Epididymal α-l -fucosidase and its possible role in remodelling the surface of bull spermatozoa

Accepted Manuscript Epididymal α-L-fucosidase and its possible role in remodelling the surface of bull spermatozoa Andrea Carolina Aguilera, Veronica Boschin, Inmaculada Robina, Pilar ElíasRodríguez, Miguel Angel Sosa PII:

S0093-691X(17)30401-6

DOI:

10.1016/j.theriogenology.2017.08.016

Reference:

THE 14236

To appear in:

Theriogenology

Received Date: 9 April 2017 Revised Date:

7 August 2017

Accepted Date: 13 August 2017

Please cite this article as: Aguilera AC, Boschin V, Robina I, Elías-Rodríguez P, Sosa MA, Epididymal α-L-fucosidase and its possible role in remodelling the surface of bull spermatozoa, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.08.016. 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.

Revised

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Running head: α-L-fucosidase in bull epididymis

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Epididymal α-L-fucosidase and its possible role in remodeling the surface of bull spermatozoa

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Andrea Carolina Aguileraa,b, Veronica Boschina, Inmaculada Robinac, Pilar Elías-Rodríguez c and Miguel Angel Sosaa,b.

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Histología y Embriología – IHEM-CONICET-FCM-UNCuyo. 5500- Mendoza, Argentina. b

Facultad de Ciencias Exactas y Naturales. Universidad Nacional de Cuyo.

Mendoza, Argentina

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c

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Sevilla. Spain.

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(‡) Correspondence: Dr. Miguel A. Sosa, IHEM-CONICET, Telephone: +54

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Departamento de Química Orgánica, Facultad de Química, Universidad de

216 4135000 (extension 2677), E-mail: [email protected].

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Laboratorio de Biología y Fisiología Celular “Dr. Franciso Bertini”. Instituto de

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Key words: α-L-fucosidase, bull epididymis, glycosidases, spermatozoa. α

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Abstract The mammalian epididymis provides an appropriate environment for sperm

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maturation. During the epididymal transit, spermatozoa undergo biochemical and

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morphological changes that lead to the acquisition of the fertilizing capacity. In this

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study we analysed the fucosylation status of membrane glycoproteins in the

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spermatozoa obtained from different regions of the bull epididymis. High amounts of

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fucose were detected on caput spermatozoa (R.F.I.= 1010 ± 20.35), mostly located in

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the post-acrosome zone. A significant decrease in the fucose levels was detected

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toward the cauda (R.F.I.= 540.5 ± 49.93) (P<0.05). This decrease was in line with the

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increased activity of α-L-fucosidase in the cauda fluid. In sperm from the cauda, the

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defucosylation occurred in some proteins, whereas others showed higher

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fucosylation rates. A significant decrease of fucose in the gametes was observed

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upon incubation of crude cauda fluid with caput spermatozoa (from R.F.I.= 1.45 ±

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0.08 to 1.06 ± 0.03) (P<0.05) indicating that the α-L-fucosidase present in the

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epididymal fluid is active on spermatozoa. Moreover, this effect was blocked with

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specific enzyme inhibitors. These results provide direct evidence that the α-L-

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fucosidase from epididymal fluid participates in the fucose removal from

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spermatozoa, as a step of sperm maturation in the bull epididymis.

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1.

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Introduction

The mammalian epididymis plays an important role in the acquisition of motility

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and the fertilizing capacity of spermatozoa [1]. During the passage through the

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epididymis, spermatozoa undergo biochemical and morphological changes, resulting

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in a gamete that is competent for fertilization. These changes include adsorption of

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epididymal proteins on the sperm surface, a molecular reorganization of the

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plasmalemma and changes in the composition of glycoconjugates on the cell surface

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[2-7]. Thus, the maturation of spermatozoa in the epididymis is the result of

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sequential interactions with proteins secreted by the epithelium along the epididymal

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duct [8-13]. Among the several proteins secreted by the epididymal epithelium, acid

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glycosidases can be found at high concentrations in the epididymal fluid of different

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mammalian species [2,5,6,14-19]. Although the pH of the epididymal lumen is not

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optimum for acid hydrolase activity [20], it has been demonstrated that some

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enzymes, such as β-galactosidase is active in the fluid of the rat epididymis [21].

