Intracellular translocation and differential ...

Hum. Reprod. Advance Access published April 12, 2013 Human Reproduction, Vol.0, No.0 pp. 1– 16, 2013 doi:10.1093/humrep/det064

ORIGINAL ARTICLE Reproductive biology

Intracellular translocation and differential accumulation of cell-penetrating peptides in bovine spermatozoa: evaluation of efficient delivery vectors that do not compromise human sperm motility

1 Molecular Pharmacology Research Group, Research Institute in Healthcare Science, University of Wolverhampton, Wolverhampton WV1 1LY, UK 2Laboratory of Molecular Biotechnology, Institute of Technology, University of Tartu, 517 Nooruse Street 1, Tartu 50411, Estonia 3Centre for Oncology and Molecular Medicine, Division of Medical Sciences, Ninewells Hospital, University of Dundee, Dundee DD1 95Y, UK 4School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK 5Department of Neurochemistry, Stockholm University, Stockholm S-10691, Sweden

*Correspondence address. Tel: +44-(0)1902-321131; Fax: +44-(0)1902-322714; E-mail: [email protected]

Submitted on September 24, 2012; resubmitted on January 17, 2013; accepted on February 15, 2013

study question: Do cell penetrating peptides (CPPs) translocate into spermatozoa and, if so, could they be utilized to deliver a much larger protein cargo?

summary answer: Chemically diverse polycationic CPPs rapidly and efficiently translocate into spermatozoa. They exhibit differential accumulation within intracellular compartments without detrimental influences upon cellular viability or motility but they are relatively ineffective in transporting larger proteins. what is already known: Endocytosis, the prevalent route of protein internalization into eukaryotic cells, is severely compromised in mature spermatozoa. Thus, the translocation of many bioactive agents into sperm is relatively inefficient. However, the delivery of bioactive moieties into mature spermatozoa could be significantly improved by the identification and utility of an efficient and inert vectorial delivery technology. study design: CPP translocation efficacies, their subsequent differential intracellular distribution and the influence of peptides upon viability were determined in bovine spermatozoa. Temporal analyses of sperm motility in the presence of exogenously CPPs utilized normozoospermic human donor samples.

materials and methods: CPPs were prepared by manual, automated and microwave-enhanced solid phase synthesis. Confocal fluorescence microscopy determined the intracellular distribution of rhodamine-conjugated CPPs in spermatozoa. Quantitative uptake and kinetic analyses compared the translocation efficacies of chemically diverse CPPs and conjugates of biotinylated CPPs and avidin. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) conversion assays were employed to analyse the influence of CPPs upon sperm cell viability and sperm class assays determined the impact of CPPs on motility in capacitated and non-capacitated human samples. main results: Chemically heterogeneous CPPs readily translocated into sperm to accumulate within discrete intracellular compartments. Mitoparan (INLKKLAKL(Aib)KKIL), for example, specifically accumulated within the mitochondria located in the sperm midpiece. The unique plasma membrane composition of sperm is a critical factor that directly influences the uptake efficacy of structurally diverse CPPs. No correlations in efficacies were observed when comparing CPP uptake into sperm with either uptake into fibroblasts or direct translocation across a & The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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Sarah Jones 1, Monika Lukanowska 1, Julia Suhorutsenko2, Senga Oxenham 3, Christopher Barratt 3, Steven Publicover 4, ¨ lo Langel 2,5, and John Howl 1,* Dana Maria Copolovici 2, U

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phosphatidylcholine membrane. These comparative investigations identified C105Y (CSIPPEVKFNKPFVYLI) as a most efficient pharmacokinetic modifier for general applications in sperm biology. Significantly, CPP uptake induced no detrimental influence upon either bovine sperm viability or the motility of human sperm. As a consequence of the lack of endocytotic machinery, the CPP-mediated delivery of much larger protein complexes into sperm is relatively inefficient when compared with the similar process in fibroblasts.

