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Biology of Reproduction 62, 1754-1762 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

In Vitro-Cultured Bovine Oviductal Cells Bind Acrosome-Intact Sperm and Retain This Ability upon Sperm Release1

Roberto Gualtieri2,a, and Riccardo Talevia

a Dipartimento di Biologia Evolutiva e Comparata, Università di Napoli "Federico II," 80134 Napoli, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian oviduct plays a key role in sperm storage, capacitation, and selection. Specific oviduct secretions and/or binding to oviductal cells are thought to be responsible for the extension of the fertile life span of sperm. In this in vitro study, a quantitative assay for sperm binding was developed to analyze the mechanisms of sperm–oviductal cell adhesion and release in the bovine species. Distribution and acrosomal status of sperm bound to in vitro-cultured ampullary and isthmic cell monolayers were followed until the time of sperm release by means of fluorescence labeling techniques. In order to understand whether release is due to surface changes of sperm or oviductal cells, double incubation experiments with unlabeled and Hoechst-labeled sperm have been performed. Main findings demonstrate that (1) only acrosome-intact sperm bind specific bovine oviductal epithelial cells; (2) acrosomes of bound sperm are preserved intact over time; and (3) release of unreacted sperm is likely to be due to changes of the sperm surface, probably triggered by capacitation. These findings support the hypothesis that binding to oviductal cells is essential for preserving the sperm fertilization competence during the interval from the onset of estrus to ovulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian oviduct is the natural site where crucial reproductive events, i.e., final gamete maturation, fertilization, and early embryo development, naturally take place [1]. In several mammals the interval from the onset of estrus to ovulation may cover several hours or even days [2]. Because sperm reaching the oviduct at mating must maintain fertilization competence until ovulation, the oviduct should provide a suitable environment that extends the fertile life span of sperm. Previous in vivo studies indicate that, after mating, sperm remain in the lower oviduct, which acts as a functional sperm reservoir, until, at the time of ovulation, they ascend to the ampulla to effect fertilization [3]. Sperm are thought to be sequestered in the lower oviduct [4] where their fertile life span may be maintained by binding to epithelial cells lining the oviduct lumen [3, 5] and/or by specific oviduct secretions [6–10]. Upon a still unknown signal, sperm may progress toward the upper oviduct for fertilization [1, 3, 11]. The first in vivo studies have shown that the fertile life of sperm is prolonged within the female reproductive tract [1, 3]. Because in vivo studies may provide only limited information about the multiple events involved in sperm storage within the oviduct, different in vitro coculture systems have been developed during recent years. Data collected suggested several hypotheses about mechanisms involved in the maintenance of sperm motility, viability, and fertility potential. Positive effects of oviductal secretions [6–10], conditioned media [12–15], specific oviduct glycoproteins [16], as well as of the binding to oviductal cells [14, 17–19], have been demonstrated. Moreover, studies with isolated fractions of oviductal plasma membrane [20–22] provided interesting data about the role played by direct membrane contact. Finally, bound sperm have been shown to be progressively released under in vitro coculture conditions, and this process should mimic what occurs in vivo in response to a still unknown physiologic signal [14, 17].

At present, it is still uncertain whether specific secretions and/or sperm binding to oviductal epithelial cells are responsible for the extension of the fertile life span of sperm. In this context, it would be interesting to analyze if the release mechanism is due to sperm capacitation or even to a complete acrosome reaction. In the present paper, distribution and acrosomal status of bovine sperm bound to homologous in vitro-cultured ampullary and isthmic cell monolayers was followed by means of fluorescence labeling techniques until the time of release. Moreover, double incubation experiments with unlabeled and Hoechst-labeled sperm were designed to understand whether the mechanism underlying sperm release is triggered by surface changes in sperm or in oviductal cells. Data obtained demonstrate that (1) only acrosome-intact sperm bind specific oviductal cells; (2) acrosomes of the bound sperm population are preserved intact at all times analyzed; and (3) release of bound sperm is likely to be due to sperm plasma membrane modifications not involving an acrosome reaction, thus supporting the hypothesis that binding to oviductal cells is essential for preserving the sperm fertilization competence during the interval from the onset of estrus to ovulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Design

Oviduct selection was done on the basis of epithelial cell ciliary beating at the time of collection. Fully confluent oviduct monolayers and a batch of frozen bovine semen pooled from three bulls were used in all experiments.

