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a Institute of Zoology, Regent's Park, London NW1 4RY, United Kingdom
b The Royal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In conclusion, the results clearly demonstrated the specific nature of the sperm-OEC interaction in the porcine species. This interaction is initiated by uncapacitated spermatozoa binding to OEC and is continued by the induction of capacitation in cocultured spermatozoa.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Attachment to oviductal epithelial explants [12–14] and cultured monolayers of oviductal epithelium [15, 16] appears to extend the life of sperm in vitro. The process of capacitation, along with the switch to the hyperactivated flagellar beating pattern [17], appears to coincide with the ability of sperm to be released from the oviductal reservoir. Noncapacitated hamster sperm injected into the oviduct bind to the epithelium, while those that have been capacitated in vitro remain free [11]. Within the transilluminated oviducts of naturally mated mice, some hyperactivated sperm were seen swimming freely while all nonhyperactivated sperm appeared tightly bound to the epithelium [18]. Moreover, Demott et al. [19] showed that fetuin interfered with hamster sperm attachment to the oviductal epithelium by binding to the acrosomal region of fresh epididymal sperm. However, fetuin did not bind to hyperactivated sperm. These results imply that there is a preferential binding of uncapacitated spermatozoa to oviductal epithelium and that capacitation or switch to hyperactivated motility allows spermatozoa in a condition appropriate for fertilization to be released from the reservoir. However, the capacitation status of spermatozoa bound to OEC and the changes in functional status of sperm bound to OEC have never been directly visualized and determined.
The main difficulty encountered in identifying the capacitation status of spermatozoa bound to OEC is that no obvious morphological changes accompany the changes in capacitation status of spermatozoa. Yet during the past few years, the availability of the fluorescent antibiotic chlortetracycline (CTC) has proven useful in this regard. Ward and Storey [20] reported that three fluorescence staining patterns could be identified on mouse sperm heads, with the population of cells in each category changing as the suspensions underwent capacitation and the acrosome reaction. The advantage of CTC analysis over other more commonly used techniques is that it not only identifies acrosome-reacted cells (AR pattern), but it also allows the acrosome-intact sperm population to be subdivided into two groups, namely the uncapacitated (F pattern) and capacitated (B pattern) spermatozoa. Since its introduction, CTC analysis has been successfully employed to identify the capacitation status of spermatozoa in several species; such studies have been done in the mouse [21], stallion [22], human [23], bull [24], boar [25, 26], ram [27], and buck [28].
In the present investigation, we hypothesized that 1) the OEC should not bind capacitated sperm—otherwise sperm transport within the oviduct would be blocked; and 2) once sperm are bound to OEC, they are likely to become capacitated before release. Therefore, 3) it follows that the sperm-OEC interaction must be specific, causing alterations of sperm functional status not achieved by other cell types in vitro. To test these hypotheses, we used CTC to study the capacitation status of boar spermatozoa in coculture with homologous OEC and cells of nonreproductive origin (LLC-PK1, porcine kidney epithelial cell line). In addition, the preferential binding of uncapacitated, capacitated, or acrosome-reacted boar spermatozoa to OEC and the other cell type was also investigated. Our approach was to vary the proportions of uncapacitated, capacitated, or acrosome-reacted boar spermatozoa in suspension using long preincubation and lysophosphatidylcholine (LPC) treatment of semen prior to a very short incubation with oviductal epithelial and LLC-PK1 cells. We predicted that if uncapacitated boar spermatozoa preferentially bind to epithelial cells, then the majority of the spermatozoa bound to the epithelial cells should be uncapacitated and no relationship should exist between the proportion of uncapacitated boar spermatozoa in suspension and of those bound to epithelial cells. Conversely, such a relationship would be observed if sperm binding to epithelial cells were nonselective.