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However, other functions for those glycosidases could be attributed from their

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interaction with specific receptors found in the plasmalemma of spermatozoa [7,22]

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Glycosidases have also been found in the epididymis of domestic animals, such as bulls [23]. Among them, α-L-fucosidase (α-FUC) appears to be important for

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reproduction in bulls, since it was prevalent in the cauda epididymal fluid of highly

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fertile animals [24]. Moreover, dogs with severe deficiency of α-FUC have shown an

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impaired sperm maturation [25].

It has been proposed that α-FUC might contribute to modify the carbohydrate

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moieties of sperm membrane glycoproteins during the epididymal transit; however,

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this hypothesis remains to be confirmed. Mammalian α-L-fucosidases are a

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ubiquitous group of a large multimeric lysosomal glycosidases involved in the

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hydrolysis of α-L-fucose from a diverse group of fucoglycoconjugates [26]. Two

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isoforms of this enzyme have been described in bull reproductive tissues, although

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only the FUCA1 isoform has been found in the cauda epididymal fluid, the seminal

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plasma and spermatozoa [27]. Other studies have suggested that, in other

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mammalian species, the enzyme detected in the epididymal fluid and seminal plasma

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differs from the sperm membrane-associated enzyme [28-30]. Several studies support the involvement of fucose and/or fucosidases in the

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fertilization process, indicating that may play an important role in the association to

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the oviduct to form a sperm reservoir [31-33] or for gamete recognition [28,29,34,35].

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In addition, the amount of fucose decrease after incubation with oviductal fluid [36].

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The addition of this monosaccharide to an in vitro fertilization assay inhibits the

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penetration into bovine oocytes [37]. The α-L-fucosidases are involved in the

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biosynthesis of fucosylated glycoproteins and, therefore, inhibitors of this enzyme are

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important tools in the study of fucose-containing glycoconjugates that participate in

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fertilization processes [29].

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Taking in account the importance of fucose after epididymal maturation, the

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aims of the present study were to determine whether the changes in the fucose

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content in the sperm surface during the epididymal transit can be taken as a

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maturation parameter, and if the secreted α-FUC is responsible of such

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modifications. Herein, we present direct evidences indicating that α-FUC from the

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cauda epididymal fluid may be responsible for the changes in the carbohydrate

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composition during sperm maturation in the epididymis.

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2.

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Materials and Methods 2.1.

Reagents

The rabbit polyclonal antibody against α-L-fucosidase (FUCA 1) was

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purchased from Santa Cruz Biotechnology (sc-134716). The FITC-conjugated UEA-I

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(cat.L9006) and biotin-conjugated UEA-I (L8262) were purchased from Sigma-Aldrich

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(St. Louis, MO). The p-nitrophenyl-α-L-fucopyranoside substrate for α-fucosidase

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(Cat. No. 3628) was also purchased from Sigma-Aldrich (St Louis, MO).

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Nitrocellulose (NC) membranes (pore size 0.45 µm) were purchased from GE Healthcare (Germany). The chemiluminescent reagent was prepared with 1.25 mM

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luminol (Sigma-Aldrich, St Louis, MO), and 198 µM p-coumaric acid in 100 mM Tris-

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HCl (pH 8.5) according to Mruk and Cheng [38]. The α-L-fucosidase inhibitors, such

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as (2S,3S,4R,5S)-2-(1-benzyl-1H-1,2,3-triazol-4-yl)-5-methylpyrrolidine-3,4-diol (C1),

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(2S,3S,4R,5S)-2-[1-((4-benzyloxycarbonyl-5-methylfuran-2-yl)-methyl)-1H-1,2,3-

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triazol-4-yl]-5-methylpyrrolidine-3,4-diol (C2) [39] and (2S,3S,4R,5S)-2-(1H-

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benzoimidazol-2-yl)-5-methylpyrrolidine-3,4-diol (C3) [40, 41] were provided by Dr.

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Inmaculada Robina (University of Seville, Spain). All the inhibitors are five membered

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iminosugars (pyrrolidines) having an L-fuco configuration, and bearing an aromatic

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moiety attached at carbon 2 of the pyrrolidine backbone.

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2.2.

Animals

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Reproductively mature bulls (Aberdeen-Angus, ∼4 years old) were used in this study. The animal’s fertility was certified by the supplying farms, where they had

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been used for reproductive purposes. Bulls were fed with diets primarily composed of

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pasture provided by farms located in the province of Córdoba (Argentina). All the

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procedures were performed according to the protocol approved by CICUAL

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(Commitee for Animal Care of the Universidad Nacional de Cuyo, Mendoza,

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Argentina). These procedures also fulfilled the policies and regulations of the

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Argentine National Institute of Health and the National Research Council.