Experiment 1 (n = 6) was designed to study (1) the maintenance of sperm motility, (2) the distribution of sperm bound to ampullary and isthmic monolayers, and (3) the ultrastructure of sperm binding cells.

Experiment 2 (n = 6) was designed to study the distribution and the acrosomal status of sperm bound to ampullary and isthmic monolayers over time, in order to determine (1) the number, distribution, and release time of sperm bound to monolayers recovered from the two different anatomical regions; and (2) the acrosomal status of both the bound and the released sperm populations. These experiments were conducted on fully confluent monolayers grown on coverslips incubated with 1 x 106 sperm/ml in a final volume of 500 µl of Tyrode's albumin, lactate, pyruvate medium (sp-TALP) according to Parrish et al. [23]. As a control, sperm suspensions were diluted in the same medium without oviductal cells. In order to reach a high number of bound sperm, incubation was performed for 1 h. The unbound sperm population was removed by washing each well five times as follows: 800 µl of fresh medium were added and then the same volume was removed, after repeated suspensions, and discarded. Monolayers with attached sperm were fixed, as specified below, 1 (time of unbound sperm removal), 3, 6, and 15 h after sperm addition. Control sperm suspension and aliquots of medium with cocultured free-swimming sperm were spotted on slides at the same times. At 1 h, free-swimming sperm in coculture were collected just before unbound sperm removal. Fixed monolayers were stained with Hoechst 33342 (Sigma Milan, Italy) for quantifying the number of bound sperm. In order to assess acrosomal status, fixed monolayers were labeled with the lectin extracted from Pisum sativum conjugated to rhodamine (PSA-TRITC; Sigma Milan, Italy) and Hoechst 33342 (see below). Released and control sperm suspensions were double labeled as well.

Experiment 3 (n = 5) was designed to understand whether 1) the uneven sperm binding observed on monolayers recovered from both anatomical regions was due to a specifically higher affinity of oviductal binding cells for sperm; and 2) release of bound sperm was triggered by changes of binding affinity or by modifications of the sperm surface. For this purpose, monolayers were incubated with 1 x 106 sperm/ml and washed free of unbound sperm at 1 h. Analysis of number of bound sperm was carried out at 1 and 16 h after sperm addition. Parallel wells at 1 and 16 h after first sperm incubation were reincubated 1 h with living Hoechst-labeled sperm (same concentration) and then observed to visualize distribution of unlabeled and labeled sperm and fixed for determination of total number of bound sperm.

Oviduct Monolayers

Oviducts, including the uterotubal junction, isthmus, ampulla, and fimbria were collected at the time of slaughter, transported to the laboratory in Dulbecco's PBS supplemented with 50 µg/ml gentamycin (Gibco, Milan, Italy) at 4°C. Upon arrival, the oviduct was dissected free from surrounding tissues, ligated at both ends, and washed three times in PBS in tissue culture dishes (Falcon 3003, Becton Dickinson, U.K.) for 2 min at 4°C. The fimbria and the uterine horn were disposed of and a 2-cm piece of the oviduct at the ampullary–isthmic junction was removed and discarded to avoid the overlap of the two epithelial cell populations. The ampullary and isthmic regions were kept separated, flushed with M199 (Gibco) supplemented with 50 µg/ml gentamycin and 1 µg/ml fungizone (Gibco), and squeezed by pressure with a glass slide in the same medium supplemented with 10% fetal calf serum (FCS; Gibco). Laminae of bovine oviduct epithelial cells (BOEC) recovered from different animals were selected on the basis of ciliary beating, washed by centrifugation at 150 x g for 5 min, and incubated in the same medium overnight at 39°C, 5% CO2 in air, in 6-cm petri dishes (Falcon, Becton Dickinson). Although, BOEC were usually collected from single individuals, in some cases BOEC recovered from two to three animals were cultured as pools. Indeed, preliminary experiments done on BOEC recovered by single or two to three individuals did not show differences in sperm adhesion (data not shown). At 24–48 h, the epithelial sheets had rearranged to form follicle-shaped structures that were frozen or directly transferred into four-well tissue culture dishes (Nunc) with or without 13-mm round coverslips on the well bottom. Freezing of BOEC was performed at 24–48 h of culture. For this purpose, pellets of BOEC recovered by centrifugation at 100 x g for 5 min were diluted 1:12 (v/v) in M199, 30% FCS, supplemented with 10% dimethyl sulfoxide (Sigma). Aliquots of 1 ml were equilibrated 30 min at 4°C, 2 h on liquid nitrogen vapors and then frozen in liquid nitrogen. Cellular plating of fresh or frozen BOEC was observed earlier on plastic compared to glass surfaces and occurred at about 24–48 h after seeding. Fresh media changes were performed every 48 h. Once cell confluence was attained, isthmic and ampullary monolayers from the same individual or from the same pool of animals were washed three times with sp-TALP medium and left in this medium until sperm addition (1–3 h).