In a previous study we demonstrated the feasibility of using OEC obtained from prepubertal gilts in examining the sperm-oviduct interaction [16]. Use of these cells in in vitro studies provides a constant and homogenous source of experimental material with a limited variation due to the age and cycle status of the donor. We used these cells in the current investigation to test further their capacity to interact with boar spermatozoa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oviducts were obtained at a local slaughterhouse from prepubertal gilts of ~120 days of age. The ovaries did not show any signs of cyclicity such as follicular growth, ovulation, or the presence of corpora lutea, and they were covered with small follicles (2–3-mm diameter). Oviducts were transported to the laboratory in PBS at room temperature (22°C). Upon arrival, the oviducts were cleaned and washed using PBS. They were trimmed from the ovaries, transferred to the washing medium containing Hanks' Balanced Salt Solution (Life Technologies, Paisley, UK) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Life Technologies), and then gently rinsed. Each oviduct was divided into three sections; one between the fimbria and the middle of the oviductal tube, containing the thicker part of the oviduct tube, was designated as ampulla. A section containing 1–2 cm of the caudal part of the uterus horn, uterotubal junction, and up to nearly the middle of the oviductal tube containing the thinner part of the oviduct tube was designated as isthmus. Finally, a section around the junction of the thin and thick part of the oviductal tube, approximately 1–2 cm long, was cut and discarded, to assure differentiation of isthmic and ampullar parts of the oviduct. The isthmic and ampullar sections of oviduct were opened longitudinally. The epithelial cells were scraped using the blunt side of a scalpel blade. The media containing scraped tissues from ampulla and isthmus were collected separately and, after initial sedimentation, were gently centrifuged for 3 min at 100 x g. The supernatants were discarded, and 5 ml of medium A, containing tissue culture medium (TCM) 199 (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B, was added to each pellet. The cells were mixed gently and were disaggregated by being passed once through a 21-gauge needle. The concentration of the epithelial cells was measured using a counting chamber. The viability was evaluated by mixing a sample of the cells with an equal volume of 4% Trypan blue (Sigma, Dorset, UK). Separate tissue culture flasks (75 cm2; Nalge Nunc International, Naperville, IL) were seeded with each preparation of isthmic and ampullar epithelial cells (1 x 106 viable epithelial cells/ml). The flasks were incubated at 39°C in 100% humidity and 5% CO2 in air. The culture medium was refreshed every 48–72 h.
Epithelial cells reached confluency after 7–14 days. At confluency, cells were rinsed several times with PBS lacking Ca2+ and Mg2+ (Life Technologies) and were detached by incubating with 3 ml of trypsin-EDTA solution (Life Technologies) containing 0.5 mg/ml trypsin and 0.2 mg/ml EDTA at 39°C for 15–20 min. The viability and concentration of the detached cells were measured, and cells were suspended in TCM 199 supplemented with 10% FBS and 10% glycerol (BDH, Poole, UK) (1 x 106 viable cells/ml). The epithelial cell suspensions were divided into 1-ml portions in cryogenic vials (Nalge Nunc International), left overnight at -70°C, and on the next day transferred to liquid nitrogen.
Establishment of Isthmic, Ampullar, and Porcine Kidney Epithelial Cell Culture in Chamber Slides
Frozen cryogenic vials of isthmic, ampullar, and LLC-PK1 (European Collection of Animal Cell Cultures, Wiltshire, UK) cells were thawed in a water bath at 37°C. Medium A (9 ml) was added to each cell type and mixed gently. The epithelial cells were centrifuged at 100 x g for 2 min. The supernatant was discarded, and a further portion of medium A (9 ml) was added to each cell sample. Cell concentration was adjusted to 5 x 104 viable cells/ml. Three wells of 4-well chamber slides (Lab-Tek II; Nalge Nunc International) were seeded with 800 µl of isthmic, ampullar, and LLC-PK1 cell suspensions (4 x 104 viable cells). The chamber slides were incubated at 39°C in 100% humidity and 5% CO2 in air. The culture medium was refreshed every 48–72 h until confluency.
