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a Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales,
b Centre de Recherche du CHUL, Université Laval, Québec, Canada G1K 7P4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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female reproductive tract, gamete biology, oviduct, sperm capacitation, sperm motility and transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many studies suggest that the physiology of bovine sperm is modified by exposure to oviductal fluids during their passage through the oviduct. Components of the oviductal fluid induce changes to the sperm membrane surface [8] and also improve sperm motility [9–11], zona binding, and fertilizing ability [12–14] and may potentate the induction of the acrosome reaction [9, 15–17]. Furthermore, bovine oviductal fluid proteins are known to bind to sperm membranes [18–20] and to improve motility, viability [21, 22], capacitation, and fertilizing capacity of sperm [12]. Studies with cultured oviduct epithelial cells (OEC) have also shown that secretions from these cells can also affect motility [10, 23, 24] and capacitation [23, 25, 26] of sperm in vitro.
Attachment of sperm to the oviduct epithelium has been observed by scanning electron microscopy (SEM) on oviducts of bred cows [27]. Specific recognition and binding between bovine sperm and homologous OEC are believed to occur in vivo and would be mediated by the interaction of a Ca2+-dependent lectin on the sperm head surface [28, 29] and fucose supported by a Lewis-a-trisaccharide [29–31] present on the apical membrane of OEC. Similar binding of bovine sperm to OEC has also been observed in vitro [26, 32–36]. This binding enhances motility, fertilizing capacity, and hyperactivation [34]. Acrosome modifications have also been observed by SEM in sperm bound to cultured oviduct epithelial cell monolayers or to oviductal explants in vitro [36]. Changes in sperm head membranes are also observed by SEM during in vivo interaction with the oviduct and are considered as a visible manifestation of the completion of capacitation [27].
The oviduct epithelial cell apical plasma membrane (OAPM) is the part of the cell surface that contacts the sperm membrane. Recently, Smith and Nothnick [37] demonstrated that a direct contact between rabbit sperm and homologous apical membrane vesicles improved sperm viability in a tissue-specific manner. Dobrinski and coauthors [38] showed that this membrane contact could contribute to the maintenance of low levels of intracellular calcium in equine sperm and could also delay capacitation. Furthermore, this contact was found to enhance motility and delay capacitation of human sperm cells [39].
Taken together, these studies suggest that the oviduct can influence sperm life span, maturation, and fertilization competence by its luminal secretions and also through direct contact between the epithelial cells and the sperm. However, the mechanisms by which sperm survive and capacitate in the oviduct are still not understood. The biochemical nature of the factor(s) located in the OAPM and implicated in sperm function modulation is still unknown. Also, studies aiming to characterize the effect of the contact of sperm with OAPM on sperm function are problematic because the coincubation creates conditions that are incompatible with computer-assisted method analyses like Computer-Assisted Semen Analysis (CASA). Therefore, the objectives of the present investigation were to 1) purify apical plasma membranes from fresh and cultured bovine oviduct epithelial cells by a technique that allows for activity measurements and for potential labeling of the active factor, 2) assess the capacity of OAPM to maintain motility in vitro, 3) verify whether this capacity is tissue specific, 4) determine whether this effect is protein dependent, and 5) test the ability of flow cytometry as a computer-assisted method to provide an accurate and complete measurement of sperm viability and calcium uptake during the coincubation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expression "fresh OAPM" refers to OAPM extracts obtained from OEC that were submitted to the apical plasma membrane-enrichment protocol immediately after their recovery from the oviduct, while the term "cultured OAPM" (see below) is associated with apical extracts derived from cultured OEC. All chemicals are from Sigma (Sigma Chemicals, St. Louis, MO) unless otherwise stated. Oviducts from cows in early estrus were collected at the slaughterhouse, maintained at 4°C during transport, and dissected from other tissues at the laboratory on ice within 