limitations, reasons for caution: It is possible that some CPPs could directly influence aspects of sperm biology and physiology that were not analysed in this study.

wider implications of the findings: CPP technologies have significant potential to deliver selected bioactive moieties and so could modulate the biology and physiology of human sperm biology both prior- and post-fertilization.

study funding/competing intersts: We are pleased to acknowledge financial support from the following sources: the Wellcome Trust, TENOVUS (Scotland), University of Dundee, Medical Research Council, NHS Tayside and Scottish Enterprise and the Research Institute in Healthcare Science, University of Wolverhampton. No conflicts of interest are reported by the authors. Key words: spermatozoa / cell-penetrating peptide / membrane translocation / cytotoxicity / motility / mitochondrion

The fully differentiated male gamete is usually a highly polarized and motile cell type specialized for the single function of oocyte fertilization. Associated with a much reduced internal volume, the mature sperm cell lacks a variety of organelles, including endoplasmic reticulum, Golgi apparatus and cytosolic ribosomes, which are not required for those processes leading up to and including fertilization (Boerke et al., 2007). Moreover, as recently reviewed (Gadella and Evans, 2011), at the time that mammalian sperm are released into seminiferous tubules the processes of genomic transcription and translation have largely been silenced; though it is likely that sperm deliver a variety of RNA species into the fertilized oocyte that may modulate early events in embryogenesis (Boerke et al., 2007). Thus, studies of sperm intracellular biology are largely restricted to cell permeable agents that modulate other biochemical events. Of particular relevance to the investigations reported herein, the sperm plasma membrane is seemingly incapable of participating in endocytotic events that require active lipid recycling (Gadella and Evans, 2011). Consequently, common and energy-dependent routes of entry that facilitate the movement of macromolecules into a majority of eukaryotic cells are severely restricted in sperm. Hence, the sperm plasma membrane is a static physical barrier to the influx of agents (e.g. peptides, proteins and chemical modifiers) capable of modulating the activities of intracellular targets and signal transduction events such as those that regulate capacitation and the acrosome reaction (Abou-haila and Tulsiani, 2009). The plasma membrane of ejaculated mammalian sperm is enriched with polyunsaturated fatty acids. Perhaps exclusively, the sperm plasma membrane contains plasmalogens, aldehydogenic lipids characterized by an ether linkage at the glycerol sn-1 position (reviewed by Lenzi et al., 1996). In an effort to overcome the physico-chemical restrictions of the plasma membrane, the common detergent triton X-100 has been widely used to produce demembranated sperm models that facilitate the direct accessibility of chemical agents to intracellular targets (Lindemann and Goltz, 1988; Leclerc and Gagnon, 1996; Ho et al., 2002). However, it is also widely documented that detergents may have a complex and often detrimental influence upon protein function (reviewed by Garavito and Ferguson-Miller, 2001). Hence, the identification of inert vectors capable of the efficient

delivery of bioactive agents into intact sperm would provide obvious advantages to the use of detergents and other invasive techniques necessary to provide access to intracellular compartments and organelles. In recent years a variety of mostly poly-cationic cell-penetrating peptides (CPPs) have been identified (see Langel, 2011 for a comprehensive recent summary) that possess utility for the efficient delivery of bioactive agents to the intracellular compartments of eukaryotic cells. It is highly likely that common CPPs, including penetratin (Derossi et al., 1994), HIV-derived tat (Vives et al., 1997) and transportan 10 (Soomets et al., 2000), gain entry to intracellular compartments using a combination of different mechanisms including direct membrane translocation and various endocytotic routes (Duchardt et al., 2007; recently reviewed by Madani et al., 2011). However, numerous studies employing inhibitors of various endocytotic pathways have failed to define a common CPP-mediated import mechanism as the process is likely to be cell-, concentration-, sequence and cargotype specific. In this regard the mammalian spermatozoon, a morphologically distinct and endocytosis-incompetent cell type, could provide a useful platform to help resolve the relative contributions of different entry mechanisms to CPP-mediated import. With this purpose in mind, coupled with the aim to identify CPP vectors that could be efficiently employed in studies seeking to address many aspects of sperm cell biology, we chose to determine the uptake efficacy of a diverse range of CPPs into bovine spermatozoa. Moreover, having identified suitable CPP vectors, additional investigations were undertaken to determine whether CPPs can deliver large protein cargoes into sperm as non-covalent complexes. The influence of the same peptides upon viability and the motility of human sperm were also determined to ensure that CPP vectors were without detrimental influence on sperm cell biology. We conclude that CPP technologies will enable the further investigation and modulation of many biochemical events that underlie sperm maturation, capacitation, fertilization and early embryogenesis