Sperm Preparation and Addition to Oviductal Monolayers

The same batch of frozen bovine semen pooled from three bulls (3 x 0.5-ml straws; approximately 50 x 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 sec, diluted into 5 ml of sp-TALP, centrifuged at 350 x g for 5 min, and resuspended in 1 ml. Swim-up was performed for 1 h at 38.5°C, 5% CO2, by layering 1 ml of sp-TALP on 250 µl of the centrifuged sperm suspension. At the end of the swim-up, 750 µl were recovered from the top of each tube, pooled, centrifuged at 350 x g for 5 min, and resuspended. Within each experiment, oviduct monolayers from one animal or a pool of individuals were incubated with the same sperm suspension at a final concentration of 1–3 x 106 motile sperm/ml in 0.5 ml of sp-TALP. At the end of coculture, oviductal cells (grown on glass coverslips) with attached sperm were fixed as described below.

In Experiment 3, oviductal monolayers, previously incubated with sperm as above, were reinoculated with Hoechst-labeled sperm. For this purpose, sperm suspensions were incubated 2 min at 38.5°C, 5% CO2 with 1 µg Hoechst 33342/ml in sp-TALP, diluted 1:100 with fresh sp-TALP medium, centrifuged at 350 x g for 5 min, and inoculated at 1 x 106 motile sperm/ml.

Quantitation of Number of Bound Sperm and Assessment of Acrosomal Status of Bound and Released Sperm

In order to test which fixative better preserved the distribution of bound sperm observed in unfixed samples, in a preliminary set of experiments (n = 4), oviductal monolayers grown on glass coverslips and incubated with the same sperm suspension were acquired at 3 h after sperm addition and then after fixation in glutaraldheyde or in paraformaldehyde. For this purpose, cocultures grown on coverslips were washed with PBS until free-swimming sperm were virtually absent (as described above) and then fixed in 2.5% glutaraldheyde or in 4% paraformaldehyde (TAAB labs, Rome, Italy) in PBS, for 2–4 h at room temperature (20–25°C), extensively washed, and mounted with the same buffer on a glass slide with cells facing up. Findings showed that glutaraldheyde fixation preserved better the distribution of bound sperm observed in living cocultures (see Results). Therefore, analysis of the number of bound sperm was carried out on cocultures fixed in glutaraldehyde, whereas, as regards visualization of acrosomal status of bound sperm, cocultures were fixed in paraformaldehyde in order to avoid glutaraldheyde autofluorescence. Following glutaraldehyde fixation, cocultures were washed in PBS, labeled for 5–10 min with Hoechst 33342 5 µg/ml PBS, and observed under half light/half fluorescence for acquisition. 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). Number of bound sperm was determined by analyzing 10 fields of 0.286 mm2 for each well. For visualization of acrosomal status, cocultures fixed in paraformaldehyde were washed in PBS and/or air dried and stored at 4°C. After washings in the same buffer, samples were permeabilized in 95% ethanol for at least 1 h at room temperature, washed in 0.05 M TRIS, 150 mM NaCl, pH 7.4 (TBS), incubated for 30 min at room temperature in the dark with tetramethyl-rhodamine isothiocyanate conjugated P. sativum agglutinin (PSA-TRITC; Sigma) 10 µg/ml in TBS, extensively washed in PBS, labeled with Hoechst 33342 5 µg/ml PBS 10 min at room temperature in the dark, washed again, mounted, and observed in a fluorescence microscope. Ten fields were alternatively acquired with filters for Hoechst (excitation: 330 nm; emission: 450 nm) and rodhamine (excitation: 535 nm; emission: 590 nm). Sperm with acrosomal region stained were considered acrosome-intact, whereas sperm with fluorescent equatorial region, as well as unstained sperm were considered acrosome-reacted. Files were superimposed using KS 300 software (Zeiss), and percentages of acrosome-intact sperm were determined by counting at least 1000 sperm per well.