10 h. Oviduct epithelial cells were obtained by stripping oviducts with a glass slide, and apical membranes were isolated according to the method of Behnke and colleagues [40]. Briefly, OEC from eight oviducts from four cows were homogenized with a polytron aggregate homogenizer (Kinematica, Lucerne, Switzerland) in 20 ml of buffer 1 (60 mM mannitol, 5 mM ethylene glycol bis(ß-aminoethyl-ether)-N,N,N',N'-tetra-acetic acid [EGTA], pH adjusted to 7.4 using a 1 M Tris- [Fisher Scientific, Fair Lawn, NJ] HCl, pH 7.4, solution). Then 200 µl of 0.1 M MgCl2 was added to the homogenate, which was maintained on ice for 30 min to agglutinate membranes of nonapical origin. A first centrifugation was performed at 3000 x g and 4°C for 15 min. The supernatant containing the apical membrane was removed and centrifuged at 27 000 x g for 30 min. The resulting supernatant was then removed and the pellet containing the membranes was resuspended in 20 ml of buffer 2 (60 mM mannitol, 7 mM EGTA, pH 7.4, with Tris base) and homogenized with a Potter S homogenizer (Fisher Scientific). The mixture was then resubmitted to the purification steps involving incubation with 0.1 M MgCl2 for 30 min and centrifugation at 3000 x g and 27 000 x g. The pellet was resuspended in 20 ml of buffer 3 (300 mM mannitol, pH 7.4, with 0.1 M Tris-HCl, pH 7.4) and again homogenized with the Potter S. The final mixture was separated into two centrifugation tubes and pelleted for a last time. One pellet was resuspended in 200 µl buffer 3 for protein assay and enzymatic analysis, while the other was resuspended in 5 ml of a modified Tyrode medium supplemented with 0.6% BSA, 0.011% pyruvic acid, and 0.005% gentamycin (TL sperm medium), centrifuged at 27 000 x g for 30 min, and resuspended again in 800 µl of the same solution to be used for sperm incubations. All preparations were kept frozen at -20°C until needed.
Preparation of Cultured OAPM
Oviducts were collected at the slaughterhouse and transported to the laboratory on ice. They were then dissected free (at 26°C) from other tissues and the epithelial cells were collected by stripping the oviduct with a glass slide. Cells were washed by allowing them to precipitate, by gravity, three times in 15 ml Hanks + 5% fetal calf serum (Medicorp, Montréal, QC, Canada) medium. The supernatant was removed carefully and a 1.1-ml volume of the final pellet was added to each of the six culture flasks containing 150 ml of TCM 199 Earle medium supplemented with 10% calf bovine serum (CBS) (ICN, Costa Mesa, CA), 0.2 mM pyruvate, and 0.005% gentamycin. Cells were incubated at 38.5°C in 5% CO2 for 2 days and the culture medium was changed prior to a final day of culture. After these 3 days of culture, cells were pelleted by gravity flow and the remaining cultured medium discarded. The pellet was resuspended with buffer 1 and cells were again allowed to pellet by gravity. The supernatant was discarded and apical membranes were purified from the cells using the protocol described above.
Preparation of Mammary Apical Plasma Membrane
Epithelial bovine mammary gland cells (graciously donated by Dr. Denis Petitclerc from the Dairy and Swine Research and Development Center, Lennoxville, Québec, Canada) immortalized using the large T antigen from simian virus SV40 were used as a control. These cells were cocultured in TCM 199 Earle medium + 10% CBS + 0.2 mM pyruvate + 0.005% gentamycin. Cells were cultured until confluence was reached in six 225-cm2 culture flasks, at which time the medium was removed. The cells were rinsed with buffer 1 and then mechanically removed from the flask. The isolated cells were submitted to the apical membrane-enrichment protocol described above.
Preparation of Boiled OAPM
Aliquots of 25 µl of fresh OAPM were prepared in sealed tubes and immersed in boiling water for 5 min just before the coincubation with sperm.
Analysis of Membrane Enzymatic Activity
The presence of apical plasma membranes was characterized by measuring the activity of 
-glutamyl transpeptidase (GGTP), an enzyme present only in polar epithelial cell membranes [41]. This enzyme activity was assayed using the Sigma diagnostic kit 545.
Protein Assay
The total protein concentration of the preparation suspended in buffer 3 was determined by the BCA protein assay (Pierce, Rockford, IL). In each coincubation experiment, an aliquot was taken from the OAPM preparation tube destined for coincubation and serial dilutions were performed on this aliquot with TL sperm medium. Membrane concentration during the coincubation was always expressed as a fraction of the total protein concentration present in the membrane extract.