Materials and Methods Materials Fmoc-protected amino acids for both manual and microwave-assisted peptide synthesis were purchased from Novabiochem (Beeston, UK).


Introduction

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Translocation of peptides into spermatozoa

AGTC Bioproducts Ltd (Hessle, UK) supplied dimethylformamide and dichloromethane for solid phase peptide synthesis. Culture medium and supplements were purchased from Sigma-Aldrich (Poole, UK) and fetal bovine serum from PAA (Yeovil, UK). Bovine semen was purchased from BULLSEMEN.COM, Inimex Genetics Ltd. UK. Avidin Texas Redw (TXR), avidin Alexa Fluorw 488, MitoTrackerw Deep Red 633, transferrin Alexa Fluorw 488, transferrin Texas Redw, dextran Texas Redw and LysoTrackerw Red were purchased from Molecular Probesw, Life technologies, (Paisley, UK). Unless otherwise indicated all other research grade chemicals were purchased from Sigma-Aldrich (Poole, UK).

Manual peptide synthesis

Microwave-assisted solid phase peptide synthesis Microwave-assisted solid phase peptide syntheses were performed using a Discover SPS Microwave Peptide Synthsizer (CEM Microwave Technology Ltd, Buckingham, UK) with fibre optic temperature control. Peptides were synthesized (0.1 mmol scale) using Rink amide MBHA resins pre-loaded with the first amino acid (AnaSpec, Inc., Cambridge Bioscience Ltd, Cambridge, UK) and employed an N-a-Fmoc protection strategy with HCTU activation. Deprotection with 7 ml of 20% piperidine was performed for 3 min at 50 W/758C A majority of AA coupling reactions were accomplished with a 4-fold molar excess of Fmoc-protected AA with HCTU and diisopropylethylamine (DIPEA), molar ratio of 1:1:2 (AA/HCTU/ DIPEA), in 4 ml for 10 min at 25 W/758C. Arg coupling was performed in two stages: 30 min 0 W/258C followed by 5 min at 17 W/758C. To reduce racemization of Cys and His, coupling conditions were 5 min at 0 W/258C followed by 6 min at 17 W/508C with the hindered base collidine (TMP) at a molar ratio of 1:1:2 (AA/HCTU/TMP; Palasek et al., 2007). Aspartimide formation was reduced by the substitution of piperidine for 5% piperazine and 0.1 M 1-hydroxybenztriazole hydrate (HOBt) in the deprotection solution (Palasek et al., 2007). Both microwave-assisted and manually synthesized peptides were purified to apparent homogeneity by semi-preparative scale high-performance liquid chromatography, and the predicted masses of all peptides used (average M + H+) were confirmed to an accuracy of +1 by matrixassisted laser desorption ionization (MALDI) time of flight mass spectrometry operated in a positive ion mode using a-cyano-4-hydroxycinnamic acid (Sigma) as a matrix (Jones et al., 2008).

