Media And Reagents
The chemicals used for the media were obtained from Sigma-Merck (Missouri, United States) and ThermoFisher Scientific (Massachusetts, USA). The HEPES-buffered Tyrode’s lactate pyruvate medium (TLP medium) contained 100 mM NaCl, 3.1 mM KCl, 0.3 mM NaH2PO4, 2.1 mM CaCl2, 0.4 mM MgCl2, 1 mM sodium pyruvate, 27.4 mM sodium lactate, 10 mM Hepes, and 1 mg/mL polyvinyl alcohol (PVA)22,40. PVA was used instead of albumin to avoid sperm capacitation41. The pH of the TLP medium was adjusted to 7.4 with NaOH 1 M and the osmolarity to 300 mOsm with NaCl. The TLP medium was stored at 4°C for a maximum of two weeks. The M199 culture medium (Medium 199, Earle’s salts; 31150-022) was supplemented with 2.5% Hepes (15630-080, ThermoFisher Scientific, Massachusetts, United States), 0.5% of gentamycin (G1272, Sigma, Missouri, United States), and 10% heat-treated fetal calf serum (A5256701, ThermoFisher Scientific, Massachusetts, United States).
The media were passed through sterile 0.22-µm filters (SLGV013SL) before use. The TLP medium was placed at 38.8°C for a minimum of 1 h before use for sperm incubation or at 4°C before use for oviduct flushing. The M199 medium was placed at 38.8°C under humidified atmosphere containing 20% O2 and 5% CO2 for a minimum of 1 h before its use for the cell culture. Unless otherwise specified, reagents were obtained from Sigma-Merck.
Culture Of Isthmic Mucosa Fragments And Selection Of Oviduct Epithelial Spheroids (Oes)
Bovine oviducts and ovaries were collected from a local slaughterhouse (Vendôme, 45 min from the laboratory) and transported on ice to avoid post-mortem modifications. Pairs of oviducts from two cows at a pre-ovulatory stage of their cycle (one follicle of 11–20 mm in diameter and a corpus albicans, i.e., corresponding to approximately days 19 to 21 of the estrous cycle) were isolated from surrounding vessels and tissues and cut at the utero-tubal and ampullary junctions to isolate the isthmus (around 5 cm long)42. The isthmic sections were rapidly washed in 70% ethanol, rinsed in NaCl 0.9%, then both extremities (around 5 mm) were cut with a sterile scalpel to avoid any contamination. The isthmus mucosa was then expelled with forceps in 10 mL of a M199 culture medium, vortexed for 1 min, then incubated at 38.8°C for a minimum of 10 min for cell sedimentation. Following the elimination of the supernatant, the pellet was resuspended in 10 mL of the culture medium and the vortex-sedimentation process was repeated. Finally, the pellet was diluted ten times in the culture medium and 50 µL of the resulting mixture was added to 450 µL of the M199 medium. The isthmic mucosa fragments were cultured for four days in four-well culture plates (176740, ThermoFisher Scientific, Massachusetts, USA) at 38.8°C in a humidified atmosphere containing 20% O2 and 5% CO2. On day 4, a cavity was formed within the mucosa fragments, forming spheroids of various sizes and shapes, with the apical side of the epithelial cells oriented outward.
Spheroids of approximately 100 µm in diameter, homogeneous in form, with vigorous cilia beating, and no surface folding were selected for the sperm binding studies (Fig. 1a).
Cell Viability And Immunostaining Of Oviduct Epithelial Spheroids (Oes)
The cell viability of the OES was assessed after four days in a M199 medium and up to 4 h in the TLP and OF media at 0.06 and 4 mg/mL of proteins (in which their incubation with spermatozoa was performed) after immunostaining with ethidium homodimer-1 (L3224, Invitrogen, Massachusetts, USA). The OES were incubated in 500 µL of the culture medium and 1 µL of ethidium homodimer-1 and Hoechst (B2261) for 45 min at 38.8°C in a humidified atmosphere containing 20% O2 and 5% CO2, before being directly observed by confocal microscopy (Zeiss LSM 700, Carl Zeiss, Oberkochen, Germany).