Experiment 1: Motility Analysis with Different Membrane Types
In order to determine if proteins from OAPM could have an effect on sperm motility and also to verify if the motility-maintaining effect was specific to oviduct epithelial cell membranes, sperm were incubated with different concentrations of fresh OAPM. The same incubation protocol was repeated with different concentrations of cultured OAPM, mammary apical plasma membrane (MAPM), and boiled OAPM. In each case, two 500-µl straws of frozen bull sperm containing 50 x 106 sperm were thawed at 37°C for 1 min. The sperm were washed twice with 5 ml of TL sperm medium by centrifugation (10 min at 250 x g and 25°C). The cell concentration was adjusted to 12.5 x 106 sperm/ml. Incubation was performed in 50-µl droplets, with each droplet containing 21 µl of fresh TL sperm medium, 25 µl of the OAPM or MAPM preparation, plus 4 µl of sperm, for a final concentration of 1 x 106 sperm/ml. After a 6-h incubation at 38.5°C in 5% CO2, motility (movement of the flagellum) percentages were determined by hemocytometer counts because the presence of OAPM vesicles precluded the analysis by the Computer Assisted Semen Analyzer. This experiment was repeated three times.
The maintained motility was obtained by dividing the motility percentage after 6 h by the initial motility percentage. The three repetitions of motility analysis were done on three different days using two pooled semen straws each day. Repetitions were handled as blocks to consider the use of different straws on different days. Each block corresponds to 1 day of experiment on two pooled semen straws. One block contained the percentage of motile sperm from these two pooled straws after 6 h of coincubation with each membrane concentration. Treatment differences were determined by the protected least-significant-difference (LSD) test (P < 0.05) and regression tests using the Statistical Analysis System (Release 6.12; SAS Institute, Cary, NC).
Experiment 2: Evaluation of Calcium Levels by Flow Cytometry
Bull sperm incubated with fresh OAPM were tested for intracellular calcium concentration using the fluorescent calcium indicator Indo-1 acetoxymethylester (Indo-1 AM; Molecular Probes, Eugene, OR). Indo-1 AM is incorporated by sperm into their cytoplasm and binds to free intracellular calcium ([Ca2+]i) in a stoichiometric ratio of 1:1. This binding changes the emission wavelength peak from 485 nm (unbound to Ca2+ = blue) to 404 nm (bound to Ca2+ = violet) [42]. The flow cytometer is able to measure the [Ca2+]i level for each sperm by standardizing the blue emission value of each sperm and then calculating the resulting violet relative emission (VRE) for each sperm. At least 10 000 sperm/sample were analyzed.
Fresh OAPM extracts were prepared as previously described. Eight 500-µl straws of cryopreserved semen containing 50 x 106 sperm were thawed at 37°C for 1 min. The sperm were washed once by centrifugation (10 min at 370 x g and 25°C) in 5 ml of a modified Tyrode medium supplemented with 0.1% polyvinyl alcohol, 1 mM pyruvate, and 0.005% gentamycin (Talp-H). The washed sperm pellet was applied to a 6-ml 45%-60%-90% percoll gradient and centrifuged for 30 min at 700 x g at room temperature. Intermediate phases of sperm were discarded and the sperm pellet was then washed twice with 5 ml of Talp-H medium by centrifugation (10 min at 250 x g and 25°C) to completely remove the percoll solution. Sperm concentration was determined by hemocytometer count and adjusted to 25 x 106 sperm/ml with Talp-H medium in the presence of 2.5 µM of Indo-1 AM followed by a 30-min incubation in the dark at room temperature. The sperm were then washed by centrifugation again, i.e., 10 min at 370 x g at room temperature once with Talp-H and once in TL sperm medium to remove any unincorporated Indo-1 AM. The supernatant was discarded and the volume adjusted to obtain 25 x 106 sperm/ml. Sperm were submitted to a 4-h incubation in TL sperm medium at 38.5°C with or without fresh OAPM (see above) in the presence or absence of 10 µg/ml heparin. Heparin is known as a capacitating agent for bull sperm [43] that increases sperm [Ca2+]i levels [44]. Analyses were performed on an Epics Elite ESP flow cytometer (Beckman Coulter, Miami, FL). Control analysis (without OAPM) was done at 0 and 4 h.