Synthesis of biotin-conjugated CPPs and bi-functional TP10 derivatives Biotin-labelled peptides, biotinyl-tat, biotinyl-penetratin and biotinyl-TP10 were synthesized on Rink-amide MBHA resin employing an N-a-Fmoc protection strategy using an Applied BiosystemsTM model 433A automated peptide synthesizer. To generate biotinyl-TP10, after orthogonal deprotection, biotin was coupled manually to the 1 group of Lys7 (Soomets et al., 2000) and was achieved by mixing biotin (4 eq.), HOBt (4 eq.), O-benzotriazole-1-yl-N,N,N ′ ,N ′ -tetramethyluronium tetrafluoroborate (TBTU, 4 eq.), DIPEA (8eq.) in DMF/DMSO (4:1) overnight at

Mammalian cell culture Swiss 3T3 cells were routinely maintained in a humidified atmosphere of 5% CO2 at 378C in DMEM supplemented with L-glutamine (0.1 mg/ml) 10% (wt/vol) fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml).

Isolation of bovine spermatozoa Bovine spermatozoa were isolated from seminal plasma by the ‘swim-up’ method as outlined by Nash et al. (1996). 150 ml of bovine semen were thawed at 378C from liquid nitrogen storage and dispensed beneath 1 ml supplemented Earl’s Balanced salt solution (sEBSS containing, 1.8 mM CaCl2.2H2O, 5.37 mM KCl, 0.81 mM MgSO4.7H2O, 26.2 mM NaHCO3, 1.0 mM NaH2PO4.2H2O, 116.4 mM NaCl, 55.6 mM D-glucose, 2.73 mM Na pyruvate, 41.8 mM Na lactate) and further supplemented with 0.3% (wt/vol) charcoal-delipidated/fatty acid free Fraction V Bovine Serum Albumin. Following incubation at 378C with 5% CO2, for 1 h only to avoid capacitation conditions, a 200 ml/sample of the upper phase of sEBSS (removing no more than 700 ml), containing 3 × 106 cells/ml, was removed and centrifuged at 1500g for 5 min. Pellets were immediately re-suspended in indicated treatments.

Human sperm cell preparation Semen samples were collected from healthy normozoospermic [as defined by the World Health Organization (WHO) 1999 guidelines] donors by masturbation into sterile plastic containers after 2 – 3 days of sexual abstinence. Sperm cells were isolated using a two-layer density gradient composed of 40 and 80% PureSperm (Nidacon Int AB, Mo¨lndal, Sweden) diluted with non-capacitating buffer (see below). Semen (maximum of 1 ml) was layered on top of each gradient and centrifuged at 300g for 20 min in 15 ml conical centrifuge tubes. The 80% fractions were transferred to clean tubes by discarding the seminal fluid and the bulk of the density gradient then retrieving the sperm pellet from the bottom of the tube taking care to avoid contamination. Sperm cells were washed in buffer then centrifuged at 500g for 5 min. The resulting pellets were re-suspended in fresh non-capacitating buffer [1.8 mM CaCl2, 5.4 mM KCl, 0.8 mM MgSO4.7H2O, 116.4 mM NaCl, 1.0 mM NaH2PO4, 5.6 mM Dglucose, 2.7 mM Na pyruvate, 41.8 mM Na lactate, 25 mM HEPES, pH 7.4] or capacitating buffer [3.0 mM CaCl2, 4.7 mM KCl, 1.0 mM MgSO4.7H2O, 106.0 mM NaCl, 1.5 mM NaH2PO4, 5.6 mM d-glucose, 1.0 mM Na pyruvate, 41.8 mM Na lactate, 1.33 mM glycine, 0.68 mM glutamine, 0.07 mM taurine, 0.01 mM NEAA, 30 mg/ml BSA, 25 mM NaHCO3, pH 7.4].


