers at the upper left corner of figures represent time in seconds. Bar = 10 μm.
Citation: Reproduction 129, 1; 10.1530/rep.1.00374
Mean [Ca2+]i increases of sperm adhering to oviductal monolayers during treatment with the pharmacologic agents (A) thapsigargin (5 μM), (B) thimerosal (50 μM), and (C) caffeine (10 mM).
Citation: Reproduction 129, 1; 10.1530/rep.1.00374
Labeling patterns of bovine sperm processed for the immunolocalization of tyrosine phosphorylated proteins. (A) equatorial segment (pattern E), (B) acrosome (pattern A), (C) equatorial segment and acrosome (pattern EA). Bar = 4 μm.
Citation: Reproduction 129, 1; 10.1530/rep.1.00374
Thanks are due to Dr A Ianora for helpful comments and critical revision of the manuscript. This research was supported by the M.I.U.R grant, ‘Proteomics of the human sperm surface’.
References
Chamberland A, Fournier V, Tardif S, Sirard MA, Sullivan R & Bailey JL2001 The effect of heparin on motility parameters and protein phosphorylation during bovine sperm capacitation. Theriogenology 55 823–835.
DeMott RP & Suarez SS1992 Hyperactivated sperm progress in the mouse oviduct. Biology of Reproduction 46 779–785.
Dobrinski I, Suarez SS & Ball BA1996 Intracellular calcium concentration in equine spermatozoa attached to oviductal epithelial cells in vitro. Biology of Reproduction 54 783–788.
Dobrinski I, Smith TT, Suarez SS & Ball BA1997 Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro. Biology of Reproduction 56 861–869.
Ellington JE, Evenson DP, Fleming JE, Brisbois RS, Hiss GA, Broder SJ & Wright RW Jr1998 Coculture of human sperm with bovine oviduct epithelial cells decreases sperm chromatin structural changes seen during culture in media alone. Fertility and Sterility 69 643–649.
Fazeli A, Duncan AE, Watson PF & Holt WV1999 Sperm-oviduct interaction: induction of capacitation and preferential binding of uncapacitated spermatozoa to oviductal epithelial cells in porcine species. Biology of Reproduction 60 879–886.
Flesch FM & Gadella BM2000 Dynamics of the mammalian sperm plasma membrane in the process of fertilization. Biochimica Biophysica Acta 1469 197–235.
Gadella BM & Harrison RA2000 The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane. Development 127 2407–2420.
Galantino-Homer HL, Visconti PE & Kopf GS1997 Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3′5′-monophosphate-dependent pathway. Biology of Reproduction 56 707–719.
Gualtieri R & Talevi R2000 In vitro-cultured bovine oviductal cells bind acrosome-intact sperm and retain this ability upon sperm release. Biology of Reproduction 62 1754–1762.
Gualtieri R & Talevi R2003 Selection of highly fertilization-competent bovine spermatozoa through adhesion to the Fallopian tube epithelium in vitro. Reproduction 125 251–258.
Harper MJK1994 Gamete and zygote transport. In The Physiology of Reproduction, pp 123–187. Eds E Knobil & JD Neill. New York: Raven Press.
Ho HC & Suarez SS2001 An inositol 1,4,5-triphosphate receptorgated intracellular Ca2+store is involved in regulating sperm hyperactivated motility. Biology of Reproduction 65 1606–1615.
Lefebvre R & Suarez SS1996 Effect of capacitation on bull sperm binding to homologous oviductal epithelium. Biology of Reproduction 54 575–582.
Parrish JJ, Susko-Parrish JL, Winer MA & First NL1988 Capacitation of bovine sperm by heparin. Biology of Reproduction 38 1171–1180.
Parrish JJ, Susko-Parrish JL, Handrow RR, Sims MM & First NL1989a Capacitation of bovine spermatozoa by oviduct fluid. Biology of Reproduction 40 1020–1025.
Parrish JJ, Susko-Parrish JL & First NL1989b Capacitation of bovine spermatozoa by heparin: inhibitory effect of glucose and role of intracellular pH. Biology of Reproduction 41 683–699.
