Nano-depletion of acrosome-damaged donkey sperm by using lectin peanut agglutinin (PNA)-magnetic nanoparticles

doi:10.1016/j.theriogenology.2020.04.011

Theriogenology

Volume 151, 15 July 2020, Pages 103-111

https://doi.org/10.1016/j.theriogenology.2020.04.011 Get rights and content

Highlights

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Biocompatible magnetic nanoparticles (MNPs) coated with lectin-PNA were synthesized.
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MNPs targeted the damaged-acrosome sperm without negative effect on magnetism.
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Nanopurification increased the percentage of intact-acrosome sperm after freezing.
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The initial quality of semen samples cannot predict nanopurification efecctiveness.
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Lectin-MNPs can be considered a suitable alternative for sperm selection.

Abstract

Lectin is considered as a suitable biomarker for nano-depletion of acrosome-damaged sperm. The aim of this study was to synthetize magnetic nanoparticles (MNPs) coated by peanut (Arachis hypogaea) agglutinin lectin (PNA) and investigate its beneficial effect in improving of sperm characteristics. MNPs were obtained by co-precipitation method, functionalized with chitosan and coated by PNA at a concentration of 0.04 mg/mL. Semen was frozen either with glycerol-based or sucrose-based extenders. Frozen-thawed straws from five donkeys (three ejaculates per donkey) were incubated with lectin-MNPs (2 mg/mL), and then exposed to an external magnet enabling the non-bound sperm to be collected as nanopurified sperm. Sperm were evaluated post-thawing (control) and after nanopurification for motility, plasma membrane integrity, acrosome integrity, morphology, DNA fragmentation and concentration. The statistical analyses were extended to investigate the correlation between the initial quality of the frozen-thawed semen samples and the effect of nanopurification after thawing. The obtained MNPs were biocompatible to the sperm and significantly improved the progressive motility (P < 0.05) for the glycerol nanopurified group (43.08 ± 3.52%) in comparison to control (33.70 ± 2.64%). Acrosome-damaged sperm were reduced (P < 0.05) in both nanopurified groups (19.92 ± 2.69 for G and 21.57 ± 2.77 for S) in comparison to control (36.07 ± 3.82 for G and 35.35 ± 3.88 for S). There were no significant changes in sperm morphology and membrane integrity after nanopurification. The average sperm recovery after nanopurification was 80.1%. Sperm quality index was significantly higher (P < 0.001) in nanopurified groups regardless of the initial quality of the frozen thawed semen samples. However, in the high sperm quality group, nanopurification significantly improved the progressive motility and membrane integrity besides the increasing of acrosome-intact sperm. Sperm nanopurification using lectin-magnetic nanoparticles can be considered as a suitable method to reduce the proportion of acrosome-damaged sperm and to increase the quality of frozen thawed donkey semen.

Introduction

Nanotechnology is the study of phenomena and manipulation of molecules less than 100 nm in diameter, known as nanoparticles [1]. In reproductive medicine, particularly in the case of sperm, nanoparticles-based protocols have been applied for sperm-mediated gene transfer [2], loading sperm with exogenous proteins [3], tagging sperm for subsequent sex- [4] or function-based sorting [5] and diet supplementation with nanoparticles such as nano-selenium could enhance the testicular function [6]. Magnetic nanoparticles (MNPs) are synthesized in controllable sizes, which can be coated with biological molecules to promote their interaction or binding to a biological entity, and then manipulated by an external magnetic field [7]. Targeting of defective sperm by MNPs coated with certain biomarkers offers a promised method for purification of mammalian semen and provides new prospects to develop efficient, easy and non-invasive techniques for mammalian sperm purification [8], including frozen-thawed semen samples [5].

Cryopreservation of semen is an important tool to maintain the genetic diversity [9]. Donkey population in Europe has dramatically diminished in the last century [10], including the Andalusian donkey breed which is considered at risk of extinction (Food and Agriculture Organisation (FAO) [11]). Semen cryopreservation causes mechanical and functional damage to sperm due to several factors, mostly caused by osmotic stress [12] and toxicity due to unequal distribution of permeable cryoprotectant agents (CPA) on the sperm cell [13]. These effects would result in a reduction on sperm motility, viability, DNA and acrosome integrity and the fertilizing capacity after thawing [14,15]. In general, the ability of jennies to conceive after artificial insemination (AI) with donkey frozen-thawed semen is lower than in mares [16]. It has been hypothesized that the toxicity of permeable CPA and in particular glycerol, negatively affects the fertility of donkey sperm, even though the dose of glycerol for donkey sperm freezing is about half that for stallion sperm [16,17]. Thus, replacement of glycerol by sucrose, a non-permeable CPA, has been successfully applied as a new strategy for donkey sperm freezing and vitrification [18,19].