A majority of peptides used in this study were manually synthesized (0.1– 0.2 mmol scale) on Rink amide methylbenzhydrylamine (MBHA) resin (Novabiochem, Beeston, UK) employing an N-a-Fmoc protection strategy with O-(6-chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU; AGTC Bioproducts, Hessle, UK) activation. Fluorescent peptides, to be used in confocal live cell imaging and quantitative uptake analyses, were synthesized by amino-terminal acylation with 6-carboxy-tetramethylrhodamine (TAMRA) (Novabiochem, Beeston, UK) as previously described (Jones et al., 2008).

room temperature. Under the same coupling conditions, biotin was coupled manually to the N-termini of tat and penetratin to generate biotinyl-tat and biotinyl-penetratin. To generate rho-biotinyl-TP10, biotinylTP10 was N-terminally extended with (5)-6-carboxytetramethylrhodamine (TAMRA) using HOBt (1eq.), N,N ′ -diisopropylcarbodiimide (DIC, 1eq) and DIPEA (3eq.) in N-Methyl-2-pyrrolidone (NMP), mixed overnight at room temperature. Biotin-labelled CPP and TP10 derivatives were purified by preparative reversed-phase HPLC (Agilent 1200) and predicted masses confirmed by MALDI-time of flight mass-spectrometry (Voyager-DETM PRO BiospectrometryTM System). Mass spectra were acquired in positive ion reflector mode using a-cyano-4-hydroxycinnamic acid as a matrix and purity was 95% as determined by analytical HPLC. Sequences, abbreviations and masses of all peptides used in this study are shown in Table I.

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Table I Peptide sequences, abbreviations and masses. Peptide (abbreviation)

Sequence

Mass

............................................................................................................................................................................................. H-RQIKIWFQNRRMKWKK-NH2

2246.8

Tata

H-GRKKRRQRRRPPQ-NH2

1719.1

C105Ya

H-CSIPPEVKFNKPFVYLI-NH2

1994.5

Mitoparan (MitP)

H-INLKKLAKL(Aib)KKIL-NH2

1608.1

Inverso mitoparan (iMitP)a

H-inlkklakl(Aib)kkil-NH2

1608.1

Inverso mastoparan (iMP)a

H-inlkalaalakkil- NH2

1479.9

Transportan 10 (TP10)a

H-AGYLLGKINLKALAALAKKIL-NH2

2182.8

Nosangiotide

H-RKKTFKEVANAVKISA-NH2

1790.2

Camptide

H-RKLTTIFPLNWKYRKALSLG-NH2

2405.9

Cyt c 5 – 13

H-KGKKIFIMK-NH2

1092.5

rV1aR102 – 113a

H-DITYRFRGPDWL-NH2

1538.7

Biotinyl-tat

biotin- GRKKRRQRRRPPQ-NH2

1945.3

Biotinyl-penetratin

biotin- RQIKIWFQNRRMKWKK-NH2

2473.4

Rho-biotinyl-TP10

TAMRA-AGYLLGKINLKALAALAKKIL-NH2 biotin

2821.0

a

a

To enable both quantitative and qualitative uptake analysis, peptides were N-terminally extended with TAMRA and subsequently, for example, designated the abbreviation of rho-tat. acids are denoted by capital letters and D-isomers are in lower case. Aib designates the known helix promoter a-aminoisobutyric acid. Peptide masses (M + H+) were confirmed by MALDI time of flight mass spectrometry. L-Amino

Ethical approval Semen donors (aged 20 – 35) were recruited at Ninewells Hospital, Dundee (HFEA Centre 004) in accordance with the Human Fertilization and Embryology Authority Code of Practice Version 8 under local ethical approval (08/S1402/6) from Tayside Committee on Medical Research Ethics.

