Ram seminal plasma proteins contribute to sperm capacitation and modulate sperm–zona pellucida interaction

Accepted Manuscript Ram seminal plasma proteins contribute to sperm capacitation and modulate sperm– zona pellucida interaction C. Luna, C. Colás, A. Casao, E. Serrano, J. Domingo, R. Pérez-Pé, J.A. CebriánPérez, T. Muiño-Blanco PII:

S0093-691X(14)00600-1

DOI:

10.1016/j.theriogenology.2014.10.030

Reference:

THE 12982

To appear in:

Theriogenology

Received Date: 17 July 2014 Revised Date:

30 October 2014

Accepted Date: 31 October 2014

Please cite this article as: Luna C, Colás C, Casao A, Serrano E, Domingo J, Pérez-Pé R, CebriánPérez JA, Muiño-Blanco T, Ram seminal plasma proteins contribute to sperm capacitation and modulate sperm–zona pellucida interaction, Theriogenology (2014), doi: 10.1016/j.theriogenology.2014.10.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Ram seminal plasma proteins contribute to sperm capacitation and modulate

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sperm–zona pellucida interaction

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Keywords: Ram spermatozoa ZBA assay Capacitation Tyrosine phosphorylation

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Departamento de Bioquímica y Biología Molecular y Celular - Instituto de Investigación en Ciencias Ambientales de Aragón (IUCA), Facultad de Veterinaria, Universidad de Zaragoza, Spain

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C. Lunaa, C. Colása,b, A. Casao, E. Serrano, J. Domingo, R. Pérez-Pé, J.A. Cebrián-Pérez, T. Muiño-Blanco*

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These authors contributed equally to this article.

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Current address: Children’s Hospital of Philadelphia, Center for Mitochondrial and Epigenomic Medicine, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Colket Translational Research Building, Room 6060, 3501 Civic Center Boulevard, Philadelphia, PA 19104-4302, USA.

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Corresponding author. Tel.: +34976761639; Fax: +34976761612; E-mail address: [email protected]

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ABSTRACT Incubation of ram spermatozoa in capacitating conditions with cAMP-elevating

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agents promotes a progressive time-dependent increase in the capacitated-sperm

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subpopulation. In this study, the fertilizing capacity of ram spermatozoa (ability to

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bind to the zona pellucida, ZBA rate) capacitated in these conditions was determined.

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The results showed an increase (P<0.001) in ZBA rate related to control samples in

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basal medium that contained BSA, calcium, and bicarbonate (1.97 ± 0.19 vs 1.31 ±

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0.09 sperm bound/oocyte, respectively). A significant correlation between protein

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tyrosine phosphorylation and ZBA rate (P<0.05, r=0.501) corroborated that

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incubation in a “high-cAMP” environment improves the fertilizing ability of ram

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spermatozoa. Likewise, the presence of two seminal plasma (SP) proteins able to

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protect sperm against cold-shock (RSVP14 and RSVP20) was evidenced in both SP

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and the ram sperm surface, and their influence in the fertilizing ability of spermatozoa

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capacitated in basal medium or with cAMP-elevating agents was determined. The

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results verified that RSVP14 and RSVP20 act as decapacitating factors given that

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their addition to SP-free sperm samples previously to capacitation maintained high

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proportions of the non-capacitated sperm pattern with no increase in protein tyrosine

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phosphorylation. However, the obtained ZBA rate in the high cAMP-containing

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samples was increased in the presence of RSVP20 (P<0.05). These findings would

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indicate that the stimulating effect exerted by this protein on the sperm-oocyte

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binding occurs downstream from the cAMP generation, and that the mechanisms by

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which RSVP20 promotes the zona-pellucida binding might be independent of protein

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tyrosine phosphorylation.

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1. Introduction

Mature spermatozoa are highly specialized cells with specific functions directed to

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fertilization. The fertilization process in mammals requires that spermatozoa bind to

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and penetrate the zona pellucida, the extracellular matrix that surrounds the oocyte.

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Although ejaculated sperm are able to bind to the zona pellucida [1, 2], their zona

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binding capacity is increased after in vitro capacitation [3]. The interaction between

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complementary molecules on the capacitated sperm surface and the zona pellucida

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starts the sequence of events leading to the acrosome reaction, an indispensible step

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for fertilization [2]. Therefore, an adequate interaction between the sperm plasma 2

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membrane and the zona pellucida is crucial for the fertilization process. Because spermatozoa are cells with a residual transcriptional activity, they

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respond to exogenous signals, including those produced by the oocyte, activating

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proteins through post-translational modifications. Protein phosphorylation is a

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post-translational modification that has been demonstrated during sperm

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capacitation in numerous mammalian species [4] and it has been suggested to be

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a prerequisite for fertilization [4].

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The involvement of the cAMP–PKA pathway in ram sperm capacitation [5] with a

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concomitant increase in protein tyrosine phosphorylation during this process [4] has

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already been shown. Bicarbonate was found necessary, whereas bovine serum

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albumin and calcium did not appear to be essential [6]. Previous results also

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suggested that the intracellular cAMP levels might be too low to initiate tyrosine

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phosphorylation of flagellar proteins, indicative of the capacitation state, which might

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be caused by unusually high levels of phosphodiesterases in ram spermatozoa [7].