Semen Preparation
Boar semen diluted and stored for 24 h in Beltsville thawing solution [29] was obtained from a commercial artificial insemination station (JSR Healthbred Limited, Thorpe Willoughby, Yorkshire, UK). Semen (10 ml) was washed three times with PBS by centrifugation and resuspension (600 x g for 10 min). After the last centrifugation the supernatant was discarded, and the pellet was resuspended in a modified Tyrode's medium ([30]) containing 2 mM CaCl2, 3.1 mM KCl, 0.4 mM MgCl2·6H2O, 100 mM NaCl, 25 mM NaHCO3, 0.3 mM NaH2PO4·2H2O, 21.6 mM sodium lactate, 10 mM Hepes, 1 mM sodium pyruvate, and 6 mg/ml BSA.
Sperm viability (used for estimation of viable sperm concentration) was assessed by adding 1 µl of 2 mM ethidium homodimer-1 (ETHD-1; Molecular Probes, Leiden, The Netherlands) and 2.5 µl of 20 µM SYBR-14 (Molecular Probes) to 1 ml of washed semen sample and incubating the suspension for 20 min at 39°C. An aliquot of this preparation was smeared on a slide and evaluated by epifluorescence microscopy. Viable spermatozoa with intact membranes excluding ETHD-1 demonstrated green fluorescence over the nucleus due to SYBR-14 staining. Spermatozoa with disrupted membranes showed red nuclear fluorescence due to ETHD-1 staining [31]. Two hundred spermatozoa were evaluated by fluorescence microscopy and classified as membrane intact (green) or membrane damaged (red).
CTC Staining
The methods used for CTC (Sigma) staining were essentially the same as those described by Wang et al. [25] for boar spermatozoa. Briefly, CTC staining solution was prepared by adding 750 mM CTC and 5 mM D,L-cysteine (Sigma) to a buffer containing 130 mM NaCl and 20 mM Tris (BDH). The solution was filtered once using a 0.22-µm filter (Millipore, Bedford, MA), and the pH was adjusted to 7.8.
CTC staining of spermatozoa bound to epithelial cells in the chamber slides was performed by adding 200 µl of CTC staining solution to each well and 30 sec later adding 200 µl of 2% paraformaldehyde in PBS (fixative). To avoid CTC uptake by epithelial cells, the CTC staining solution and the fixative were removed after 2 min and replaced with 200 µl of fixative. Slides were mounted with an antifade material (Citiflour; Agar, Stansted, UK) and examined under an epifluorescent microscope (x40 objective). At least 200 viable spermatozoa (ETHD-1 negative) were assessed for various CTC patterns. The CTC staining of unbound spermatozoa in coculture with epithelial cells was performed by addition of 200 µl of CTC staining solution to the recovered spermatozoa. Thirty seconds later, 200 µl of fixative was added. The spermatozoa were examined by fluorescence microscopy as described above.
Experimental Design
Determination of the capacitation status of boar spermatozoa in coculture with homologous epithelial cells The question of the capacitation status of boar sperm in coculture with homologous epithelial cells was addressed in a 4 x 4 x 4 factorial experiment as described below. Four sets of 4-well chamber slides were used in each experiment. One milliliter of semen preparation (3 x 106 viable sperm/ml) containing 0.4 µl of 2 mM ETHD-1 was used to replace the culture medium in each well of the 4-well chamber slides; the wells contained 1) no epithelial cells (blank), 2) isthmic, 3) ampullar, and 4) LLC-PK1 cells. Chamber slides were incubated at 39°C in 100% humidity and 5% CO2 in air. After 15, 60, 120, or 240 min of coincubation, one chamber slide (4 wells) was removed from the incubator. Medium containing unbound spermatozoa was removed, and wells of the chamber slides were washed vigorously three times each with 1 ml PBS. After the last wash the bound spermatozoa were processed for CTC staining as described above.