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2.3.

Biological samples

All procedures were carried out according to Aguilera et al. [7]. Briefly, after

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slaughtering, epididymides were removed and transported to the laboratory and

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processed immediately. Organs (one per animal) were carefully dissected and the

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caput, corpus and cauda were processed separately. To obtain epididymal tissue

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ACCEPTED MANUSCRIPT and spermatozoa, each organ (the three regions) was cut into small pieces with a

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stainless steel blade. Samples were suspended (1:3 w/v) in Hank´s solution.

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Incubations were carried out, at 37 ºC for 30 min with gentle manual agitation to

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allow for spermatozoa release. The minced tissue was settled for 10 min at 4 ºC and

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the resulting supernatant (containing fluid and spermatozoa) was centrifuged at 400 x

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g for 5 min and spermatozoa were separated from the fluid. The spermatozoa were

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washed three times with Hank´s solution and finally pelleted at 1000 x g for 10 min.

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The epididymal fluids were stored at -20 °C until u sed. The remaining epididymal

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tissue was homogenized with buffer H (10 mM Tris–acetate, pH 7.2, containing 0.25

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M sucrose, 1% EDTA, 1 mM PMSF, 0.02% sodium azide, and 5 mM

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glycerophosphate) employing a Teflon/glass tissue homogenizer and centrifuged at

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800 x g for 20 min at 4ºC. The post-nuclear supernatant was stored at -20ºC until

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used.

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2.4.

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All the procedures were carried out according to Aguilera et al. [19]. Briefly, 45

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Immunoblotting

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µg of proteins from the epididymal tissue or fluid were run on SDS–PAGE gels (10%

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acrylamide/bisacrilamide) and electrotransferred to nitrocellulose (NC) membranes at

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250 V for 1 h. After transferring, the non-specific binding sites were blocked for 1 h

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with 6 % skimmed milk in phosphate-buffered saline containing 0.05% Tween 20

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(PBS-T). Membranes were then washed three times with PBS-T and incubated for 2

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h with anti-FUCA 1 diluted 1:250 in PBS-T. Membranes were washed and incubated

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with the biotinylated anti-rabbit IgG antiserum (diluted 1:5000 in PBS-T) for 2 h. After

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washing as above, NC membranes were incubated with peroxidase-conjugated

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streptavidin (1:10000 in PBS-T) for 1 h and washed again. Specific bands were

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detected by the enhanced chemiluminescence method, and quantified by

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densitometric scanning of membranes using an Image Quant LAS 4000 scanner (GE

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Healthcare, Uppsala, Sweden). NC membranes were stained with Ponceau S as

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loading and transference controls. 2.5.

Detection of fucose on nitrocellulose immobilized glycoproteins

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Proteins from caput or cauda sperm membrane were extracted with a

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hypoosmotic solution according to Jeyendran et.al. [42]. Briefly, spermatozoa (up to

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1x107spermatozoa/ml) were mixed with a hypoosmotic solution (25 mM sodium

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citrate dihydrate and 75 mM fructose) and incubated for 2 h at 37°C. Gametes were

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sonicated over ice until spermatozoa tails were separated from the rest of the cell

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and centrifuged at 4000 g for 15 min. Pellets, corresponding to intact spermatozoa,

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were discarded and the supernatants (SN1) were centrifuged at 10000 x g for 30

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min. Pellets (enriched-membrane fractions) were resuspended in 0.05 M Tris-HCl

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buffer (pH 7.2), containing 0.5% saponin, 50 mM EDTA and 1 mM PMSF. Proteins

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(45 µg) were run on 10% SDS-PAGE gels under non-reducing conditions and

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electrotransferred to NC membranes for 90 min at 250 V. After transferring, non-

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specific binding sites were blocked for 1 h with 6% skimmed milk in PBS-T.

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Membranes were then washed three times with PBS-T and incubated for 2 h with the

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biotin-conjugated lectin UEA-I (5 µg/ml in PBS-T). Membranes were washed as

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described above and incubated with peroxidase-conjugated streptavidin (1:10000 in

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PBS-T) for 1 h. After washing with PBS-T, the lectin-reactive bands were detected

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with the enhanced chemiluminescence method and quantified by densitometric

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scanning on the membranes using an Image Quant LAS 4000 scanner.

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2.6.