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Seminal plasma (SP) of mammals is a key modulator of sperm functionality

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(reviewed by [8-10]) which exerts a decapacitating effect as proved by in vitro

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fertilization

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modifications can be prevented by the addition of SP, which accounts for an

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improvement in sperm quality parameters [12-14] and fertility following artificial

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insemination in ram [15], boar [16] and stallion [17]..SP proteins are able to protect

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[18] and repair the ram sperm membrane damage induced by cold-shock [12, 19] or

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detergent treatment. [20]. Conversely, several SP proteins have been described as

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infertility factors in horse [21], bull [22] and human [23]. Likewise, the binding of

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certain SP proteins to spermatozoa may reduce capacitation-related events [11, 24]

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and delay the subsequent acrosome reaction [25]. These proteins have been

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described as ‘‘decapacitation factors’’, and they must be removed, modified, or

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masked before spermatozoa undergo the acrosome reaction [2, 25], an essential

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process to successful fertilization.

with

boar

spermatozoa

[11].

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cryoinjury

sperm

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assays

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In a recent study, we have shown that SP proteins support survival of ram

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spermatozoa acting not only at the plasma membrane but also by inhibition of

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capacitation, and resulted in higher fertilizing ability of ram spermatozoa determined

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as ZBA (zona pellucida binding) rate [26]. In order to go further into the knowledge of

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the protective mechanism of SP proteins, this study was conducted to: 1) determine

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whether capacitation of ram spermatozoa in a medium with high intracellular cAMP 3

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levels influences ZBA rate; 2) evaluate the role of specific seminal plasma proteins

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(RSVP14 and RSVP20) in the fertilizing capacity (ZBA) of in vitro capacitated ram

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spermatozoa.

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2. Materials and methods

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2.1. Sperm preparation

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All the experiments were carried out with fresh semen obtained from eight

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mature Rasa Aragonesa rams (2–4 yr old), using an artificial vagina. All the rams

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belonged to the National Association of Rasa Aragonesa Sheep Breeders

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(ANGRA) and were housed under uniform nutritional conditions at the

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Experimental Farm of the University of Zaragoza in compliance with the

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requirements of the European Union Directive for Scientific Procedures. The sires

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were kept apart, and semen was collected every 2 days, in two successive

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matings each day. Under these conditions, and using second ejaculates, individual

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differences are very low, as already reported [27], and pooled ejaculates provide a

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good quality, uniform sperm sample suitable for representative studies of ram

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semen.

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A seminal plasma-free sperm population was obtained by a dextran/swim-up

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procedure [28] performed by using a swim-up medium (SM) consisted of 200 mM

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sucrose, 50 mM NaCl, 18.6 mM sodium lactate, 21 mM HEPES, 10 mM KCl, 2.8 mM

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glucose, 0.4 mM MgSO4, 0.3 mM sodium pyruvate, 0.3 mM K2HPO4, 5 mg/mL of

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BSA 1.5 IU/mL penicillin, and 1.5 mg/mL streptomycin, pH 7.2 (adjusted by NaOH

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addition), and devoid of CaCl2 and NaHCO3 [6]..

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2.2. Assessment of standard semen parameters Sperm concentration was calculated in duplicate using Neubauer’s chamber

(Marienfeld, Lauda-Königshofen).

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Cell viability (membrane integrity) was assessed by flow cytometry analysis on

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a Beckman Coulter FC 500 (IZASA, Barcelona, Spain) with CXP software,

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equipped with two lasers of excitation (Argon ion laser 488 nm and solid state

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laser 633 nm) and five filters of absorbance (FL1-525, FL2-575, FL3-610, FL4-675

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and FL5-755; ±5 nm each band pass filter). At a minimum, 20000 events were

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counted in all samples. The sperm population was gated for further analysis on 4

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the basis of its specific forward (FS) and side scatter (SS) properties and other

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non-sperm events were excluded.

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second was used. Three microliters of each stain, carboxyfluorescein diacetate

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(CFDA; 1 mM; Sigma Chemical Co., Madrid, Spain) and propidium iodide (PI;

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0.75 mM; Sigma Chemical Co., Madrid, Spain) were added to 400 µl of sperm

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samples (final concentration of 8 x 106 cell/mL), based on a modification of the

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procedure described by Harrison and Vickers [29]. Samples were incubated at

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37 °C in darkness for 15 min. The argon laser and filters of 525 and 675 nm were

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used to avoid overlapping.

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(CFDA) and FL4 (PI).

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A flow rate stabilized at 200-300 cells per

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Monitored parameters were FS log, SS log, FL1

The capacitation status was determined by means of the chlortetracycline

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(CTC) fluorescence assay, previously validated for the evaluation of capacitation

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and acrosome reaction-like changes in ram semen [6]. Three sperm types were

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estimated [30]: noncapacitated (NC, even distribution of fluorescence on the head,

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with or without a bright equatorial band), capacitated (C, with fluorescence in the

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anterior portion of the head), and acrosome-reacted cells (AR, showing no

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fluorescence on the head). The samples were examined within 12 hours using a

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Nikon Eclipse E-400 microscope under epifluorescence illumination with a V- 2A

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filter. All samples were processed in duplicate, and at least 150 spermatozoa were

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scored per slide. No fluorescence was observed when CTC was omitted from the

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preparation.