The media samples collected from wells were centrifuged for 2 min at 600 x g. The supernatant was discarded, and the pellet containing recovered unbound spermatozoa was processed for CTC staining as described above.
Experiments were replicated four times, using semen samples of a different boar on each occasion.
Do homologous epithelial cells preferentially bind uncapacitated, capacitated, or acrosome-reacted boar spermatozoa? To investigate the binding of uncapacitated, capacitated, and acrosome-reacted boar spermatozoa to homologous epithelial cells, boar sperm suspension containing 0.4 µl/ml of 2 mM ETHD-1 was preincubated at 39°C in 100% humidity and 5% CO2 in air. At three irregular time intervals (up to 8 h), two aliquants, each of 2 ml, were removed from this preparation. One was incubated for 10 min with 2 ml of TALP containing 250 µg/ml LPC (diluted in 1:1 mixture of methanol and chloroform; Sigma) and the other for 10 min with 2 ml of TALP medium alone. Thereafter, 1 ml of samples treated with LPC was used to replace the culture medium in each well of one chamber slide containing different epithelial cells or control (without epithelial cells). The same procedures were performed using samples treated with TALP medium alone and another chamber slide. The chamber slides were incubated for 2 min at 39°C; then the medium containing unbound spermatozoa was removed and slides were washed vigorously three times each with 1 ml PBS. After the last wash, spermatozoa bound to epithelial cells were stained with CTC and assessed as described above. Spermatozoa in the control well were collected, and after centrifugation for 2 min at 600 x g, the supernatant was discarded and the pellet was stained with CTC and assessed as described above.
The experiments were replicated three times using semen from different boars and a total of six sets of chamber slides for each replicate.
Statistical Analysis
The data were expressed as mean ± SEM. Where appropriate, data were transformed using arcsin transformations. ANOVA and linear regression analysis were used for the statistical analysis of the data. In the first part of the study the results were examined for the effect of type of coculture (isthmic, ampullar, LLC-PK1 cells, or blank without epithelial cells), incubation time, and the interaction between these factors. The level of significance was considered p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The three patterns observed after CTC staining of boar spermatozoa were the same as those reported by Wang et al. [25]. These patterns were uniform fluorescence over the whole head (F), fluorescence-free band in the postacrosomal region (B), and almost no fluorescence over the whole head except for a thin band of fluorescence in the equatorial segment (AR). To avoid CTC uptake by epithelial cells, the CTC staining duration was limited to 30 sec, which was followed by immediate exposure of epithelial cells to the fixative. Further improvement of the staining quality was achieved by fixative removal after 2 min and its replacement with fresh fixative. This procedure resulted in distinguishable CTC staining patterns of boar spermatozoa and minimal background fluorescence of epithelial cells. Owing to the orientation of spermatozoa on the epithelial cells, it was not possible to distinguish the CTC staining patterns of 30–40% of spermatozoa bound to epithelial cells.
Capacitation Status of Boar Spermatozoa in Coculture with Homologous Epithelial Cells
Figure 1 demonstrates the CTC staining patterns of unbound spermatozoa in coculture with and without (blank) epithelial cells during incubation. Sixty minutes after the start of the incubation, unbound spermatozoa in coculture with isthmic and ampullar epithelial cells showed a significant increase in the proportion of spermatozoa demonstrating pattern B and a significant decrease in those demonstrating pattern F. By the end of the incubation (240 min), a gradual increase in pattern B and a gradual decrease in pattern F unbound spermatozoa were detected. The relative proportions of CTC staining patterns of unbound spermatozoa in coculture with LLC-PK1 cells and in the blank group (except for the 240-min interval of incubation) remained constant during the course of incubation.
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Sixty minutes after the start of incubation, there were a significant increase in the proportion of pattern B spermatozoa bound to OEC and a significant decrease in those demonstrating pattern F (Fig. 2). Thereafter until the end of the incubation, the relative proportions of various CTC staining patterns in OEC-bound spermatozoa were constant. There was no change in the proportion of CTC staining patterns in LLC-PK1-bound spermatozoa during the course of incubation.