Fluorescence microscopy

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To evaluate the fucose amount on the sperm surface, the spermatozoa

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obtained from caput and cauda (as described above) were resuspended in PBS/PVP

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ACCEPTED MANUSCRIPT (0.1% polyvinylpyrrolidone in PBS) and fixed with 2 % p-formaldehyde (PAF) in PBS

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for 10 min. Cells were then placed on slides (previously treated with 10 mM poly-

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lysine for 30 min) to perform the immunofluorescence method. To this end, slides

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containing spermatozoa were blocked for 1 h at room temperature with 5 % horse

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serum in PBS/PVP and then incubated with 100 µl UEA-I lectin (10 µg/ml) in

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PBS/PVP containing 1% horse serum. After three washes with PBS/PVP, slides were

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mounted on glass coverslip with UltraCruzTM mounting medium (USA). Slides were

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then examined with in a Nikon 80 fluorescence microscope (Japan).

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2.7.

Flow cytometry

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Gametes from caput and cauda were washed three times with Hank’s buffer

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and centrifuged at 1000 x g for 10 min. Samples were fixed with 2% PAF in PBS/PVP

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for 10 min at room temperature and then blocked for 1 h at 37 °C with 5 % horse

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serum (in PBS/PVP) and washed three times with PBS/PVP. Each sample (5 x 105

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cells) was incubated with FITC-conjugated UEA-I (5 µg/ml) in 100 µl final volume for

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2 h at room temperature in the dark. The specificity of lectin binding was controlled

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by incubation in the presence of 0.5 M fucose. The sugar concentration was

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determined in previous experiments. Gametes were then washed twice with

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PBS/PVP, and the number of the lectin reactive sperm was analyzed on a BD

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FACSAria III (USA) flow cytometer and the FACS Diva Software. In all cases, a gate

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was established using dots plots of forward scatter (FSC) vs. side scatter (SSC)

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plots, since the same cellular and physical properties (size and granularity) was

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obtained for all treatments. After excitation at 488 nm, the emission was detected

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employing a 525 nm band pass filter. The percentage of the spermatozoa that

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reacted with FITC-conjugated lectins and the mean fluorescence intensity was

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analyzed using the FlowJo software version 7.6.2.

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2.8.

Incubation of caput sperm with cauda fluid

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Caput spermatozoa were obtained as described above and the crude cauda epididymal fluid was obtained by gentle retrograde perfusion with Hank’s buffer from

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the deferent duct until a drop with the luminal content was observed. Four drops of

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each epididymis were collected in Eppendorf tubes. After centrifugation at 2000 rpm,

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supernatants were recovered and used in the assay. For the assay, gametes were

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previously washed three times with Hank’s buffer at 33 °C. Spermatozoa (5 x 10 5

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cells) were then mixed with 100 µl of the crude epididymal fluid and incubated for 3 h

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at 33 °C. To confirm the enzyme specificity, the as says were carried out in the

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presence of the following FUCA 1 inhibitors at the final concentrations of 0.75 mM of

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the benzyltriazole pyrrolidine (C1), 0.1 mM of the benzyloxicarbonyl triazole

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pyrrolidine (C2), 0.43 mM of the imidazolil pyrrolidine (C3). These conditions had

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previously been determined in previous works according to the IC50 values [39-41].

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As controls, gametes were incubated with either Hank´s buffer alone or with

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inactivated fluid (15 min at 70 °C). After incubati on, gametes were washed three

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times with Hank’s buffer and prepared for subsequent lectin binding and flow

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cytometry.

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2.9.

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The activity of α-Fuc was measured using p-nitrophenyl -α-L-fucopyranoside

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Enzymatic assays

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substrate according to Barrett and Heath [43]. One unit of enzymatic activity

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catalyzes the release of 1 nmol of p-nitrophenol. Different buffer solutions were used

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depending on the experiment: 0.2 M citrate buffer was used to determine the activity

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at low pH values (4.5, 5 and 5.5) and 0.2 M phosphate buffer was employed to the

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determine the activity at high pH values (6, 6.5 and 7). The protein concentration was

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determined according to Lowry [44].

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Data were analyzed for normality distribution before a t test or one-way

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ANOVA was performed. A Dunnett´s multiple comparisons test or a Tukey-Kramer’s

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multiple comparisons test was used after one-way ANOVA as indicated in each

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experiment. The level of significance was set at p< 0.05.