Suspensions of sperm in culture medium (control) or spontaneously released by oviductal monolayers were spotted on glass slides, air dried, processed for assessment of acrosomal status, and analyzed as mentioned above.

Scanning and Transmission Electron Microscopy Analysis

Monolayers grown on glass coverslips or polycarbonate cell-culture inserts (Falcon) were incubated with sperm and washed as described above, fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate (TAAB lab.), pH 7.4, 2–4 h at room temperature, washed 3 x 10 min in buffer, postfixed in 1% osmium tetroxide (TAAB lab.), 1 h, on ice and washed again. Glass coverslips were critical point dried and sputter coated with gold for observations at the scanning electron microscope (SEM), whereas membrane inserts were dehydrated with ethanol and embedded in Embed 812 (Polysciences, Milan, Italy), for transmission electron microscopy (TEM). Serial ultrathin sections mounted on copper grids were triple-stained according to Daddow [24] with uranyl acetate and Reynold's lead citrate and observed in a Philips CM12 EM operating at 80 kV.

Statistical Analysis

A randomized block design was employed to analyze data from preliminary experiments designed to assess effect of fixation on preservation of bound sperm distribution, as well as from Experiments 1, 2, and 3. Data were analyzed by one-way ANOVA followed by pairwise comparisons of means with Tukey's post test using GraphPad Instat (GraphPad Software, San Diego, CA). Data were not transformed because they passed tests of normality and homogeneity of variances.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Epithelial sheets (Fig. 1a), collected from the oviduct lumen and incubated as described in the Materials and Methods, rearranged to form solid cell clumps with actively beating cilia within 24 h (Fig. 1b). During the next day of culture, most cell clumps cavitated and developed into follicle-shaped structures (Fig. 1b). Both cords and follicles actively swam in culture medium and started to attach and spread on the surface of plastic dishes at about 48 h (Fig. 1c,d). Proliferating cell monolayers showed typical epithelial features with well-packed polygonal cells that do not overlap each other. At this stage, SEM analysis showed a higher number of ciliated cells on the surface of plating follicles compared to the adjacent spreading cells (Fig. 1d). Confluence of monolayers was observed at 5–7 days on plastic dishes and 7–10 days on glass coverslips. At this stage ciliated cells were rarely observed.



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  		FIG. 1. Phase-contrast (a–c) and scanning electron micrographs (d) of oviductal monolayer formation. (a) Ciliated epithelial sheets at the time of collection. Bar = 20 µm, 550x. (b) BOEC cords (triangle) and follicles at 24 h of culture. Bar = 100 µm, 150x. (c,d) BOEC attachment and proliferation of monolayer at 48 h of culture. (c) Bar = 100 µm, 150x. (d) Bar = 20 µm, x550

In a first set of experiments, oviductal monolayers grown on glass coverslips and incubated for 3 h with the same sperm suspension were first analyzed in the living condition and then after fixation in glutaraldheyde or in paraformaldehyde. The mean number of sperm bound was not different between replicate wells (glutaraldehyde: P = 0.9, n = 4; paraformaldehyde: P = 0.6, n = 4; living: P = 0.9, n = 4). Number of bound sperm per 0.05 mm2 of oviductal monolayer analyzed before (mean ± SD: 52 ± 4) or after glutaraldehyde fixation (mean ± SD: 49 ± 5) were not significantly different (P > 0.05), whereas it was significantly lower after fixation in paraformaldehyde (mean ± SD: 24 ± 3) (P > 0.001).

Experiment 1

At inoculation, sperm immediately began to bind epithelial monolayers, and motility of bound sperm, as monitored by flagellar beating, was maintained in coculture until release that occurred within 24–36 h after sperm addition. During this time, there were no differences in the maintenance of sperm motility on ampullary and isthmic monolayers. In sharp contrast, the motility of control sperm in culture medium dropped to 53% at 3 h and to 21% at 6 h. Confluent monolayers of both anatomical regions had a limited number of sperm binding cells (SBC). Moreover, observations on bound sperm distribution revealed some differences between ampullary and isthmic monolayers. In fact, at all times analyzed, sperm were bound to most cells of ampullary monolayers (Fig. 2a), and only to a few cells, larger in size, in isthmic monolayers (Fig. 2b). A higher number of sperm bound ampullary compared to isthmic monolayers and their ratio was relatively constant (Fig. 3) (sperm 3 and 1.5 x 106/ml, P < 0.05; sperm 0.6 x 106/ml, P > 0.05; n = 6). TEM analysis of sperm–oviductal cell interaction showed that SBCs were joined to the surrounding cells by means of desmosomes and interdigitations (Fig. 4a); microvilli of the cell apical surfaces were closely apposed to the plasma membrane of the sperm head acrosomal region (Fig. 4a). The SEM analysis showed that (1) isthmic and ampullary monolayers have a different distribution of bound sperm (Fig. 4b,c); (2) binding is mediated by cell microvilli that closely adhere to the plasma membrane of the sperm head acrosomal region (Fig. 4d). SBCs and surrounding cells had similar ultrastructural features.