To further characterise the OES, immunostaining for pan-cytokeratin (epithelial cell marker), vimentin (stromal cell marker), and acetylated alpha-tubulin (marker of ciliated cells) was performed as previously described43,44. Briefly, the OES were fixed (4% paraformaldehyde in PBS supplemented with 1% of bovine serum albumin (PBS-BSA), 1 h at 37°C), washed three times in PBS-BSA, and incubated in a blocking solution (10% goat serum in PBS-BSA, 30 min, room temperature). The OES were then incubated overnight at room temperature with the primary antibody (anti-cytokeratin, C2931; anti-vimentin, V6630; anti-acetylated tubulin, T7451; final concentrations of 30, 100, and 2 µg/mL, respectively). Immunostaining for acetylated alpha-tubulin (T7451; final concentration of 2 µg/mL) was performed on the sperm-OES complexes to assess the proportions of sperm bound to the ciliated cells. The control OES were incubated with IgG1 (M9269) at the same concentration as the primary antibody. After washing in PBS-BSA, the OES were incubated with the secondary antibody coupled with Alexa Fluor 488 (A11001, Invitrogen, Massachusetts, USA; final concentration at 2 µg/mL) for 1 h at room temperature under agitation.
After washing in PBS-BSA, the OES or sperm-OES complexes were stained with Hoechst (B2261, 10 µg/mL) and observed under confocal microscopy. For each antibody, a total of 100 OES from five different replicates were evaluated.
Preparation Of Spermatozoa, Assessment Of Sperm Motility, And Membrane Integrity
A pool of semen from the same ejaculates of two Normande and one Holstein bulls (Union Evolution, Noyal-sur-Vilaine, France) and previously frozen in Optidyl extender (IMV technologies, L’Aigle, France) were used for all experiments. Semen samples were thawed 3 min in a water bath at 37°C then washed on a 45%-90% Percoll gradient by centrifugation (700×g, 20 min, 25°C). An aliquot of the sperm pellet was used to assess the sperm concentration in a Thoma cell counting chamber. The sperm were then washed in 1 mL of the TLP medium for 10 min at 300×g at 25°C prior to their incubation with spheroids. Just before and at the end of each incubation, the sperm motility was assessed using computer-assisted sperm analysis (CASA, IVOS II, IMV technologies). The proportions of motile spermatozoa before and after incubation typically exceeded 70 and 60%, respectively (Supplementary Data S1). In some experiments, aliquots of spermatozoa were analysed by flow cytometry (MACSQuant Analyser 10, Miltenyi Biotec, Germany) following their incubation for 10 min at room temperature in 200 µL of TLP with 7.5 µM propidium iodide (PI, Sigma 81845) and 1 µM peanut agglutinin fluorescein (PNA, Vector FL-1071) to assess the sperm membrane integrity and acrosome reaction, respectively.
Sperm Addition To Spheroids And Quantification Of Bound Sperm Density
Two hours before the sperm addition, the OES were briefly washed in a M199 medium then transferred into 500 µL of the TLP medium at 38.8°C (with no CO2 to avoid the formation of bicarbonate).
During the Percoll centrifugation (30 min prior to sperm addition), groups of 20 OES in 10 µL of TLP medium were transferred to 50 µL of the medium (TLP or treatment) in a sterile 96-well culture plate at 38.8°C. Sperm were then added to 5 µL of TLP so that the final volume of sperm-OES incubation was 65 µL. Preliminary data using increasing concentrations of spermatozoa added to 20 OES in the TLP medium showed that the density of bound sperm reached a plateau at 1 million of spermatozoa per mL. Therefore, the final sperm concentration added to a group of 20 OES was 1 million spermatozoa per mL in a final volume of 65 µL. After the incubation, the sperm-OES complexes were gently washed three times in 500 µL of TLP medium at 38.8°C using a glass pipette (400 µm in diameter) in order to eliminate slightly attached spermatozoa, then immediately fixed in 2.5% glutaraldehyde diluted in 0.1 M sodium cacodylate (27746.180, Prolabo, France) for 1 h at 37°C. After three washes in PBS-BSA, the sperm-OES complexes were kept at 4°C overnight until further analyses. The preliminary experiments showed that this procedure did not induce sperm detachment from the OES. The nuclei of both the sperm and OES were stained with Hoechst (B2261; 10 µg/mL) for 30 min under gentle agitation at room temperature. After two washes in PBS-BSA, the OES were placed on a Superfrost slide (J1800AMNZ, Epredia, Breda, Netherlands), mounted under a coverslip, and analyzed by confocal microscopy (Zeiss LSM 700, Oberkochen, Germany). In order to visualise all sperm heads, both sides of the OES were observed under a 405 nm excitation wavelength and pictures were saved. The total number of bound spermatozoa per OES and areas of OES were quantified using the ImageJ software (version 1.53t). The bound sperm density was calculated as the total number of sperm bound on the two sides of the spheroids divided by the total area of spheroids.