Quantitative analyses of peptide translocation Translocation efficacies of CPPs and CPP-mediated intracellular delivery of avidin were determined by quantitative uptake analysis of fluorescently labelled moities and based upon the method previously described by Holm et al. (2006). Somatic Swiss 3T3 cells were transferred to six-well plates and grown to 80% confluence. After washing in phenol red-free DMEM, cells were incubated with 5 mM TAMRA-labelled CPP (in phenol red-free DMEM) for 1 h, or as otherwise indicated, at 378C in a humidified atmosphere of 5% CO2. For the formation of biotinylated CPP-fluorescent avidin complexes, 1 mM biotinylated CPP was mixed with either avidin TXR or avidin Alexa Fluorw 488 at a 3:1 molar ratio for 30 min prior to incubation with cells. Cells were then washed four times with phenol-red free Hank’s balanced salt solution (HBSS), detached with 1% (wt/vol) trypsin at 378C, collected by centrifugation at 3000g and lysed in 300 ml 0.1 M NaOH for 2 h on ice. 250 ml of each sample cell lysate were transferred to a black 96-well plate, and analysed using a ThermoFischer Scientific Fluoroskan Ascent FL flurorescence spectrophotometer (lAbs 544 nm/lEm 590 nm). Isolated bovine spermatozoa were re-suspended and incubated using the above treatments and conditions, replacing phenol red-free DMEM for sEBSS. Trypsinization was followed by four washes in sEBSS with cell lysis and analysis as above. To determine the kinetics of CPP uptake, sperm and Swiss 3T3 cells were incubated with fluorescent CPPs for various time periods (see figure legends) and processed as described above.

Data were normalized using GraphPad Prism 5, whereby the mean intracellular fluorescence of tat at a 60 min time point in each experiment was assigned a value of 1.0.

Live cell imaging Live cell imaging analyses was performed using a Carl Zeiss LSM510Meta confocal microscope with live cell imaging chamber. Isolated bovine spermatozoa were re-suspended and incubated with 5 mM TAMRA-labelled CPP or the intracellular probes, MitoTrackerw Deep Red 633 (500 nM), transferrin Alexa Fluorw 488 (50 mg/ml), transferrin Texas Redw (50 mg/ml), dextran Texas Redw (10 mM) and LysoTrackerw Red (75 nM) for 1 h at 378C in a humidified atmosphere of 5% CO2. The formation of biotinylated CPP-fluorescent avidin complexes was performed as described above using a 3:1 molar ratio (CPP: protein). All treatments were dissolved in phenol red-free sEBSS. Following gentle centrifugation at 1500g for 5 min, pellets were immediately re-suspended in 250 ml sEBSS and transferred to 5% (wt/vol) poly-D-lysine hydrobromide (mW 30 000 – 70 000)-coated glass bottom 35 mm Petri dishes (PAA) for live confocal cell imaging. Swiss 3T3 cells were transferred to glass bottom 35 mm Petri dishes and grown to 80% confluence. Cells were treated with intracellular probes and biotinylated CPP-fluorescent avidin complexes as above, replacing sEBSS with phenol red-free DMEM. Immediately prior to confocal analysis, cells were gently washed (8×) with phenol-red free HBSS and transferred to phenol red-free DMEM.

Cell viability assays Bovine spermatozoa cell density was adjusted to 6 × 105 cells/ml in sEBSS. Spermatozoa were transferred to 96-well plates at 90 ml/well and treated with CPP (1 – 30 mM) for 1 h in a humidified atmosphere of 5% CO2 at 378C. Spermatozoa viability was measured using the CellTiter 96w AQueous Non-Radioactive Cell Proliferation Assay (Promega) according to manufacturer’s guidelines. The reduction of tetrazolium compounds has previously been used as a reliable and