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Except for the 15-min interval for isthmic and ampullar cocultures and the 120-min interval for ampullar cocultures, in all time intervals the proportions of different CTC staining patterns in the bound and unbound spermatozoa in coculture with epithelial cells were similar.
Preferential Binding of Uncapacitated, Capacitated, or Acrosome-Reacted Boar Spermatozoa to Homologous Epithelial Cells
Treatment of preincubated sperm suspension with TALP, with or without LPC, yielded semen samples with different ranges of proportions of F (99–25%), B (42–2%), and AR (36–0%) CTC staining patterns. Using these semen samples for 2-min coincubation with epithelial cells, most bound spermatozoa demonstrated the F pattern. The proportion of F pattern spermatozoa in the control semen samples and that of the spermatozoa bound to isthmic or ampullar cells were unrelated (r2 = 0.03, p = 0.53, and r2 = 0.03, p = 0.49, respectively). In contrast, there was a highly significant relationship between the proportion of spermatozoa demonstrating F pattern in the control semen samples and that of the spermatozoa bound to LLC-PK1 cells (r2 = 0.43, p < 0.005) (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During incubation of mouse [34], human [23], and bovine [24] sperm suspensions under conditions known to support capacitation and fertilization in vitro, there are sequential alterations from the F to the B pattern and then to the AR pattern of CTC-stained spermatozoa. These changes in CTC patterns provide information on the functional state of spermatozoa: the F pattern is characteristic of uncapacitated, acrosome-intact spermatozoa; the B pattern is characteristic of capacitated, acrosome-intact spermatozoa; and the AR pattern is characteristic of acrosome-reacted spermatozoa. In the present study, these three distinct patterns, which were essentially the same as those described in the other studies for boar spermatozoa [25, 26], were consistently observed.
One hour after the start of incubation, the proportion of capacitated spermatozoa increased significantly in unbound spermatozoa in coculture with OEC. From then until the end of the incubation, there was a slight but gradual and significant reduction in the proportion of uncapacitated spermatozoa and a gradual increase in the proportion of capacitated spermatozoa. In contrast, there was no significant change in the CTC staining patterns of unbound spermatozoa in coculture with LLC-PK1 epithelial cells. The only increase in the proportion of capacitated spermatozoa in the blank group was observed at the end of the incubation (240 min). This suggests a specific capacitation-enhancement effect of porcine OEC on boar spermatozoa. Similar results were reported by Kervancioglu et al. [35], who studied the effect of human sperm coculture with human OEC and Vero cells (derived from green monkey kidney epithelium) on the movement characteristics of human spermatozoa. The OEC enhanced the incidence of hyperactivated motility after 4, 24, and 48 h of coculture, which was interpreted as capacitation. This effect was not observed with cocultures containing Vero cells. Similarly Gutierrez et al. [36] demonstrated that capacitation/acrosome reaction of ram spermatozoa took place in vitro when spermatozoa were incubated with oviductal cell monolayers. Ram spermatozoa coincubated with IBRS-2 (a commercial porcine kidney epithelial cell line) monolayers showed better viability and progressive motility compared to preparations incubated without these cells, but there was no effect on the occurrence of capacitation or the acrosome reaction. Furthermore, enhancement of capacitation has been also reported in stallion [37, 38] and bull [39] spermatozoa in coculture with OEC. In these studies, no comparison was made with spermatozoa cocultured with cells of nonreproductive origin.
The proportion of capacitated spermatozoa bound to OEC increased significantly after 60 min of coculture and remained constant until the end of the incubation. In contrast, there was no significant change in the capacitation status of spermatozoa bound to LLC-PK1 cells during the course of incubation. This parallel increase of capacitated spermatozoa in both the unbound and OEC-bound sperm populations—an increase not induced by the LLC-PK1 cells—emphasized the specific capacitation-enhancement effect of porcine OEC on boar spermatozoa.