Collection And Preparation Of Oviduct Fluid (Of) Samples
Bovine OF were collected from adult cyclic cows at a local slaughterhouse. Pairs of oviducts and ovaries were immediately placed on ice, transported to the laboratory, and classified according to their ovarian morphology as previously described42. Oviducts which were ipsilateral and contralateral to ovulation at either the pre-ovulatory, post-ovulatory (presence of an early corpus luteum consisting of red, loosely organised tissue of less than 1 cm in diameter, approximately days 1 to 4 of the cycle), or luteal stage of the cycle (presence of a well-formed brown or orange corpus luteum of more than 1 cm in diameter) were used. The ovaries, surrounding tissues, and infundibulum were removed before flushing with the TLP medium at 4°C. For most experiments, the whole oviducts were flushed with 500 µL of TLP. In some cases, the oviducts were cut at the ampullary-isthmus junction and each section (isthmus and ampulla) was flushed separately with 200 µL of TLP. The cells and cellular debris were discarded through two successive centrifugations (2000×g for 10 min at 4°C then 12,000×g for 10 min at 4°C). An OF aliquot was used for protein concentration using the Uptima BC Assay kit (Interchim, Montluçon, France). The protein concentrations of the OF at the collection time ranged from 1 to 4 mg/mL. The OF samples used for incubation with OES and sperm consisted of pools of OF collected from ipsi- and contralateral oviducts from two cows at the same stage of their estrous cycle and these were stored at -80°C until use (one freezing-thawing cycle before use). The final protein concentration was adjusted at the time of thawing.
To obtain the OF filtration fractions, 500 µL of pre-ovulatory OF at a final protein concentration of 1 mg/mL were centrifuged on a Vivaspin® ultrafiltration unit (3-kDa cut-off; VS0191, Sartorius, Göttingen, Germany) at 12,000×g for 1 h at 4°C. The fractions at the top (above 3 kDa) and at bottom (below 3 kDa) of the unit were collected, assayed for protein concentration, and stored at -20°C until use. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to check the efficiency of the ultrafiltration.
For protein denaturation, pre-ovulatory OF with a 1 mg/mL concentration of proteins was treated for 10 min at 100°C, then kept at room temperature for 20 min before its use. For protein digestion, pre-ovulatory OF with a protein concentration of 1 mg/mL was incubated in a TLP medium supplemented with 200 µg/mL of proteinase K (A3830, AppliChem, Germany) overnight at 56°C.
The proteinase K was discarded from the OF through ultrafiltration (Vivaspin® 50 kDa cut-off, VS0132, Sartorius, Göttingen, Germany) for 10 min at 12,000×g. A Bradford protein assay (Interchim, Montluçon, France) and SDS-PAGE were performed to assess the efficiency of the digestion and elimination of proteinase K. The proteinase K-free digested OF fraction below the 50 kDa cut-off was stored at -20°C before use.
In order to eliminate the HS from the OF, pre-ovulatory OF with 1 mg/mL of proteins was incubated in a TLP medium supplemented with a mixture of heparinases I, II, and III (H3917, H6512, v/v) with a final concentration of 0.1 UI/mL for 5 h at 37°C.