The flow cytometry results were analyzed in three steps. Step 1 was to establish the sperm population that would be included for analysis of the treatment effect on [Ca2+]i. To accurately assess [Ca2+]i levels, three sperm populations were excluded, which left only sperm that had analyzable VRE. The agglutinated population was first excluded because, if more than one sperm is analyzed at a time by the flow cytometer, the value obtained may be erroneous (Fig. 1). The damaged sperm population was also excluded (gate X) since Indo-1 is incorporated well only by those sperm that have a functional plasma membrane; thus, gate X sperm have a nonreliable VRE value (Fig. 1). The gate Y population (Fig. 1) was plotted as violet relative emission vs. time (Fig. 2), wherein the Ca2+-overwhelmed sperm (VRE > 64) were excluded since these sperm contain so much [Ca2+]i that the VRE value from flow cytometry is not accurate. Thus, the [Ca2+]i levels evaluated by flow cytometry and expressed as VRE are reliable only in a limited sperm population that excluded agglutinated, damaged, and [Ca2+]i-overwhelmed sperm.
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Step 2 was to characterize the sperm populations that were excluded from the first analysis, including viability assessment. The agglutinated population remained excluded (Fig. 1). Gates X and Y were fused together, creating gate XY (Fig. 1), to include damaged sperm. The gate XY population was plotted as violet relative emission vs. time (Fig. 3), and the percentage of [Ca2+]i-overwhelmed sperm was determined in each treatment. In a separate experiment (no OAPM), sperm were incubated in the same conditions and gated the same way. For this separate experiment, three repetitions were done and 5 µl of propidium iodide (PI) per milliliter were added to each tube 1 min before flow cytometry. This enabled us to study the viability level of the [Ca2+]i-overwhelmed sperm population. Thus, agglutinated sperm remained excluded and the total percentage of [Ca2+]i-overwhelmed sperm was determined. As their [Ca2+]i level cannot be assessed, the viability level of these sperm was verified using PI.
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Step 3 was to verify that the percentage of agglutinated sperm from each treatment did not skew the previous analysis (steps 1 and 2). As an example, a massive agglutination of dead sperm could lead to the measurement of false high viability percentages since agglutinated sperm are excluded from step 1 and step 2 analyses. At least this "control gating" assures a stable proportion of agglutination events through the treatment.
For the flow cytometry study, a factorial experiment was performed with oviductal protein concentration and presence of heparin as cofactors. The three repetitions of the flow cytometry study were done on three different days using eight pooled semen straws each day. Repetitions were handled as blocks to consider the use of different straws on different days. Each block corresponds to 1 day of experiment on eight pooled semen straws and contains sperm parameters for these pooled straws after 4 h of coincubation with each membrane concentration. Differences between treatments were determined by protected LSD tests (P < 0.05) and a least-significant-means procedure (due to a missing value) using the Statistical Analysis System.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The GGTP enzyme served as a marker for the presence of apical plasma membranes. The protocol used to obtain OAPM from bovine oviducts created an enrichment of the GGTP activity per milligram of total protein. The fresh OAPM preparation used to assess the effect on sperm motility had a GGTP activity of 11 153 U/mg, while the freshly extracted-cells lysate from which the fresh OAPM extract was derived was presenting an activity of only 4265 U/mg. The cultured OAPM preparation used to assess the effect on sperm motility had a GGTP activity of 11 720 U/mg, while the cultured-cells lysate from which the cultured OAPM extract was derived was presenting an activity of only 2188 U/mg. The fresh OAPM preparation used to assess the effect on sperm [Ca2+]i had a GGTP activity of 5622 U/mg, while the freshly extracted-cells lysate from which the fresh OAPM extract was derived was presenting an activity of only 1312 U/mg (Table 1). On the other hand, no GGTP activity was found in the epithelial bovine mammary gland-cell lysate in the extract resulting from the apical plasma membrane-enrichment protocol. This suggests that the enzyme was absent from the cell line used in this experiment. Under light microscopy, the MAPM vesicles observed looked very similar to, indeed even exactly the same as, OAPM vesicles.
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Experiment 1: Motility Analysis with Different Membrane Types
Motility was strongly influenced by the OAPM vesicles prepared from fresh OEC. The presence of OAPM maintained up to 65% of the initial motility of sperm after 6 h, while the control exhibited only 17% of the initial motility. Motility was best maintained by an OAPM treatment corresponding to a total protein concentration of 40 µg/ml (see 1/2 in Fig. 4A). The response of sperm to exposure to OAPM was dose dependent with these total protein concentrations (Fig. 4B).