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2.11. Experimental design

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Experiment 1. Qualitative and quantitative evaluation of fucose in caput and cauda epididymal spermatozoa. For fucose content, FITC labelled lectin UEA-I

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followed by fluorescence microscopy and flow cytometry was used. In addition, we

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analysed the pattern of fucosylated proteins by incubating biotin labelled lectin UEA-1

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with sperm proteins immobilized in Nitrocellulose membrane.

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Experiment 2. Evaluation of α-FUC content in the epididymis. The expression of α-FUC was studied by western blot on proteins from the tissue and fluid of the

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caput, corpus and cauda epididymis. Moreover, we tested the activity of this enzyme

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by spectrophotometrically, using a specific colorimetric substrate (p-nitrophenyl -α-L-

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fucopyranoside).

Experiment 3. Effect of the cauda epididymal fluid on fucose content of caput

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spermatozoa. To examine the ability of the cauda fluid to remove fucose from the

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caput spermatozoa, we incubated a crude cauda fluid with the spermatozoa under

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conditions of epididymal environment. Afterwards, we evaluated the fucose content

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by flow cytometry. As control, a heat inactivated (70º C) cauda fluid was used.

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Experiment 4. Effect of α-FUC inhibitors on the fucose content of caput

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spermatozoa. To corroborate the involvement of the soluble cauda epididymal α-Fuc

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in the fucose removal of the caput spermatozoa, we incubated the samples in the

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presence of specific inhibitors for the enzyme. The fucose content was then

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evaluated by flow cytometry.

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3.

Results In this study, we analysed the presence and location of α-L-fucose in sperm

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plasma membrane in bulls during their epididymal transit. By using FITC labelled

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lectin UEA-I, we observed a post-acrosomal fucose detection in the caput

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spermatozoa, which becomes lower (or weaker) towards the cauda gametes (Figure

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1A). These results were quantified by flow cytometry and we observed a significant

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decrease of fucose content in the surface of epididymal spermatozoa from the caput

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to cauda (Figure 1 B). The specificity of the lectin was confirmed by competitive

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inhibition with the sugar L-fucose (Figure 1B). In addition, the pattern of fucosylated

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proteins was evaluated by binding of biotinylated UEA-I to sperm proteins

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immobilized on nitrocellulose membranes (see materials and methods). In agreement

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with the previous results, a decrease in the fucosylation level was observed in most

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of the proteins of cauda spermatozoa (in the range of 75-110 and 30-20 kDa) when

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compared with the caput spematozoa, although an increase in other protein of lower

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Mr (~50 kDa) was observed in cauda spermatozoa (Figure 2). The decreased

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fucosylation in the cauda spermatozoa may be related to an increase of α-FUC

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activity along the epidiymal duct. Therefore, we evaluated the presence and the

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activity of α-FUC in the bull epididymis to find that the enzyme is mostly expressed in

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the caput and cauda and also in the corpus though at a lesser extent (Figure 3 A).

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Interestingly, the enzyme was mostly detectable in the fluid obtained from the cauda

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epididymis (Figure 3 B). These results were in agreement with the measured

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enzymatic activity in each epididymal region (Figure 3 C). For these reasons, we

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responsible of fucose remotion under the conditions of epididymal environment. First,

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we observed that epididymal α-FUC is active against synthetic substrates at the

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epididymal pH (6.5) although only 50 % of the optimum (pH 4.5) (data not shown).

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Then we developed an assay in which the crude cauda fluid was incubated with

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caput spermatozoa at 33 °C.

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After incubation, the fucose content was evaluated by flow cytometry, and we

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observed a significant decrease in the sugar amount (Figure 4), indicating that the

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enzyme can remove fucose from the caput spermatozoa. This effect was not

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observed when heat inactivated epididymal fluid (70 °C) was used (Figure 4). To

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corroborate that α-FUC activity from the epididymal fluid was responsible for the

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decrease in the fucose content, the cauda spermatozoa were incubated with the

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caput fluid in the presence of specific inhibitors, such as C1, C2 and C3 (Figure 5 A).

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As shown in the Figure 5 B, the three compounds were able to inhibit the sperm

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defucosylation caused by the epididymal fluid, thus confirming that an α-FUC is

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involved in this phenomenon. In addition, the specificity of the inhibitors was

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confirmed by evaluating enzyme activity with a synthetic substrate (Figure 5 C).

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Discussion

The mammalian epididymis provides the proper molecular environment for the

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sperm maturation. During this process, the plasma membrane undergoes several

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modifications that include changes in the carbohydrate composition. In this study, we

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present evidences indicating that the fucose content on the sperm surface is modified

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during the epididymal transit and that this phenomenon could be related to sperm

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maturation in bulls.