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  		FIG. 2. Half light/half fluorescence phase-contrast micrographs of sperm (triangle) bound to ampullary (a) and isthmic (b) monolayers after glutaraldheyde fixation and Hoechst 33342 staining. Bar = 50 µm, x225



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  		FIG. 3. Effect of sperm concentration on sperm binding to ampullary (AMP) and isthmic (IST) monolayers. Line shows the ratio between number of sperm bound to ampullary and isthmic monolayers (AMP/IST) at the different sperm concentrations. Mean ± SD bound sperm/0.05 mm2 monolayer; n = 6



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  		FIG. 4. Transmission (a) and scanning (b–d) electron micrographs of sperm bound to oviductal monolayers. (a) Sperm bound to oviductal cell microvilli (arrows). Bar = 1 µm, x19 000. Inset: higher magnification of the junctional region indicated by the triangle in a. Bar = 150 nm, x43 000. (b) Sperm bound to isthmic monolayer. Bar = 20 µm, x750. (c) Sperm bound to ampullary monolayer. Bar = 20 µm, x1000. (d) Oviductal cell microvilli bound to the sperm head rostral region. Bar = 2 µm, x10 000

Experiment 2

In the second set of experiments, ampullary and isthmic monolayers incubated with 1 x 106 sperm/ml were extensively rinsed 1 h after sperm addition in order to eliminate unbound sperm. In both ampullary and isthmic monolayers, number of bound sperm at 3 and 6 h after sperm addition was not significantly different from that at 1 h (P > 0.05, n = 6). On the other hand, at 15 h after sperm addition, number of sperm bound to ampullary and isthmic monolayers was 71% and 81% lower compared to those bound at 1 h, the time of unbound sperm removal (P < 0.001, n = 6) (Fig. 5a). Parallel ampullary and isthmic wells at 1, 3, 6, and 15 h were fixed and labeled for assessment of acrosomal status of bound sperm as described in the Materials and Methods. Percentages of acrosome-intact sperm bound to monolayers of both anatomical regions ranged between 98 and 100% at all time points analyzed (Fig. 6). In order to understand whether bound sperm were released in response to acrosome reactions, percentages of acrosome-intact sperm were first determined on sperm suspensions incubated for 1 h in coculture and in medium alone and then on sperm released by ampullary and isthmic monolayers at 3, 6, and 15 h. As control, percentages of acrosome-intact sperm in medium alone were analyzed at the respective time points. Percentages of acrosome-intact sperm were not significantly different among treatments (P > 0.05) (Fig. 5b). Values analyzed over time were significantly different at 1 versus 6 and 15 h (P < 0.05) (Fig. 5b).



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  		FIG. 5. Sperm bound to ampullary and isthmic monolayers (a) and percentages of acrosome-intact sperm (b) after 1, 3, 6, and 15 h of incubation in ampullary (AMP) and isthmic (IST) cocultures or in medium alone (sp-TALP). (a) Mean ± SD bound sperm/0.05 mm2 monolayer; n = 6. (b) mean ± SD percentage acrosome-intact sperm; n = 6



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  		FIG. 6. Fluorescence micrograph of sperm bound to oviductal monolayer labeled with Hoechst 33342 and PSA-TRITC for assessment of acrosomal status. Triangles indicate acrosome-reacted sperm. Bar = 20 µm, x1000

Experiment 3

The third set of experiments was designed to study the specificity of SBCs and the causes of sperm release. In ampullary and isthmic monolayers incubated with unlabeled sperm for 1 h and reincubated with Hoechst-labeled sperm 1 or 16 h after first sperm addition, labeled and unlabeled sperm were colocalized on SBCs (Fig. 7). Moreover, data on parallel wells indicate that at 16 h after first sperm addition (bound sperm/0.05 mm2 = 24 ± 2; n = 5) there was a release of 65% of sperm bound at 1 h (bound sperm/0.05 mm2 = 69 ± 8; n = 5) (P < 0.001). On the other hand, at 17 h after first sperm addition, i.e., 1 h after addition of labeled sperm, total numbers of bound sperm (bound sperm/0.05 mm2 = 60 ± 7; n = 5) was not significantly different (P > 0.05) from those detected in parallel wells fixed at 1 h after first sperm addition (bound sperm/0.05 mm2 = 69 ± 8; n = 5).