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Motility was also strongly influenced by apical extracts from cultured bovine OEC (Fig. 5A). Initial motility was maintained at 84% by a membrane treatment corresponding to a total protein concentration of 200 µg/ml compared with 23% in the control droplet treatment after 6 h. In this experiment, sperm continued to show a dose-dependent response to the presence of membrane (Fig. 5B). Even if sperm seemed to interact closely with bovine MAPM, these vesicles did not improve sperm motility (23%) after 6 h when compared with the controls (Fig. 6).
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When aliquots of a fresh OAPM preparation were boiled before adding to the coincubation droplet, the motility-maintaining effect was lost (21%) (Fig. 7). After 6 h of coincubation with boiled apical extracts, no significant effect was detected compared with the control.
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Experiment 2: Evaluation of Calcium Levels by Flow Cytometry
There was no effect of heparin on the three parameters analyzed (ratio, % of V/B >1, or agglutination) by flow cytometry. There was no interaction between heparin and protein, so data with or without heparin were pooled within each block for further analysis (unpooled data not shown).
Step 1 reveals that, when incubated with fresh OAPM, sperm had a lower violet relative emission value than the controls and the difference was significant in the 1.4 mg/ml total protein treatment (Fig. 8). It was also revealed that the percentage of analyzed sperm was relatively low and was varying through this first analysis (data not shown).
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Step 2 was to measure the percentage of [Ca2+]i-overwhelmed sperm and the viability level of this population. The presence of OAPM caused a clear reduction in the percentage of [Ca2+]i-overwhelmed sperm (Fig. 9) after a 4-h incubation compared with the control (no OAPM). At 0 or 4 h, [Ca2+]i-overwhelmed sperm showed less than 1% viability, while those with a VRE value lower than 64 demonstrated between 75.3% and 86.5% viability (data not shown). Sperm presenting a VRE value higher than 64, [Ca2+]i-overwhelmed sperm, were therefore considered as dead or dying. This shows that the OAPM treatment reduced the number of dead or dying sperm.
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Step 3 was to analyze the percentage of agglutination events throughout all OAPM treatments. In Figure 10, we can see that all treatments with OAPM showed lower agglutination than the control after 4 h. Agglutination is similar in all OAPM treatments.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many in vitro studies describe the effect of epithelial cells alone or epithelial cells with their secretions on sperm [26, 34]. However, these studies may create confounding conditions in which to study the individual contribution of the secretions and the cell. If the cells are used alone, they may secrete soluble factors rapidly either spontaneously or in response to the sperm, preventing a chemical analysis of the contact effect. As a result, our first goal was to isolate and purify apical plasma membranes from OEC, allowing us to study the effect of direct contact with OEC apex on sperm.
For this study, early estrous cycle oviducts were used because periovulatory apical membrane vesicles appeared to be most efficient at maintaining the viability of rabbit sperm [37]. Although our experiments were based on OEC recovered from the stripping of whole oviducts, in vivo, the ampulla and isthmus may have differential roles in the maintenance of sperm until fertilization. Previous results from our laboratory have shown that isthmic and ampullary conditioned media had additive effects on penetration rates during in vitro fertilization [26]. The zona-binding ability of sperm is greater when they are incubated in isthmic fluid compared with ampullary fluid [13], and it has been suggested that macromolecules produced by the isthmus at estrus play a major role in capacitation [23]. In addition, it has been demonstrated that flushings from the ampulla at the follicular stage of the estrous cycle are more active in maintaining sperm viability and motility [10]. Therefore, it appears that the oviduct has dual roles toward sperm: maintaining their viability until fertilization is possible but at the same time creating capacitating conditions that will lead to their eventual success or demise.
For control analysis, we chose mammary gland cells on the basis of an expected low toxicity because milk is often used as a bull semen diluent for artificial insemination. Also, to our knowledge, this cell type has never been used for coincubation with sperm and could add evidence that no other apical membrane is better for sperm function than oviduct epithelial cell apical membranes.