Penetratina

5


of endocytosis and viewed by live confocal cell imaging analysis at 378C. As a marker of clathrin-mediated endocytosis, Swiss 3T3 cells and bovine spermatozoa were treated with Transferrin Alexa Fluorw488 (50 mg/ml) and Transferrin Texas Redw (50 mg/ml), respectively. As a marker of macropinocytosis, both cell types were treated with Dextran Texas Redw (10 mM). To visualize lysosomal formation, Swiss 3T3 cells and bovine spermatozoa were treated with LysoTrackerw Red DND-99 (75 nM). Though LysoTracker stains have been utilized at much higher concentrations (≥333 nM; Thomas et al., 1998) as acrosomal markers, a maximum application concentration of 75 nM, as recommended by the manufacturers, gives no discernible intracellular labelling of bovine spermatozoa. In contrast, application of 75 nM LysoTrackerw Red DND-99 strongly labels lysosomal vesicles in Swiss 3T3 cells. To refine these results, both cell types were treated with two LysoSensorTM probes, LysoSensorTM Green DND-189 pKa 5.2 (1 mM) and LysoSensorTM Green DND-153 pKa 7.5 (1 mM), which exhibit a pH-dependent increase in fluorescence intensity. Most significantly and in accordance with an absence of endocytotic machinery, there is no evidence of vesicularized ultrastructures in LysoSensor-treated spermatozoa as is observed in Swiss 3T3 cells. Both LysoSensors do demonstrate diffuse labelling of the head regions of bovine spermatozoa and signal intensity is enhanced with LysoSensorTM Green DND-189 pKa 5.2. However, this acidotropic probe does not specifically label acrosomal regions (Castro-Gonza´lez et al., 2010). Fluorescent distributions of endocytotic markers (first and third columns) are also presented merged with images taken under differential interference contrast (second and fourth columns) so as to assist with the visualization of subcellular distribution. Scale bars: for Swiss 3T3 cells incubated with transferrin, dextran and LysoTrackerw Red DND-99, bars ¼ 10 mm. For Swiss 3T3 cells incubated with LysoSensorTM Green DND-189 and LysoSensorTM Green DND-153, bars ¼ 5 mm. For spermatozoa incubated with transferrin and dextran, bars ¼ 20 mm. For spermatozoa incubated with LysoTrackerw Red DND-99, bars ¼ 50 mm and for spermatozoa incubated with with LysoSensorTM Green DND-189 and LysoSensorTM Green DND-153, bars ¼ 10 mm.

rigorous assessment of spermatozoa viability (Iqbal et al., 2010). The novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt

and the electron coupling reagent phenazine methosulfate (Promega) were chosen as a convenient ‘in-solution’ method for the assessment of viable spermatozoa.


Figure 1 Endocytosis incompetent bovine spermatozoa. Swiss 3T3 cells and bovine spermatozoa were treated with fluorescently labelled markers

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Lipid-PAMPA assay The parallel artificial membrane permeability assay (PAMPA, Millipore) was used as a non-cell-based assay to assess passive permeability of


Figure 2 Intracellular accumulation and differential subcellular localization of CPPs in bovine spermatozoa. (A) Differential subcellular distributions of the CPPs Rho-penetratin, Rho-tat and Rho-C105Y. Isolated bovine spermatozoa were incubated for 1 h with 5 mM TAMRAlabelled peptides and viewed by live confocal cell imaging. Whilst Rhopenetratin demonstrates a more diffuse and generalized distribution (i), Rho-tat is confined to the spermatozoa head, with a predominant fluorescent labelling of the acrosomal region (ii). Rho-C105Y accumulates predominantly and strongly within the post-equatorial and equatorial subdomains of the head, the midpiece and posterior ring and neck (iii, iv) and panel (iv) clearly demonstrates an absence of rhodamine-labelled peptide within the interior head and acrosome. (B) Subcellular distributions of mitoparans and related analogues. Isolated bovine spermatozoa were treated for 45 min with both 5 mM TAMRA-labelled peptides and 500 nM MitoTrackerw Deep Red 633 prior to live confocal cell imaging analysis (i, iv, v, vi, vii). Rho-MitP (i, ii) co-localizes strongly with the mitochondrial midpiece. Panel (ii) represents a bovine spermatozoon solely labelled with Rho-MitP and compares closely to a spermatozoon solely labelled with MitoTrackerw Deep Red 633 (iii). Mitochondrial midpiece labelling by Rho-MitP is purposefully shown here in the absence of MitoTracker staining so as to eliminate any bleed-through artefacts. Rho-TP10 (iv, v) demonstrates a similar subcellular distribution to that of Rho-MitP, whereas the subcellular distributions of Rho-iMP and Rho-iMitP are more generalized (vi, vii). All treatments were performed at 378C including live cell imaging analysis. (C) The TAMRA-labelled non-penetrant peptide, rV1aR102 – 113 was used as a negative control and bovine spermatozoa were treated as above. The confocal image (i) is also presented here merged with an image taken under differential interference contrast (DIC) so as to better visualize sperm morphology (ii). Scale bars: Panel A (i) and (iii) ¼ 20 mm, (ii) and (iv) ¼ 10 mm. Panel B (i) ¼ 20 mm, (ii) and (iii) ¼ 10 mm, (iv) ¼ 100 mm, (v) ¼ 10 mm, (vi) ¼ 20 mm and (vii) ¼ 20 mm. Panel C (i) and (ii) ¼ 10 mm.