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  		FIG. 7. Half light/half fluorescence phase-contrast micrograph of labeled and unlabeled sperm colocalized on SBCs. Hoechst-labeled sperm (gray heads) were added 16 h after incubation with unlabeled sperm (black heads). Dotted lines at the level of sperm tails are caused by sperm movements during the exposure time required to visualize the fluorescent sperm heads. Bar = 40 µm, x500


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In recent years, several studies have dealt with the role of the mammalian oviduct in sperm storage, capacitation, and selection. However, it is still not well understood whether specific oviduct secretions and/or binding to oviductal cells are responsible for the extension of the fertile life span of sperm. In this in vitro study, a quantitative assay for sperm binding was developed to analyze the mechanisms of sperm–oviductal cell adhesion and release. Main findings demonstrate that (1) only acrosome-intact sperm bind specific BOEC, (2) their acrosomes are preserved intact over the time, and (3) release of unreacted sperm is likely to be due to changes of the sperm surface probably triggered by capacitation. Moreover, the ability of BOEC to bind sperm is retained intact after sperm release. Taken together, these findings suggest that sperm–oviduct binding plays a key role in the maintenance of sperm fertilizing ability.

General results showed that oviductal monolayers were able to bind sperm, maintain their motility compared to unattached sperm populations, and progressively release them. Moreover, the ultrastructural analysis demonstrated that (1) SBCs were epithelial and (2) BOEC microvilli selectively adhered to the rostral sperm head region. These data are in agreement with results reported by others authors [18, 25, 26] and validate the use of oviductal monolayers to study the sperm oviduct interaction in vitro.

In order to study sperm adhesion to BOEC and to determine the molecular basis of this interaction, an assay to quantitate large numbers of sperm bound to oviductal monolayers was needed. Several methods of counting bound sperm have been reported in the literature. Indirect counts have been done by subtracting number of sperm recovered from coculture wells from the number in control wells [14, 15, 18, 27]. Direct counts have been performed on bovine, porcine, and human explants or monolayers, in living or fixed cocultures [19, 28–33]. During the development of the present assay on bovine cocultures, three main points were considered: (1) to perform analysis of a large number of sperm on many parallel wells within the same experiment; (2) to find a simple and quick fixation method that preserves sperm binding in such a way to reflect the living condition; and (3) to find a culture stage allowing better bound sperm visualization and consequently more reliable counts. Our data showed that sperm–oviductal monolayer cocultures, fixed in glutaraldheyde and labeled with Hoechst, allowed reliable and reproducible bound sperm counts.

The mammalian oviduct consists of distinct anatomical and functional regions where crucial reproductive events occurs. As regards sperm binding, it has been suggested that the caudal isthmus acts as a sperm reservoir in vivo [11, 34–37]. Although regional differences have been found in vitro in various species [28, 29, 38], the bovine, ampullary, and isthmic explants bind sperm in a comparable way [19]. Our findings showed that ampullary monolayers are able to bind sperm and maintain their motility under in vitro conditions. Moreover, SBCs were more numerous in ampullary than in isthmic monolayers. The apparent contrast between in vivo and in vitro studies in the ability of ampullary cells to bind sperm may be a consequence of the accessibility of such cells to high sperm numbers in vitro versus in vivo. In fact, because infusion of sperm at both ends of the bovine oviduct resulted in equal numbers of bound sperm in ampulla and isthmus, it has been suggested that the reservoir is found only in the caudal isthmus in vivo, because this is the first region reached by sperm [19]. In our experimental conditions, ampullary monolayers had a higher ability to bind sperm compared to the equal binding detected on ampullary and isthmic explants by others [19]. At present it is not known whether this discrepancy arises from experimental differences. However, because a very limited number of sperm reach the ampulla in vivo, it can be suggested that sperm binding to ampullary cells occurs and may also have important functions in vivo.