Intact functional sperm acrosome is an essential prerequisite for oocyte fertilization [20]. Sperm with damaged acrosomes or that have undergone premature acrosome reaction after freezing-thawing, lose their ability to bind to the zona pellucida and are no longer capable of fertilization. In other words, acrosome-reacted sperm have a short life span [21] and, eventually, the increase in the proportion of such sperm will negatively affect the fertilizing capacity of the semen doses [22].

It has been shown that sperm with damaged acrosomes show definite ligands, which can be detected by a special biomarker and then removed from the intact sperm population [23]. A variety of lectins are used to evaluate the acrosomal status for their ability to bind to the glycoconjugates of the outer acrosomal membrane or the acrosomal matrix [24,25]. Purified bull semen using MNPs coated with peanut agglutinin from Arachis hypogaea lectin (PNA) achieved conception rates after AI equal to those of unpurified semen at half the sperm dose [5]. Field trials using nanopurified semen did not negatively affect the fertility and enhanced the reproductive outcomes of inseminated gilts by enrichment of the semen doses with high quality sperm [26]. All of these previous studies using lectin-functionalized MNPs differed significantly from each other in methodology.

Other strategies have been also proposed to improve the efficiency of sperm cryopreservation in donkeys, including purification of semen samples after thawing by sperm selection techniques. In this sense, colloid-single layer centrifugation (SLC) has been shown to improve the quality of frozen thawed donkey semen [27]. However, SLC results in a low recovery rate [28] and do not target any intrinsic sperm characteristics that may impact the fertilization potential [29]. The use of nanoparticle-based intracellular markers allows the extension of magnetic-activated cell sorting (MACS) applications [30,31]. Thus, we proposed the possible application of the labelled magnetic nanoparticles by lectin as an alternative technique for purification of frozen-thawed donkey sperm.

Therefore, the aims of this study were to: 1) validate lectin coated MNPs for donkey sperm nanopurification; 2) investigate the effect of nanopurification with lectin coated MNPs in post-thaw sperm quality of donkey semen frozen with glycerol-based extender or sucrose-based extender; and 3) evaluate the relationship between the initial quality of the frozen-thawed semen samples and the effect of nanopurification after thawing.

Section snippets

Ethics statement

All the experiments were approved by the Ethical Committee for Animal Experimentation of the University of Córdoba, in compliance with the Regional Government of Andalusia and the Spanish law for animal welfare and experimentation (RD 53/2013).

Reagents and samples

All the reagents required for the synthesis and functionalization of magnetic nanoparticles were purchased from Sigma-Aldrich (Madrid, Spain), unless otherwise stated. The magnetic core was prepared using ferric chloride (FeCl₃·6H₂O), ferrous chloride

Synthesis and characterization of lectin-magnetic nanoparticles

Transmission electron microscopy (TEM) revealed that the bare Fe₃O₄ nanoparticles were of nearly spherical shape (Fig. 1a), and the shape was not affected by the subsequent functionalization steps (Fig. 1b). However, after functionalization, the size of the inner core of MNPs has not changed, but the formation of clusters of nanoparticles are observed covered by the chitosan and lectin layers, which could be dispersed via sonication. The average size of the bare magnetic nanoparticles

Discussion

Sperm selection methods are routinely applied before assisted reproductive techniques (ART) for various species [40]. These methods are particularly used to remove the undesirable defective sperm [41,42]. Recently, MNPs coated with special biomarker could be one of the best choices to target the defective sperm for semen purification. Lectin is considered as a suitable biomarker for nano-depletion of acrosome-damaged sperm. In the present study, Lectin- MNPs were prepared via a three-step

CRediT authorship contribution statement

M.S. Yousef: Formal analysis, Writing - original draft, Writing - review & editing. A.I. López-Lorente: Writing - original draft. M. Diaz-Jimenez: Formal analysis. C. Consuegra: Formal analysis. J. Dorado: Formal analysis, Project administration. B. Pereira: Formal analysis. I. Ortiz: Writing - review & editing. S. Cárdenas: Formal analysis. M. Hidalgo: Formal analysis, Writing - original draft, Project administration.

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgment

This study was supported by the University of Cordoba, grant PP. 2018, Mod. 4.1. The authors would like to thank Dr. J.M. Lopez Martin for his help in the acquisition, analysis and interpretation of data concerning the flow cytometric assays, Beckman-Coulter company for providing the Cytoflex equipment and the Central Support Service for Research of University of Cordoba, Spain (SCAI, UCO) for the services provided to characterize the nanoparticles.