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2.3. In vitro capacitation with RSVP14 and RSVP20 For the induction of in vitro capacitation, aliquots of 1.6 x 108 cells/mL were

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incubated for 3 hours at 39 ºC in a humidified incubator with 5% CO2 in air.

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Incubations were performed in complete TALP medium [31] containing 100 mM

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NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.3 mM NaH2PO4, 21.6 mM Na lactate, 3 mM

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CaCl2, 0.4 mM MgCl2, 10 mM HEPES, 1 mM Na pyruvate, 5 mM glucose, and 5

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mg/mL bovine serum albumin, pH 7.2 (adjusted using NaOH). This sample was used

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as control of capacitation.

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To evaluate the role of the cAMP-PKA pathway, the effects of a cocktail already

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proved for capacitating ram spermatozoa [7] composed of dibutyryl-cAMP (db-cAMP,

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Sigma Chemical Co., Madrid, Spain; 1 mM), caffeine and theophylline (both inhibitors

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of phosphodiesterases, Sigma Chemical Co., Madrid, Spain; 1 mM each), okadaic 5

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acid (OA, a broad spectrum phosphatase inhibitor, Sigma Chemical Co., Madrid,

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Spain; 0.2 µm), and methyl-β-cyclodextrin (M-β-CD, Sigma Chemical Co., Madrid,

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Spain; 2.5 mM) were tested. To analyse the involvement of two specific ram seminal plasma protein fractions

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(RSVP14 and RSVP20) on the ability to bind to the zona pellucida (ZBA rate), we

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added 400 µg and 800 µg of each fraction to the control and high cAMP-containing

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samples (final volume and sperm concentration of aliquots as indicated above). The

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protein fractions were added immediately after preparing the aliquots (0 hours), and

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the samples were incubated for 3 hours simultaneously with the controls.

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Ovaries of lamb ewes were collected in the slaughterhouse and transported to the

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laboratory in sodium chloride 0.9% at room temperature (RT). In the laboratory,

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ovaries were freed from surrounding tissues and blood vessels, washed twice in

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saline and frozen at -20 ºC until use.

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Lamb ewes ovaries were used given their high availability in the slaughterhouse,

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and because there are not significant differences in terms of fertilization between

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adult and prepubertal ewe oocytes [32] and the mean number of oocytes collected by

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a single ovary is significantly higher in prepubertal ewes than in adult ewes [33].

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Although there is not influence in the final result of the binding assay [34], all the

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ovaries were from lambs of the same farmer.

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The day of the assay, ovaries were thawed at RT, and washed again in saline

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buffer. Oocytes were collected by slicing techniques: ovaries were placed in a Petri

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dish and covered partially with handling medium (Hepes buffered TCM-199, 0.1%

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polyvinyl alcohol (PVA), 0.04% NaHCO3, 25 UI/mL of heparin and 100 UI/mL of

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penicillin G and 100 µg/mL of streptomycin sulphate), and all ovary surface was

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sliced with an scalpel blade. The rest of ovaries and tissues were removed, and

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oocytes were identified and transferred to another Petri dish containing handling

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medium.

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Oocytes without cumulus cells and intact zona pellucida were selected, and

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washed twice in handling medium and once in pre-equilibrated bicarbonate-buffered

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synthetic oviduct fluid medium (SOF, [35] with 2% FBS added (fertilization medium).

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Oocytes were randomly distributed in groups, and placed in wells of a four-well

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Petri dish, with 400 µl of fertilization medium, between 10 – 15 oocytes per well, and

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kept at 39 ºC and 5% CO2 in an humidified atmosphere until fertilization.

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2.5. Zona binding assay Sperm samples were diluted in fertilization medium (5 x 105 spermatozoa/mL) and

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100 µl were added to the oocytes. Wells with the mixture of gametes were covered

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with mineral oil, and kept for one hour in an humidified atmosphere, with 5% CO2 at

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39 ºC.

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After incubation, oocytes were placed in a Petri dish with handling medium, and

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washed by gentle pipetting to remove unattached spermatozoa [36]. Oocytes were

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washed again in handling medium, and fixed in glutaraldehyde 1.5% for 15 minutes,

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then washed in saline and stained with Hoechst 33342 (1 µg/mL) for 15 minutes at 37

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ºC. Oocytes were washed again in saline, to remove the excess of stain, and groups

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of 5–6 oocytes were placed in a slide under a coverslip and examined with a

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fluorescence microscope at 400X. The number of zona pellucida attached

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spermatozoa per oocyte were counted and recorded.

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2.6. Seminal plasma protein preparation and exclusion chromatography

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Seminal plasma (SP) was obtained by spinning 1 mL of semen at 12 000 xg for

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5 minutes at 4 ºC. The supernatant was centrifuged again, and 400 µl of undiluted

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seminal plasma were taken and, after filtering through a 0.22-µm membrane, kept

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at -20 ºC.