In order to study the role of sperm bound to oviductal epithelium and their fate at release, it is important to assess sperm acrosomal status. In fact, some authors reported that acrosomes of sperm bound to oviductal epithelium were intact, whereas others observed a general progressive vesiculation of the bound sperm head both in vivo and in vitro [5, 17, 18, 26]. However, such studies were performed by scanning electron microscopy, a technique that does not allow the unequivocal assessment of the acrosomal status of a large number of attached sperm. As regards the status of released sperm, Chian and Sirard [14] pointed out that it remains to be determined whether changes in acrosomal status of attached sperm are related to the gradual release from oviductal epithelial cell monolayers. This could be an important point for understanding the role of sperm released by the oviductal epithelium. In fact, although spermatozoa may undergo the acrosome reaction at any place along the female tract, the fertilizing spermatozoon does not initiate the acrosome reaction until it binds to the egg zona pellucida [39]. To our knowledge, the present study is the first in which an assay for visualizing the acrosomal status of a large number of attached sperm has been developed. Data from Experiment 2 showed that only acrosome-intact sperm bind oviductal monolayers in vitro, their status does not change over time, and finally, sperm release is not likely to be due to an acrosome reaction. In fact, the percentage of attached acrosome-intact sperm ranged from 98 to 100 at all times analyzed. Interestingly, under our experimental conditions, although the great majority of sperm was released between 6 and 15 h after sperm addition, the analysis of acrosomal status of released sperm did not reveal any significant increase of acrosome-reacted sperm compared to the control sperm suspension. This finding supports the hypothesis that sperm release is not triggered by the acrosome reaction. On the other hand, if the acrosome reaction was the trigger for sperm release, the whole population should be acrosome-reacted. The quota of reacted sperm detected in the released population might be explained by acrosome reactions occurring after release. It is worthwhile mentioning that work in progress in our laboratory (Talevi and Gualtieri, unpublished data) demonstrates that the addition of molecules capable of triggering sperm release from cocultures caused massive release of acrosome-intact sperm. The finding that progressive release of bound sperm by the oviduct is not due to acrosome reactions is in good agreement with data from Experiment 3. In fact, double incubation experiments with unlabeled and Hoechst-labeled sperm demonstrated that (1) in monolayers reinoculated at 1 or 16 h (after initial sperm addition) with labeled sperm, only the cells identified as SBCs at the first sperm addition were able to bind sperm; and (2) SBCs at reinoculation bound sperm with the same efficiency, as detected by quantitative analysis. These findings demonstrate that SBCs fully retain their affinity for sperm after release, and, therefore, indirectly support the conclusion that release of acrosome-intact sperm from SBCs is due to sperm head surface changes leading to a decrease in the affinity toward the oviductal cell surface, probably related to a capacitation process.

The present findings allow us to hypothesize that the presence of SBCs in other locations than the caudal isthmus may also play a key role in the maintenance of sperm fertile life. SBCs at different locations along the oviduct might function as "filling stations" to provide substrates or signals that preserve the sperm during its migration toward the eggs. Questions arise about the nature of molecules involved in sperm binding and maintenance of motility, as well as about physiologic signals triggering sperm release. As regards the first point, in vitro binding to oviductal apical plasma membrane vesicles has been shown to maintain sperm motility in the mammalian species studied so far [20–22]. Moreover, oviductal cell surface carbohydrates seem to be involved in sperm adhesion in different species [40–42]. On the contrary, the nature and source of molecules acting as release signals remains open for future research.

In conclusion, sperm interaction with oviductal monolayers in vitro may be a useful model for studying sperm physiology and fertilization within the oviductal environment. Because it has been shown that BOEC are able to bind [32, 33] and select higher quality human sperm [43] in vitro, this coculture system could be a useful research tool in human in vitro fertilization technologies.


   ACKNOWLEDGMENTS
 
We thank Mr. G. Falcone for printing and Mr. G. Cafiero and the staff of the CIRUB (Via Foria 222, Napoli) for use of the EM facilities.


   FOOTNOTES
 
First decision: 23 September 1999.

1 Supported by Italian C.N.R. and M.U.R.S.T. grants. Back

2 Correspondence: R. Gualtieri, Dipartimento di Biologia Evolutiva e Comparata, Università di Napoli "Federico II," Via Mezzocannone 8, 80134 Napoli, Italy. FAX: 081 2528902; gualtier{at}dgbm.unina.it Back

Accepted: January 18, 2000.

Received: July 22, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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