The heparinases were discarded from the OF via ultrafiltration (Vivaspin® 50 kDa cut-off, VS0132, Sartorius, Göttingen, Germany) for 10 min at 12,000×g at 4°C. SDS-PAGE was performed to assess the elimination of heparinases from the OF. The treated samples were stored at -20°C before use.
Observation Of Sperm-cilia Interactions By Scanning Electron Microscopy
Sperm-OES complexes were immersed in a fixative solution (2,5% glutaraldehyde in 0.2 M sodium cacodylate buffer, pH 7.4) and stored at 4°C until processing. The fixative was removed, and samples were rinsed in the sodium cacodylate solution (pH 7.4). They were deposited on sterile cover-glasses discs (Marienfeld, VWR, France). The samples underwent progressive dehydration by soaking in a graded series of ethanol (50 to 100%) before critical-point drying under CO2.
Samples were mounted on aluminum SEM sample stubs (15 mm diameter x 6 mm M4, Micro to Nano, Haarlem, Netherlands) with carbon adhesive discs (Agar Scientific, Oxford Instruments SAS, Gometz-la-Ville, France) and sputter coated with Gold-Palladium (Polaron SC7640, Milexia, Verrières-le-buisson, France) for 220 s at 10 mA. Samples were visualized by field emission gun scanning electron microscopy (SEM FEG). They were viewed as secondary electron (2 kV) with a Hitachi S4500 instrument (Milexia, Verrières-le-buisson, France). Samples preparation and scanning Electron Microscopy analyses were performed at the Microscopy Platform of the INRAE PACA center, Avignon, France.
Experimental Design
The general design of the experiments is presented in the Supplementary Figure S1. For all experiments, sperm motility was assessed by computer-assisted sperm analysis (CASA) at 0 and 60 min of incubation with native OF and the TLP medium as controls.
Experiment 1 evaluated the kinetics of sperm binding to OES under control conditions and with OF. The sperm and OES were incubated for 5, 15, 30, and 60 min in the TLP medium or in the presence of pre-ovulatory OF at a final protein concentration of 1 mg/mL.
Experiment 2 evaluated the effect of the stage of the cycle (pre-ovulatory vs. luteal phase of cycle) and anatomical region of the oviduct (ampulla vs. isthmus) from which the OF originated on sperm binding. For each experiment, the sperm and OES were incubated for 60 min in the presence of OF with a final protein concentration of 1 mg/mL.
Preliminary experiments showed that more than 99% cells in the OES were viable (showed a negative signal for ethidium homodimer) after 60 min in TLP and OF. The TLP medium was used as the control.
Experiment 3 evaluated the effect of ultra-filtrated fractions OF on sperm binding. The sperm and OES were incubated for 60 min in > and < 3 kDa fractions of pre-ovulatory OF. The TLP medium was used as the control.
Experiment 4 evaluated the effect of the OF proteins on sperm binding. The sperm and spheroids were incubated for 60 min with pre-ovulatory OF with 0.06, 0.125, 0.25, 0.5, 1, 2, or 4 mg/mL concentrations of proteins. The sperm and OES were incubated for 60 min in pre-ovulatory OF in which the proteins were denatured by heating or digested by proteinase K. The TLP medium and native OF were used as the controls.
Experiment 5 evaluated the effect of HA and HS on sperm binding. The sperm and OES were incubated for 60 min in pre-ovulatory OF treated with heparinases I, II, and III (OF-HS). In addition, purified HS (H7640) and HA (53747) diluted to 10, 100, and 1000 µg/mL in the TLP medium were tested. The TLP medium and native OF were used as the controls.
Each experiment was repeated at least three times with OES and OF samples from different cows.
Statistical analysis
Unless specified otherwise, the data are presented as the mean ± SEM of bound sperm density per mm2 relative to the control group in the TLP medium. The statistical analysis was performed in the GraphPad Prism software (version 8.1.1). The bound sperm densities and motility parameters for different conditions were compared using ANOVA and Tuckey’s post-tests for pair-wise comparisons. The proportions of spermatozoa observed by SEM were compared with Chi-square tests. A P-value < 0.05 was considered significant.