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Whole SP proteins were obtained [12] by filtering the seminal plasma through

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Microsep microconcentrators (Filtron Tech, Northborough, MA) of a 3-kDa

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molecular weight cut-off, spinning for 6 hours at 3 000 xg at 4 ºC. The obtained

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sample was diluted with five volumes of a medium containing 0.25 M sucrose, 0.1

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mM EGTA, 4 mM sodium phosphate (pH 7.5), 10% (v/v) of 10 x buffer stock

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Hepes (50 mM glucose, 100 mM Hepes, 20 mM KOH) and then centrifuged again,

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after which the seminal plasma proteins were recovered and stored at -20 ºC.

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Protein concentration was assessed according to the method described by

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Bradford [37].

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For Sephacryl-100 HR Chromatography, 40 mg of SP proteins were loaded (0.1

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mL/minute) on a 1.6- x 90-cm Sephacryl-100 high-resolution column (XK 16/100 7

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phosphate buffer (pH 8) containing 0.2 M NaCl. The column was then washed at

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0.1 mL/minute with 120 mL of equilibration buffer. Fractions (1 mL) were collected

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at the same flow rate. During purification, protein concentration in each fraction

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was estimated by monitoring the absorbance at 280 nm. Pooled fractions were

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thoroughly dialyzed against distilled water containing 0.1% sodium azide and kept

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frozen (dried) until use.

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2.7. Extraction of ram sperm proteins

Aliquots of 3.2 x 107 cells of control or cAMP-capacitated samples were

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resuspended in 100 µl of the same extraction medium previously used [7] composed

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of 2% SDS (w/v), 0.0626 mM TRIS-HCl (pH 6.8), 0.002% bromophenol blue in 10 %

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glycerol (final glycerol concentration 1%), and protease and phosphatases inhibitors

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(Sigma Chemical Co., Madrid, Spain), and immediately incubated for 5 minutes at

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100 ºC. After centrifugation at 7500 xg for 5 minutes at RT, the supernatant was

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recovered and 2-mercaptoethanol and glycerol were added to a final concentration of

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5% and 1%, respectively. The protein concentration was determined using the

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Bradford assay [37] and lysates were stored at -20 ºC.

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2.8. SDS–PAGE and immunoblotting Sperm extracted proteins (20 µl) were separated in one dimension on 14%

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SDS–PAGE for tyrosine phosphorylated proteins detection and on 18% for

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identification of seminal plasma proteins RSVP14 and RSVP20, following the

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Laemmli method [38] using a mini protean II vertical slab gel electrophoresis

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apparatus (Bio-Rad, Hercules, CA). The samples containing 15 µg of proteins

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were diluted 4:1 v/v with the sample buffer (10% glycerol, 3% SDS, 0.045 M Tris-

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HCl [pH 8.0], 5% 2-mercaptoethanol, 0.8 mM EDTA, and 0.004% bromophenol

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blue) and heated for 5 minutes at 95 ºC. Electrophoresis was performed for 1 h

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and 30 min at 130 V at 4 ºC. A mixture of prestained molecular weights ranging

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from 10 to 250 kDa (Bio-Rad, Hercules, CA) was used as a standard. Gels were

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stained with 0.1% Coomassie R (Serva, Heidelberg, Germany).

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For western-blot analysis, separated proteins were blotted onto 0.2 µm

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polyvinylidene fluoride (PVDF) membranes (BioRaD, Hercules, CA) at 2.5 A

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constant up to 25 V, 10 minutes, using the Trans-Blot Turbo unit (Trans-Blot®

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TurboTM Transfer System, BioRad, Hercules, CA). For the detection of phosphorylated proteins, the blots were incubated as

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previously described [39]. Non-specific sites on the membranes were blocked for 1

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hour with 5% BSA (w/v) in phosphate-buffered saline (PBS, 136 mM NaCl, 0.2 g/l

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KCl, 1.44 g/l Na2HPO4, and 0.24 g/l KH2PO4, pH 7.4) at RT. Then, the blots were

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incubated with the mouse monoclonal anti-phosphotyrosine antibody (Monoclonal

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Antibody, clone 4G10®, Millipore, Temecula, CA), diluted 1:1000, overnight at 4 ºC,

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followed by incubation for 1 hour at RT with a secondary anti-mouse horseradish

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peroxidase HRP-conjugated IgG antibody (1/40000; GE Healthcare-Amersham, Little

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Chalfont, UK). After extensive washing, the proteins that bound the antibody were

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visualized by chemiluminescence procedures (Pierce® ECL Western Blotting

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Substrate; ThermoFisher Scientific, Waltham, MA USA). Western-blot images were

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quantified using Quantity One software (Bio Rad, Hercules, CA, USA) to determine

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the peak intensity of the tyrosine-phosphorylated protein bands. The total intensity

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signal of each lane was evaluated as the summatory of the peak intensity of all bands

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detected in the lane. To avoid the high differences due to the western development of

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each individual experiment, the corresponding controls were always included in each

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blot. The experiment was performed four times, and presented data are means ±

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standard error of relative intensity units.

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To identify seminal plasma proteins, non-specific binding sites on membranes

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were blocked for 1 hour with 5 % BSA (w/v) in phosphate-buffered saline (PBS).