Parrish JJ, Susko-Parrish JL, Uguz C & First NL1994 Differences in the role of cyclic adenosine 3′,5′-monophosphate during capacitation of bovine sperm by heparin or oviduct fluid. Biology of Reproduction 51 1099–1108.
Patel R, Wright EM & Whitaker M1997 Caffeine overrides the S-phase cell cycle block in sea urchin embryos. Zygote 5 127–138.
Paula-Lopes FF, de Moraes AAS, Edwards JL, Justice JE & Hansen PJ1998 Regulation of preimplantation development of bovine embryos by interleukin-1. Biology of Reproduction 59 1406–1412.
Petrunkina AM, Friedrich J, Drommer W, Bicker G, Waberski D & Topfer-Petersen E2001 Kinetic characterization of the changes in protein tyrosine phosphorylation of membranes, cytosolic Ca2+concentration and viability in boar sperm populations selected by binding to oviductal epithelial cells. Reproduction 122 469–480.
Petrunkina AM, Simon K, Gunzel-Apel AR & Topfer-Petersen E2003 Regulation of capacitation of canine spermatozoa during co-culture with heterologous oviductal epithelial cells. Reproduction in Domestic Animals 38 455–463.
Rossato M, Di Virgilio F, Rizzuto R, Galeazzi C & Foresta C2001 Intracellular calcium store depletion and acrosome reaction in human spermatozoa: role of calcium and plasma membrane potential. Molecular Human Reproduction 7 1606–1615.
SAS/STAT User’s Guide1988 Release 6.03 edn. Cary, NC: Statistical Analysis System Institute.
Smith TT1998 The modulation of sperm function by the oviductal epithelium. Biology of Reproduction 58 1102–1104.
Smith TT & Yanagimachi R1990 The viability of hamster spermatozoa stored in the isthmus of the oviduct: the importance of sperm-epithelium contact for sperm survival. Biology of Reproduction 42 450–457.
Smith TT & Yanagimachi R1991 Attachment and release of spermatozoa from the caudal isthmus of the hamster oviduct. Journal of Reproduction and Fertility 91 567–573.
Talevi R & Gualtieri R2001 Sulfated glycoconjugates are powerful modulators of bovine sperm adhesion and release from the oviductal epithelium in vitro. Biology of Reproduction 64 491–498.
Talevi R & Gualtieri R2004 In vivo vs in vitro fertilization. European Journal of Obstetrics and Gynecology 115 S68–S71.
Thastrup O1990 Role of Ca2(+)-ATPases in regulation of cellular Ca2+signalling, as studied with the selective microsomal Ca2(+)-ATPase inhibitor, thapsigargin. Agents and Actions 29 8–15.
Thomas PGA, Ball BA & Brinsko SP1994 Interaction of equine spermatozoa with oviduct epithelial cell explants is affected by estrous cycle and anatomic origin of explant. Biology of Reproduction 51 222–227.
Thomas PGA, Ball BA & Brinsko SP1995 Changes associated with induced capacitation influence the interaction between equine spermatozoa and oviduct epithelial cell monolayers. Biology of Reproduction Monographs 1 697–705.
Visconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P & Kopf GS1995 Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development 121 1129–1137.
Visconti PE, Galantino-Homer H, Moore GD, Bailey JL, Ning X, Fornes M & Kopf GS1998 The molecular basis of sperm capacitation. Journal of Andrology 19 242–248.
Visconti PE, Westbrook VA, Chertihin O, Demarco I, Sleight S & Diekman AB2002 Novel signaling pathways involved in sperm acquisition of fertilizing capacity. Journal of Reproductive Immunology 53 133–150.
Walensky LD & Snyder SH1995 Inositol 1,4,5-triphosphate receptors selectively localized to the acrosomes of mammalian sperm. Journal of Cell Biology 130 857–869.
Wilmut I & Hunter RHF1984 Sperm transport into the oviducts of heifers mated early in estrus. Reproduction, Nutrition, Development 24 461–468.