Recently Dobrinski et al. [40] demonstrated that the induction of capacitation was delayed in stallion spermatozoa coincubated with apical plasma membrane (APM) preparations of mare isthmic oviductal epithelium. This seems to be in contrast with the results of our study. However, one should note the differences between these investigations. Apart from the fact that these studies were performed in different species and that species differences may exist, in the present investigation we utilized OEC monolayers rather than APM preparations of these cells. Although APM provide a simple model for the study of sperm oviduct interactions, they cannot represent the whole interaction involved in this process. It is likely that the secretory products of OEC or changes in composition or organization of OEC plasma membrane after sperm binding to OEC are also involved in sperm-oviduct interactions.
In the present investigation the capacitation-enhancement effects of isthmic and ampullar cocultures were similar, irrespective of the sperm binding status. Whether this was due to the short coincubation interval for spermatozoa and OEC (up to 4 h), or to a property of the porcine species, requires further investigation.
The temporal sequence of events leading to capacitation in coculture is unclear. Does OEC-binding induce capacitation or do the OEC preferentially bind those spermatozoa that are already capacitated by OEC secretory products in coculture? To address this question, semen samples with different proportions of uncapacitated, capacitated, or acrosome-reacted spermatozoa were prepared by preincubation of spermatozoa in TALP medium, known to support capacitation and in vitro fertilization. In addition, at intervals, preincubated spermatozoa were also treated with LPC, a lysophospholipid capable of inducing capacitation and the acrosome reaction in spermatozoa in vitro [26, 41–43]. These functionally heterogeneous preparations of spermatozoa were incubated for a very short period (2 min) with OEC prior to CTC staining to avoid any potential influence of OEC on bound spermatozoa. Irrespective of the proportions of uncapacitated, capacitated, or acrosome-reacted spermatozoa in the control droplets, the majority of spermatozoa that were bound to the isthmic and ampullar cells were uncapacitated, and no significant correlation existed between the proportion of uncapacitated spermatozoa in the control droplet and that of bound spermatozoa. This supports the hypothesis that OEC do not bind capacitated sperm. This hypothesis seems logical because otherwise sperm transport within the oviduct would be blocked and fertilization inhibited. Although the majority of spermatozoa bound to LLC-PK1 cells were also uncapacitated, a correlation existed between the proportion of uncapacitated spermatozoa in the control droplet and that of spermatozoa bound to LLC-PK1 cells. These results support the view that sperm-OEC binding is not simply a random event but is probably mediated by specific mechanism(s) employed by OEC and/or spermatozoa. In support of the present investigation, other studies have demonstrated that capacitation treatments significantly reduced the capacity of bovine [44], equine [45], and hamster [11] spermatozoa to bind to oviductal explants. These results justify the view that capacitation diminishes the capacity of sperm binding to OEC.
In conclusion, the results of the present investigation clearly demonstrated the specific nature of the sperm-OEC interaction in the pig. This interaction is initiated by uncapacitated spermatozoa binding to OEC and is continued by induction of capacitation in cocultured spermatozoa. More studies are required to confirm these results in vivo. In addition, in the present investigation CTC was used as a marker of capacitation. Since CTC does not reflect all the changes associated with capacitation (such as hyperactivation), further studies should be directed toward investigating these changes and the mechanisms underlying the preferential binding of uncapacitated spermatozoa to OEC.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 44 171 5862870; arfazeli{at}ucl.ac.uk
Accepted: November 13, 1998.
Received: June 17, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 M. S. Chen, K. S. K. Tung, S. A. Coonrod, Y. Takahashi, D. Bigler, A. Chang, Y. Yamashita, P. W. Kincade, J. C. Herr, and J. M. White Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin alpha 6beta 1: Implications for murine fertilization PNAS, October 12, 1999; 96(21): 11830 - 11835. [Abstract] [Full Text] [PDF]
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