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Incubations with the primary antibody (rabbit anti-RSVP14 and rabbit anti-RSVP20,

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diluted 1/60000 with 0.5 % BSA [40]), were performed overnight at RT, followed by

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incubation for 1 hour at RT with the secondary anti-rabbit horseradish peroxidase

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(HRP)-linked antibody, diluted 1/15000 with 0.5 % BSA. After primary and secondary

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antibody incubations, extensive washes were carried out to eliminate unspecific

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binding. The proteins that bound the antibody were visualized and quantified as

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described above for phosphotyrosine blots.

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To prove that the signal was specific, western blotting omitting either primary or secondary antibodies was performed.

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2.9. Statistical analysis Results are shown as mean ± SEM of the number of samples indicated in each

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case. Statistical analyses were performed to determine whether there were

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significant differences between samples. Data distribution was analyzed by the

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Kolmogorov-Smirnov test. Differences between experimental groups were analyzed

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by means of ANOVA, and post hoc comparisons were made using the Student-

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Newman-Keuls Multiple Comparisons Test. A p value of ≤ 0.05 was considered

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statistically significant. The GraphPad InStat software (3.01; San Diego, CA, USA)

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was used for CTC staining and tyrosine phosphorylation blots, whereas the SPSS

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software 15.0 was used for analysis of zona pellucida-binding assays.

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Correlations between tyrosine phosphorylation and zona pellucida-binding were calculated using Pearson’s correlation test (GraphPad InStat software).

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3. Results

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3.1. In vitro capacitation of ram spermatozoa increases the zona pellucida-binding

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ability

The sperm number bound per oocyte (ZBA rate, Table 1) of high cAMP-

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capacitated samples was significantly higher than controls after 3 h of incubation,

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while no significant difference was found at the beginning of incubation (0 h). Longer

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incubation time resulted in decreased zona pellucida binding ability of both samples,

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with a higher effect in the high cAMP-containing samples.

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Viability assays revealed that 4 h of incubation resulted in a significant decrease in

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membrane integrity in the high-cAMP containing samples, while no significant

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decrease was found in control samples (Table 2). Therefore, 3 h of incubation was

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established for further experiments.

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3.2. Protein tyrosine phosphorylation correlates with zona pellucida-binding rate

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Protein tyrosine phosphorylation was associated to the fertilizing ability as a

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positive correlation (r=0.5014) between the western-blot total signal quantified in

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each lane in all assayed samples (Fig.1) and ZBA rate was found (Fig. 2).

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3.3. Identification of RSVP14 and RSVP20 in seminal plasma and sperm surface Fig. 3A shows SDS-PAGE analysis of two fractions isolated from SP by

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exclusion chromatography, described as able to revert the cold-shock sperm

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membrane damage, F6 (RSVP20) and F7 (RSVP14) [41]. In order to evidence the

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presence of these SP proteins in both seminal plasma and the ram sperm surface,

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western-blot assays were carried out. Fig. 3B and 3C show representative

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membranes of several assays carried out in different months along the year, which

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proved that the presence of PRSV14 and RSVP20 in the ram sperm surface is

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fairly constant (data not shown). The average value of total peak intensity

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quantified by densitometry was not significantly different in reproductive and non-

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reproductive seasons, 168.85 ± 5.25 and 159.77 ± 15.70 for RSVP14, and 129.11

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± 13.57 and 171.01 ± 30.03 for RSVP20 (n=3).

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3.4. Effect of specific SP protein fractions on zona pellucida-binding ability In vitro capacitation with cAMP-elevating agents resulted in higher sperm number

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bound per oocyte (ZBA rate) (Table 3) with only a tendency to significance (P=0.052).

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The addition of RSVP20 before capacitation resulted in increased ZBA rate in the

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high-cAMP containing samples, while no significant effect was found with RSVP14.

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CTC analysis revealed that both RSVP14 and RSVP20 were able to maintain a

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higher proportion of non-capacitated sperm pattern during incubation in capacitating

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conditions of both controls and with cAMP-elevating agents samples (Fig. 4A).

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However, the addition of these SP proteins did not result in any significant change in

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either the phosphorylation of specific proteins (Fig. 4B) or the average value of total

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peak intensity.

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4. Discussion

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vitro capacitation have been extensively studied in several mammalian species [5,

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42, 43]. It has been already proved that the cAMP–PKA pathway is at least

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partially involved in ram sperm capacitation, and that protein tyrosine

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phosphorylation is associated to this process [6, 24]. Furthermore, changes in the

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content and localization of proteins phosphorylated at serine, threonine and

Regulation of sperm protein tyrosine phosphorylation and changes during in

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ACCEPTED MANUSCRIPT tyrosine residues during in vitro ram sperm capacitation and acrosome reaction

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have been shown [44]. However, in that study the capacitation medium contained

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NaHCO3, CaCl2 and BSA, as the one used for the control samples in the present

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study (basal conditions) in which a more effective capacitation was achieved using

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a cocktail containing cAMP-elevating agents and methyl-β-cyclodextrins (M-β-CD)

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as already reported [7, 20, 39, 45, 46]. Given that M-β-CD treatment did not