We showed that sperm motility is modulated in a dose-dependent manner by coincubation of sperm with the OAPM. Our hypothesis is that this effect is simply caused by a relatively high number of sperm in the droplet that saturates the binding sites on OAPM. In this case, increasing the membrane quantity allows for more spermatozoa to bind and to benefit from the contact-dependent motility-maintaining effect. Also, there is a point at which the positive effect on sperm motility is moderated (Fig. 4A) although a positive dose-response occurs (Fig. 4B). If really significant, it is possible that this moderation (Fig. 4A) could be induced by the presence of excessive capacitating factors in the concentrated OAPM preparations. Because the oviduct seems to play contradictory roles (i.e., sperm survival and capacitation), it is logical to speculate that, at higher concentrations, the capacitating factor begins to surpass the effect of the motility-maintaining factors and leads to premature cell death. Even if the incubation with MAPM tends to confirm that the apical membrane-preparation protocol is not toxic to sperm, a potential toxicity of the membrane preparation cannot be totally dismissed at this time, and further purification should clarify this aspect.
In previous studies, apical membrane preparations with a motility-maintaining effect on sperm were obtained [37]. Nevertheless, for the first time, we succeeded in repeating our experiments with low membrane concentrations from cultured OEC and observed the desired maintenance of sperm motility.
The response of sperm motility was also dose dependent with cultured OAPM treatment, but no decrease in motility was observed even at the highest concentration. It is possible that the culture could change the protein production pattern of OEC [46], which could decrease the toxicity or capacitation effect associated with the membrane-to-membrane contact or favor the production of sperm-protection products. Also, OAPM from cultured cells were isolated only from the OEC that formed swimming vesicles in vitro. If this subpopulation is not totally representative of the cell population used to prepare fresh OAPM, it could explain the different effects observed on sperm motility generated by these two OAPM types (Figs. 4 and 5).
Boiled OAPM samples did not maintain motility of the sperm, strongly indicating that the effect observed is protein dependent. MAPMs were not able to maintain sperm motility either when using the same apical protein concentration as with cultured OAPM, supporting the idea that the motility-maintaining effect of the OAPM is not present in all epithelial cells [37]. Also, the fact that the preparation of apical membranes could contain proteinic factors that may not be from apical origin was considered. Using the same protocol with a second type of epithelial cells eliminated the possibility that intracellular organelle proteins present in both cell types could have had an effect by contaminating our extract. This kind of control still does not totally eliminate a potential effect of subcellular components but reduces the probabilities to a reasonable level. Therefore, we consider that the effect was caused by a cell surface-located protein. Further characterizations of the active factor should clarify this aspect.
Also unique to this study is the use of flow cytometry to measure the intracellular calcium levels in sperm coincubated with OAPM vesicles. The flow cytometer system allowed for the analysis of more than 10 000 sperm per repetition of each treatment, providing highly representative results that cannot be achieved with other techniques. Also, the use of the Indo-1 AM provides a relative violet emission value for each individual sperm cell, thereby eliminating intersperm variations that are not related to intracellular calcium differences. Moreover, to our knowledge, no flow cytometry approach to analyze [Ca2+]i levels in sperm submitted to any treatment was able to consider all subpopulations from the entire population submitted to the treatment. Sperm were analyzed for their [Ca2+]i levels when possible, and the double-staining technique with Indo and PI allowed us to reveal the viability level of the population excluded from the first analysis. These studies were validated by a stable agglutinated population through the OAPM treatment.
As previously shown, the poor fertility of cryopreserved sperm in vivo can partially be explained by a premature capacitation [47]. Recent models of capacitation have established that intracellular calcium concentration control plays an important role in bovine sperm function [44, 48]. Also, low levels of [Ca2+]i are associated with better in vivo fertility in the bovine [49]. To understand how motility is maintained in capacitating conditions, we examined the intracellular calcium concentrations using Indo-1 AM.
First, in the flow cytometry experiment, sperm present in gate Y (Fig. 1) were the only population analyzed for calcium content. Discarding [Ca2+]i-overwhelmed sperm, [Ca2+]i levels were lower in OAPM-treated sperm, suggesting that OAPM contains factors capable of delaying capacitation or cellular damage.