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Herein, we observed that most of the fucosylated proteins are located along the post-acrosomal region in the caput spermatozoa. Despite the total fucose content

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on sperm membrane decreases from the caput to the cauda, some proteins increase

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their fucosylation status. This phenomenon could be the result of the balance

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between the activities of α-L-fucosidase (α-FUC) and fucosyl-transferase existing in

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the epididymal fluid. The presence of glycosyl-transferases has been documented in

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the mammalian epididymis [2,6,45]. Moreover, it is known that the mammalian

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epididymis cells synthesize and secrete acid glycosidases into the lumen, which

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could be responsible for the changes observed in the sperm surface [2,5,6,14-19].

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However, there were few direct evidences that demonstrate a role of the enzymes in

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this process. In previous studies, we have described the presence of some of these

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enzymes in the epididymal lumen and also observed that some of them interact with

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specific receptors on the sperm plasma membrane [7]. However, α-L-fucosidase

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mostly found in the soluble form, but not associated to spermatozoa neither within

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membrane bounded vesicles from epididymal fluid, likely epididymosomes

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(unpublished data).

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Curiously, the pH of the epididymal environment would not be favourable for the

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enzymatic activity, since the glycosidases are lysosomal enzymes. In this work, we

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have focussed our study on the role of α-FUC in the remodelling of the sperm surface

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and have provided direct evidences indicating that the α-FUC present in the cauda

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epididymal fluid is active at conditions similar to those of the epididymal environment,

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being able to remove fucose from the surface of bull spermatozoa. In previous

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studies, the α-FUC activity was associated with the reproductive capacity, since this

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enzyme increases in the epididymis of reproductively mature animals with respect to

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immature ones, and it is mostly redistributed to the epididymal fluid in the the cauda

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ACCEPTED MANUSCRIPT region (Aguilera et al., unpublished data). These evidences allow suggesting a role of

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this enzyme in the fertility development in bulls. Furthermore, these results were in

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accordance with those of Moura et.al [24], who have demonstrated the existence of a

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high content of α-FUC in the cauda fluid of bulls with high fertility capacity. By means

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of a polyclonal antibody against α-FUC, we detected a single band of 56 kDa,

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suggesting the existence of one isoform in the epididymal fluid as in other species

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[29,30]. However, the presence of other isoforms cannot be ruled out, since α-FUC

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associated to sperm membrane has been described in bulls and in other mammalian

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species [7,28-30,46,47]. The fact that the expression and activity of α-FUC are

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detected in the cauda epididymis may indicate that most of the defucosylation

333

process occurs in this region of the organ. The epididymal α-FUC also exhibits some

334

properties that are similar to those from other tissues, such as an optimal acidic pH

335

and sensitivity to synthetic specific inhibitors [29,39,40]. However, the epididymal α-

336

FUC is also active, although at a lesser extent, at a pH that is similar to the

337

epididymal environment. Taking into account this phenomenon, we performed an in

338

vitro assay in which we incubated spermatozoa from the caput with the crude fluid

339

from the cauda. Under these experimental conditions, we clearly demonstrated that

340

α-FUC from the fluid is able to remove fucose from the sperm surface. The

341

involvement of the enzyme in this process has been corroborated by the use of

342

specific inhibitors which are active against the epididymal enzyme. It has been

343

suggested that fucose may play an important role in fertilization, since the addition of

344

this monosaccharide or fucoidan to the fertilization medium causes a reduction in the

345

penetration rates to porcine and bovine oocytes [37,48]. Other studies suggest that

346

fucose is involved in the formation of the sperm reservoir in the oviduct [31-33]. It is

347

also likely that sperm surface undergo additional changes in carbohydrate

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14

ACCEPTED MANUSCRIPT 348

composition once they leave the epididymis. This could be the result of the enzymatic

349

activity of glycosidases that have been described in reproductive fluids, such as

350

oviductal fluid [49]. These data provide, for the first time in bulls, direct evidence that the soluble

351

α-FUC in the epididymal fluid participates in the remodelling of the sperm surface, as

353

a step in the maturation of gametes, and that the fucosylation status of sperm

354

proteins contributes, in part, to that maturation process. Thus, the activity of α-FUC

355

and the state of fucosylation of sperm proteins can be considered as a parameter of

356

maturation. The latter finding might be useful in future studies assessing dysfunctions

357

related to male infertility.