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stimulate major increases in protein tyrosine phosphorylation that takes place

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independently of cholesterol efflux [7], in this study the cocktail is designated as

353

“cAMP-elevating agents” or “high-cAMP containing samples”. The results obtained

354

show that capacitation of ram spermatozoa in this medium resulted in increased

355

(P<0.001) sperm ability to bind to the zona pellucida. Sperm-zona pellucida

356

binding assay has been well established as an indicator of the fertilizing capacity

357

of spermatozoa [47, 48]. The effective binding can reflect multiple sperm functions

358

such as viability, motility, morphology, acrosome status, and the ability to

359

penetrate the oocyte [49], and it has also been associated with increased embryo

360

quality after in vivo fertilization [50]. Our data support an association between

361

protein tyrosine phosphorylation during capacitation and the ability of ram

362

spermatozoa to bind to the oocyte. This relationship is consistent with previous

363

results in human [51, 52] and mouse [53] spermatozoa that reported the indirect

364

involvement of phosphotyrosine expression in sperm-egg recognition. These

365

findings suggest that tyrosine phosphorylation can be a good indicator of the

366

sperm ability to bind to the zona pellucida and enhance the significance of tyrosine

367

phosphorylation in sperm-oocyte binding.

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The results of western-blot analysis revealed the presence of two SP protein

369

fractions able to protect sperm against cold-shock (RSVP14 and RSVP20, [41]) in

370

seminal plasma and in sperm extracted-proteins. The antibodies used were raised

371

in our lab against these proteins recovered from a non-denaturing gel, and their

372

specificity is not absolute. The ”doublet” of 20-22 kDa recognised by the anti-

373

RSVP20 antibody is consistent with results of previous studies showing that the

374

20- and 22- kDa bands tended to migrate together and were recovered untidily by

375

electroelution from the gel to obtain the band initially identified as P20 [40] and

376

lately named RSVP20 [41].

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It has been postulated that due to the presence of FN2 domains (Fibronectin

378

Domain Type II), certain ram SP proteins may take part in the protein structure 12

ACCEPTED MANUSCRIPT surrounding the spermatozoa [10, 40]. Recent studies have shown that the

380

incubation of ram spermatozoa freed from seminal plasma by swim-up with whole

381

SP proteins stabilizes the sperm membrane and results in lower ZBA rate [26]. In

382

this study, the influence of RSVP14 and RSVP20 on the fertilizing ability of ram

383

spermatozoa was determined using samples capacitated in a basal medium (with

384

calcium, bicarbonate and BSA) or in a medium with cAMP-elevating agents. The

385

results verified that RSVP14 and RSVP20 act as decapacitating factors given that

386

their addition previously to capacitation maintained high proportions of non-

387

capacitated sperm pattern with no change in protein tyrosine phosphorylation.

388

However, the ZBA rate was increased in the presence of RSVP20 in the high-

389

cAMP containing samples (P<0.05), while no significant increase with RSVP14

390

was found. The increase in ZBA rate is apparently inconsistent with the CTC

391

results. The possibility that RSVP14 and RSVP20 might interfere with the CTC

392

staining must be ruled out given that no increase in protein tyrosine

393

phosphorylation was either found. The explanation for the RSVP20 ability to

394

increase ZBA rate despite of no increase in phosphorylation might be the dual

395

effect of SP proteins that would maintain the sperm membrane structure until it

396

receives the adequate effector stimuli that would trigger the physiological

397

processes leading to oocyte binding. Thus, capacitation would not occur until the

398

appropriate time, nearby the oocyte. When this happens, SP proteins would also

399

be involved in changes related with capacitation, as it was deduced in previous

400

studies that showed the partial release and relocation of these proteins from the

401

membrane during in vitro capacitation [40], in agreement with previous results [54].

402

Taken together, the results obtained suggest that these SP proteins might

403

stabilize the sperm plasma membrane, delaying their progression towards the

404

acrosome-reacted state, thus maximising the possibility of binding to the oocyte,

405

and that a procapacitating environment gives rise to changes in the SP protein-

406

sperm-oocyte interactions. These data corroborate our previous results, which

407

showed that the addition of SP proteins to ram spermatozoa resulted in better

408

survival rates, and that the protective effect of SP proteins is related to the sperm

409

membrane capacitation status [26]. This appreciation is consistent with previous

410

studies which postulated that ram SP proteins may bind to the sperm surface at

411

ejaculation, acting as decapacitating factors, stabilizing membrane phospholipids;

412

and later, in the female tract, they could participate in membrane modification

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ACCEPTED MANUSCRIPT during capacitation [10, 40], as also suggested for BSP proteins [55]. The

414

stimulating effect exerted by RSVP20 on sperm-oocyte binding in high-cAMP

415

capacitated samples, with no further increase in either capacitated pattern or

416

tyrosine phosphorylation would indicate that it occurs downstream from the cAMP

417

generation. Furthermore, our results suggest that although the acquisition of

418

sperm-oocyte binding competence is associated with an increase in protein

419

tyrosine phosphorylation, the mechanisms by which RSVP20 promotes the zona

420

binding might be independent of protein tyrosine phosphorylation. The possibility

421

that adding a high concentration of Fibronectin Type II proteins might increase

422

non-specific binding between sperm and the zona-pellucida cannot be excluded.