Second, it was considered that a large number of sperm was excluded from the initial analysis of [Ca2+]i levels. So gates X and Y (Fig. 1) were pooled for a viability analysis, creating gate XY. Using this method, it was discovered that the percentage of [Ca2+]i-overwhelmed sperm in the entire population declined in the presence of OAPM. To better understand the characteristics of these sperm, a control analysis of viability was done using the same techniques with Indo-1 and gates afterward. For this control test, viability was also assessed with PI. It was revealed that [Ca2+]i-overwhelmed sperm (gate XY+) are dying. They are not dead because they do include Indo-1, but they are dying because they are unable to exclude PI. In fact, this double-staining technique indicates an [Ca2+]i-overwhelmed population for which the cytoplasmic membrane reached a threshold. This second part of the flow cytometry experiment analysis has two implications. First, this observation tends to confirm that a high level of intracellular calcium in cryopreserved semen implicates capacitation and cell death [44, 47]. Second, it shows that OAPM can slow down the process by which this occurs. The goal of gating sperm with a VRE value above 64 apart from others was to distinguish between sperm that can be analyzed for [Ca2+]i levels and those that cannot be. In spite of this, the sperm population having a VRE value below 64 also contains some dead sperm cells. Consequently, further studies on sperm should consider this fact.
The variation in the percentage of [Ca2+]i-overwhelmed sperm cannot be explained by a variation in the agglutination population. Then it can be considered that the viability effect shown here is directly caused by OAPM, not by a leak of sperm to unanalyzed populations.
No heparin effect was observed after the 4-h incubation. This observation is similar to other studies in our laboratory [49]. To our knowledge, the lowest incubation time that leads to bovine sperm capacitation with heparin is 4 h [43, 44]. Thus, it is logical to think that a longer incubation could have had an effect. The absence of the heparin effect does not contradict the results obtained by Talevi and Gualtieri [50], who demonstrated that heparin and other sulfated glycoconjugates inhibit sperm binding to cultured monolayers of OEC and are able to induce sperm release. The OAPM vesicles cannot be used to study the characteristics of sperm adhesion and release from the oviduct epithelium because, in this model, no response from the oviduct cell is possible and sperm are not immobilized by the contact, i.e., they are free to swim with the OAPM vesicles and so a weaker interaction is sufficient to maintain close contact. As a consequence, any treatment that could affect the strength of the binding between the sperm and the OEC plasma membrane or that could affect motility characteristics of the sperm may be observable with sperm bound to the oviductal cells without being observable with the OAPM vesicles.
Capacitated sperm show less affinity to oviduct epithelial cells than do uncapacitated sperm [31, 33]. It would appear that in vivo contact of uncapacitated sperm with OEC plays an important role in the fertilization process. This may be particularly true with preovulatory matings, in which the oviduct could reduce death of sperm and maintain low calcium levels in attached sperm while unattached capacitated cells die in the oviduct fluid waiting for the oocyte.
It can be concluded that OAPM has a dual effect: to diminish the number of dead sperm after a 4-h period and to maintain a lower intracellular calcium concentration in sperm that are still viable. This suggests that OAPM contains factors capable of influencing calcium flux in the sperm. These results could help to explain why sperm motility was improved after 6 h in droplets containing OAPM and why sperm viability is enhanced. The restricted calcium influx is likely responsible for the sperm survival.
Even if our results demonstrate that OEC can influence the survival and maintenance of lower [Ca2+]i concentrations in viable sperm by products anchored in their surface, they do not eliminate a potential response (induced or not) from the oviduct to the sperm. However, our data indicate the presence of readily available factors on the OEC surface that favor sperm function through simple contact.
In conclusion, proteins from the apical membrane of oviduct epithelial cells are important players in maintaining a population of sperm with optimal fertilization characteristics at the time of ovulation. We are presently attempting to identify these proteins of the OAPM that promote sperm motility and limit Ca2+ influx.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Marc-André Sirard, Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Pavillon Paul Comtois, Université Laval, QC, Canada G1K 7P4. FAX: 418 656 3766; marc-andre.sirard{at}crbr.ulaval.ca
Received: 24 May 2001.
First decision: 9 July 2001.
Accepted: 29 April 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E. Sostaric, C. H.A. van de Lest, B. Colenbrander, and B. M. Gadella Dynamics of Carbohydrate Affinities at the Cell Surface of Capacitating Bovine Sperm Cells Biol Reprod, February 1, 2005; 72(2): 346 - 357. [Abstract] [Full Text] [PDF]
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M. Boilard, C. Reyes-Moreno, C. Lachance, L. Massicotte, J.L. Bailey, M.-A. Sirard, and P. Leclerc Localization of the Chaperone Proteins GRP78 and HSP60 on the Luminal Surface of Bovine Oviduct Epithelial Cells and Their Association with Spermatozoa Biol Reprod, December 1, 2004; 71(6): 1879 - 1889. [Abstract] [Full Text] [PDF]
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