359

5.

Funding

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This work was supported by Secretaria de Ciencia, técnica y Posgrado

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(SECTyP,) Universidad Nacional de Cuyo-Mendoza (Argentina) [grant number:

362

06/J459]

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363 6.

Acknowledgements

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Ms. Andrea Lafalla Manzano and Dr. Clara García Samartino (Flow Cytometry

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Facility, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo) provided

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assistance and consultation during flow cytometry experiments.

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fucose-binding protein from bull sperm and seminal plasma that may be responsible

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for formation of the oviductal sperm reservoir. Biol Reprod 2001;64:1806–11.

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carbohydrate affinities at the cell surface of capacitating bovine sperm cells. Biol

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competes with a fucosylated neoglycoprotein for binding sites on the human sperm

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surface. Fertil Steril 1993;60:344-50.

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containing epitope potentially involved in gamete interaction on the human zona

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pellucida. Hum Reprod 1994;9:1532-38.

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bovine spermatozoa mediated by oviductal fluid: a flow cytometric study using

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lectins. Int J Androl 2007;30:108-14.

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Vogel P, et al. Synthesis of novel pyrrolidine 3,4-diol derivatives as inhibitors of

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alpha-L-fucosidases. Org Biomol Chem 2009;7:1192-2202.

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[41] Moreno-Vargas AJ, Carmona AT, Mora F, Vogel P, Robina I. Stereoselective

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synthesis of (2S,3S,4R,5S)-5-methylpyrrolidine-3,4-diol derivatives that are highly

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selective alpha-L-fucosidase inhibitors. Chem Commun. 2005; 4949-51.

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[42] Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG, Zaneveld LJ.

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Development of an assay to assess the functional integrity of the human sperm

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membrane and its relationship to other semen characteristics. J Reprod Fertil

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1984;70:219-28.

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[43] Barrett AJ, Heath MF. Lysosomes: A Laboratory Handbook. In: Dingle J, editor.

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Amsterdam: Elsevier/North Holland and Biomedical Press; 1977, p. 118-20.

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[48] Song XX, Park CK, Piao, YJ, Niwa K. Effect of monosaccharide L-fucose and

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[49] Carrasco LC, Coy P, Avilés M, Gadea J, Romar R. Glycosidase determination in

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Fertil Dev 2008;20:808-17.

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8.

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Figure 1. α-L-fucose in bull epididymal spermatozoa. A) Distribution of the sugar in

504

spermatozoa from caput and cauda using FITC-conjugated UEA-I and assessed by

505

fluorescence microscopy. B) Analysis of the fluorescence by flow cytometry, and C)

506

the corresponding quantification. Aut: autofluorescence of cauda spermatozoa alone;

507

Cau Sp: cauda spermatozoa; Cau Sp + F: cauda spermatozoa plus 0.5 M fucose;

508

Cap Sp: caput spermatozoa. Bars represent the means of relative fluorescence

509

intensity (R.F.I.) ± SD (n = 12). Data were analysed by t test, (***) significantly

510

different from cauda (P<0.05).

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Figure legends

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Figure 2. Patterns of membrane fucosylated proteins from caput (Cap Sp) and cauda

513

(Cau Sp) spermatozoa, detected with biotin-conjugated UEA-I on nitrocellulose

514

membranes. As control, the membrane was stained with Ponceau S.

515

21

ACCEPTED MANUSCRIPT Figure 3. Detection of α-L-fucosidase (α-FUC) in bull epididiymis. Immunoblot for

517

detection of α-FUC in epididymal tissue (A) and fluid (B), and the corresponding

518

quantifications from the epididymal regions as indicated. Bars represent the means of

519

relative optical densities (R.O.D.) ± SD (n = 3). Ponceau S stain was used as loading

520

and transference controls. (*) Significantly different from corpus and caput (P<0.05),

521

and (**) significantly different from corpus (P<0.05 C). α-FUC activity measured in

522

epididymal tissue and fluid from the three epididymal regions (C). Bars represent the

523

means ± SEM of specific activity (n=5). (*) Significantly different from corpus and

524

caput (P<0.05) and (**) significantly different from corpus (P<0.05). In all the cases,

525

data were analysed by one-way ANOVA followed by a Tuckey-Kramer´s multiple

526

comparison test.