423

Differences in the RSVP14 and RSVP20 structure might substantiate the different

424

effect found in ZBA rate.

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In conclusion, the results of this study indicate that RSVP14 and RSVP20 are

426

able to stabilize the sperm plasma membrane and that RSVP20 is involved in

427

gamete interaction. Whether this SP proteins effect is only mediated by plasma

428

membrane events or they can also exert a direct interaction with intracellular

429

targets still remains to be determined. Characterization of the molecular pathway

430

involved in the dual SP protein effect, membrane stabilizing and zona pellucida

431

binding stimulating, may help to understand the biochemical mechanisms involved

432

in the action of seminal plasma and its components, sperm capacitation and,

433

ultimately, fertility.

434

Acknowledgments

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Supported by grants CICYT-FEDER AGL 2011–25850 and DGA A- 26FSE.

436

C. L. was financed by FPU AP2009-1298 and E. S. by FPI BES-2012-053094

437

fellowships (MEC). The authors thank ANGRA for supplying the sires and S.

438

Morales for the collection of semen samples.

439

Competing interests

440

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There is no conflict of interest of any kind whatsoever in this work.

441

References

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[3] Topper EK, Killian GJ, Way A, Engel B, Woelders H. Influence of capacitation and fluids from the male and female genital tract on the zona binding ability of bull spermatozoa. J Reprod Fertil. 1999;115:175-83. [4] O'Flaherty C, de Lamirande E, Gagnon C. Positive role of reactive oxygen species in mammalian sperm capacitation: Triggering and modulation of phosphorylation events. Free Radical Bio Med. 2006;41:528-40. [5] Sakkas D, Leppens-Luisier G, Lucas H, Chardonnens D, Campana A, Franken DR, et al. Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding. Biol Reprod. 2003;68:1463-9. [6] Grasa P, Cebrian-Perez JA, Muino-Blanco T. Signal transduction mechanisms involved in in vitro ram sperm capacitation. Reproduction. 2006;132:721-32. [7] Colas C, James P, Howes L, Jones R, Cebrian-Perez JA, Muino-Blanco T. Cyclic-AMP initiates protein tyrosine phosphorylation independent of cholesterol efflux during ram sperm capacitation. Reprod Fertil Dev. 2008;20:649-58. [8] Caballero I, Parrilla I, Alminana C, del Olmo D, Roca J, Martinez EA, et al. Seminal Plasma Proteins as Modulators of the Sperm Function and Their Application in Sperm Biotechnologies. Reprod Domest Anim. 2012;47:12-21. [9] Leahy T, Gadella BM. Capacitation and Capacitation-like Sperm Surface Changes Induced by Handling Boar Semen. Reprod Domest Anim. 2011;46:7-13. [10] Muiño-Blanco T, Perez-Pe R, Cebrian-Perez JA. Seminal plasma proteins and sperm resistance to stress. Reproduction in domestic animals = Zuchthygiene. 2008;43 Suppl 4:18-31. [11] Suzuki K, Asano A, Eriksson B, Niwa K, Nagai T, Rodriguez-Martinez H. Capacitation status and in vitro fertility of boar spermatozoa: effects of seminal plasma, cumulus-oocyte-complexes-conditioned medium and hyaluronan. Int J Androl. 2002;25:84-93. [12] Barrios B, Pérez-Pé R, Gallego M, Tato A, Osada J, Muiño-Blanco T, et al. Seminal plasma proteins revert the cold-shock damage on ram sperm membrane. Biol Reprod. 2000;63:1531-7. [13] Colas C, Junquera C, Perez-Pe R, Cebrian-Perez JA, Muino-Blanco T. Ultrastructural study of the ability of seminal plasma proteins to protect ram spermatozoa against cold-shock. Microsc Res Tech. 2009;72:566-72. [14] Maxwell WMC, de Graaf SP, Ghaoui RE-H, Evans G. Seminal plasma effects on sperm handling and female fertility. Soc Reprod Fertil Suppl. 2007;64:13-38. [15] Maxwell WMC, Evans G, Mortimer ST, Gillan L, Gellatly ES, McPhie CA. Normal fertility in ewes after cervical insemination with frozen-thawed spermatozoa supplemented with seminal plasma. Reprod Fert Develop. 1999;11:123-6. [16] Rozeboom KJ, Troedsson MH, Hodson HH, Shurson GC, Crabo BG. The importance of seminal plasma on the fertility of subsequent artificial inseminations in swine. J Anim Sci. 2000;78:443-8. [17] Alghamdi AS, Foster DN, Troedsson MH. Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri. Reproduction. 2004;127:593-600. [18] Perez-Pe R, Cebrian-Perez JA, Muino-Blanco T. Semen plasma proteins prevent cold-shock membrane damage to ram spermatozoa. Theriogenology. 2001;56:425-34. [19] García-López N, Ollero M, Cebrián-Pérez JA, Muiño-Blanco T. Reversion of thermic-shock effect on ram spermatozoa by adsorption of seminal plasma

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ACCEPTED MANUSCRIPT [53] Asquith KL, Baleato RM, McLaughlin EA, Nixon B, Aitken RJ. Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition. Journal of Cell Science. 2004;117:3645-57. [54] Desnoyers L, Manjunath P. Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid. The Journal of biological chemistry. 1992;267:10149-55. [55] Manjunath P. Gonadotropin-Release Stimulatory and Inhibitory Proteins in Bull Seminal Plasma. J Steroid Biochem. 1984;20:1585-.

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ACCEPTED MANUSCRIPT Table 1. Effect of cAMP-elevating agents on zona pellucida-binding ability. Sperm number bound per oocyte (mean ± SEM). nº indicates the number of oocytes in each assay, n=3. Significant differences related to the same sample at 0 h (*, P<0.05; **, P<0.01). Significant difference between control and high-cAMP conditions at 3 h (


, P<0.001). 0h 3h 4h nº

nº

nº

Control

52 0.99 ± 0.12

41 1.31 ± 0.09 **

52

1.22 ± 0.11

High-cAMP

56 1.11 ± 0.09

40 1.97 ± 0.19 *,




56

1.55 ± 0.21

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ACCEPTED MANUSCRIPT 611 612 Table 2. Effect of incubation in capacitating conditions on sperm membrane integrity in control and cAMP-containing samples. Mean value ± SEM, n=7. Significant differences related to the same sample at 0 h (**, P<0.01). Significant difference between control and high-cAMP conditions (
P<0.05).

0h

3h

Control

60.33 ± 3.25

56.16 ± 2.99

High-cAMP

66.66 ± 3.53

43.83 ± 6.17**,


50.05 ± 3.61

36.28 ± 3.62**,


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ACCEPTED MANUSCRIPT Table 3. Effect of specific SP protein fractions on the sperm fertilizing potential (zona pellucida binding assay). 800 µg of RSVP14 or RSVP20 were added to control and high-cAMP containing samples before incubation in capacitating conditions for 3 hours. Sperm number bound per oocyte (mean ± SEM). nº indicates the number of oocytes in each assay, n=3. Significant difference (within a column, * P<0.05) between control and high-cAMP conditions with 800 µg of RSVP20. A tendency to significance (within a column, P=0.052) between control and high-cAMP conditions in the absence of SP proteins. No SP protein RSVP14 RSVP20 nº

nº

nº

Control

47

0.95 ± 0.20

48

2.35 ± 0.56

49

1.08 ± 0.24

High-cAMP

52

1.88 ± 0.39

48

2.40 ± 0.49

50

2.52 ± 0.48*

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619 620 621 622 623 624 625

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Figure Legends

628

Fig. 1. Phosphotyrosine proteins evaluated by western blotting (A) and quantified

629

by densitometry (B). Mean values of total peak intensity in each lane ± SEM (n=6).

630

Significant differences related to the same sample at 0 h or to control at 3 h: *** P

631

< 0.0001.

632

Figure 2. Correlation between ZBA rate (sperm bound/oocyte) and total tyrosine

633

phosphorylation (expressed as total peak intensity in each lane) in all assayed

634

samples (controls and after incubation in capacitating conditions), (n=16).

635

Fig. 3. A) Coomassie brilliant blue-stained protein bands in ram SP fractions

636

separated by SDS-PAGE. Each lane was loaded with 15 µg of protein. Lane 1:

637

molecular weight markers; lane 2: F6 (RSVP20); lane 3: F7 (RSVP14).

638

Identification of RSVP14 (B) and RSVP20 (C) in seminal plasma (lane 1) and in

639

sperm extracted-proteins (lane 2) by western-blot with polyclonal antibodies raised

640

against RSVP14 and RSVP20, respectively.

641

Fig. 4.- Effect of RSVP14 and RSVP20 on the capacitation state, evaluated by

642

CTC staining (A) Control and high-cAMP containing samples were incubated with

643

400 or 800 µg of either RSVP14 or RSVP20 in capacitating conditions for 3 h.

644

Mean values (%) ± SEM (n=5). Percentage of on capacitated (NC), capacitated

645

(C) and acrosome reacted (AR) spermatozoa. Significant differences in the

646

percentage of non capacitated spermatozoa in the presence of RSVP14 or

647

RSVP20 related to the same condition (control or high-cAMP) with no fraction

648

added:

649

and P < 0.001. Protein tyrosine phosphorylation (B) quantified by densitometry

650

(C): Average values of the total peak intensity signal of each lane ± SEM (n=4).

651

a

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P < 0.05; bP < 0.01 and cP < 0.001 and to the control at 0 h:

d

P < 0.05

e

652

22

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ACCEPTED MANUSCRIPT HIGHLIGHTS

We evidence two seminal plasma (SP) proteins, RSVP14 and RSVP20, in SP and the ram sperm surface.

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The addition of RSVP20 increases the sperm ability to bind to the zona pellucida (ZBA rate).

The results verify that RSVP14 and RSVP20 act as decapacitating factors. We propose a dual SP protein effect, membrane stabilizing and ZBA rate stimulating.

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This study may help to understand the molecular mechanisms involved in the action of SP.