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Figure 4. Determination of α-L-fucose in caput spermatozoa after the incubation with

529

cauda fluid. Flow cytometry analysis of the fucose content using the FITC labelled

530

lectin UEA-I after the incubation of caput spermatozoa (Sp) with cauda fluid. As

531

control, cauda inactivated fluid (I Fluid) (heated for 15 min at 70 ºC) and buffer

532

(Hank`s) were used. Bars represent the means of the R.F.I ± SEM (n = 12) of each

533

treatment. Data were analysed by one-way ANOVA followed by a Dunnett´s multiple

534

comparison test. (**) Significantly different from Buffer and I Fluid (P<0.05).

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Determination of α-L-fucose in caput spermatozoa (Sp) after the

536

Figure 5.

537

incubation with cauda fluid, in the absence or presence of the α-FUC inhibitors. A)

538

Molecular structure of the synthetic inhibitors (C1, C2 and C3) of α-L-fucosidase (α-

539

FUC). Ki represents the inhibition constant for each compound. B) Flow cytometry

540

analysis, the profiles are representative of all the conditions employed and C)

22

ACCEPTED MANUSCRIPT quantification of R.F.I. Data were normalized to fluid alone (R.F.I.=1). Bars represent

542

the means of RFI ± SD (n = 12). Data were analysed by one-way ANOVA followed by

543

a Dunnett´s multiple comparison test. (*) Significantly different from all the conditions

544

used (P<0.05). D) The effect of the inhibitors (at 0.3 µM, 17 nM and 80 nM for C1, C2

545

and C3, respectively) on the α-FUC activity of cauda epididymal fluid (n = 5). Bars

546

represent the means ± SEM of specific activity.

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ACCEPTED MANUSCRIPT

C

1500

***

1000

R.F.I.

M AN U

Aut Cau Sp + F Cau Sp Cap Sp

SC

B

RI PT

Caput

Cauda

A

500

Cap Sp

Cau Sp

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Aguilera et al., Figure 1



ACCEPTED MANUSCRIPT

UEA-I biotin Cap Sp

Cau Sp

Cap Sp

Cau Sp

AC C

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Mr (kDa)

Ponceau S

Aguilera et al., Figure 2

Epididymal Tissue

A

Caput

Corpus

Cauda 4

56 kDa

**

3

R.O.D.

α-FUC

2 1 0

Caput

Corpus

Cauda

20

56 kDa

Corpus

Cauda

*

15

R.O.D.

α-FUC

Caput

SC

B

Epididymal Fluid



RI PT

Ponceau S

10

M AN U

5

Ponceau S

0

C

Caput

Corpus

Cauda

α-FUC activity

)

2000 1000 0

Cauda

Caput

Corpus

Cauda

4000

*

2000

U/g protein (10 2)

**

3000

EP

U/g protein (10 2)

*

Fluid

2500

U/g protein (10 2)

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Tissue 4000

1500 1000 500 0

Caput

Corpus

Cauda

AC C

us

ACCEPTED MANUSCRIPT



Aguilera et al., Figure 3



3000 2000 1000 0

Capu

ACCEPTED MANUSCRIPT Sp + Buffer

2.0

M AN U TE D EP AC C





Aguilera et al., Figure 4



id

d

IF lu

ui

+

Fl

SC

Sp

+

B

uf

fe

r

0.0

+

0.5

RI PT

**

1.0

Sp

R.F.I.

1.5

Sp

Sp + Fluid Sp + I Fluid

ACCEPTED MANUSCRIPT

A

C3

Ki =0.024 µM (C)

Ki =0.004 µM (C)

Ki =0.080 µM (C)



RI PT

C2

C )

Sp + Buffer Sp + Fluid

2.0

Sp + Fluid + C1

SC

R.F.I.

1.5

*

1.0

M AN U

0.5

1500

AC C

500

EP

1000

Aguilera et al., Figure 5

3 Fl

ui

d

+

C

2 Fl

ui

d

+

C

1 C

+

d

Fl

ui

Fl

ui

d

0

C 3 +

Sp

+

Fl ui d

+

C 2

C 1 Fl ui d +

Sp

+F lu id

+

Fl Sp

+

Sp

Sp

+

B

uf

ui

fe

d

r

0.0

2

) D

TE D

B

C1

U/g protein U/g protein (10 (102))

)

ACCEPTED MANUSCRIPT

Highlights •

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• •

Fucose content of sperm membrane decreases toward the cauda of bull epididymis. An increased activity of α-L-fucosidase is found in the fluid of cauda epididymis. Defucosylation of glycoproteins would be related to epididymal sperm maturation.