Figure 2 Continued

CPP across an artificial membrane. Using a 96-well MultiScreen Permeability plates, the assay measures the ability of compounds to diffuse from a donor to acceptor compartment separated by a PVDF membrane pre-treated with a lipid-containing organic solvent. 4% (wt/vol) l-a-phosphatidylcholine in dodecane was added at 5 ml/well to donor plates. Within 10 min, 5 mM TAMRA-labelled peptide, dissolved in PBS containing 5% DMSO (vol/vol), was also added at 150 ml/well to the

7



Figure 3 Quantitative analysis of peptide translocation. (A) Comparative analysis of CPP translocation efficacies into bovine spermatozoa. Bovine spermatozoa were incubated with TAMRA-labelled CPP (5 mM) for 1 h at 378C. (B and C) Temporal-dependent intracellular uptake of C105Y into bovine spermatozoa and Swiss 3T3 cells. Bovine spermatozoa (B) and Swiss 3T3 fibroblasts (C) were incubated at 378C with TAMRA-labelled peptides (5 mM) for the times indicated. Data are the mean + s.e.m. of three experiments performed in triplicate and are expressed as mean fluorescence (minus background) + s.e.m. from three experiments normalized so that the mean value for tat is equal to 1.0 (see the section Materials and methods).

donor plates. 300 ml PBS containing 5% DMSO was added to each well of the acceptor plates and donor plates placed directly on top. Following 6 h of incubation at room temperature, the contents of the acceptor plates were transferred to a black 96-well plate and analysed using a ThermoFischer Scientific Fluoroskan Ascent FL fluorescence spectrophotometer (lAbs 544 nm/lEm 590 nm). Apparent permeability (cm/s) was calculated using the following equation (Seo et al., 2006):

Papp =

Vr [dCr /dt] ACd

whereby Vr is the volume of the acceptor well (0.30 cm3), dCr is the change in concentration of the acceptor compartment, dt is the change in time (seconds), A is the area of the filter membrane corrected for

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Figure 4 Verification of CPPs accretion in intracellular structures of bovine spermatozoa. (A) Degradation of possible surface-associated CPP. To establish that detected fluorescence was not attributable to surface-associated CPP, isolated bovine spermatozoa were incubated for 1 h with 5 mM TAMRA-labelled CPP and subsequently treated with 1% (wt/vol) trypsin at 378C prior to visualization using live confocal cell imaging. Intracellular accumulation of TAMRA-labelled CPPs, presented here, demonstrates no discernible difference compared with bovine spermatozoa treated with TAMRA-labelled CPP in the absence of trypsin. (B) To further establish that our fluorescently labelled CPPs were not merely surface associated we extended our investigations of polycationic CPP to include any observed co-localizations with established intracellular probes. Isolated bovine spermatozoa were incubated for 1 h with Rho-tat (5 mM) and LysoTrackerwGreen DND-26 (2 mM) and subsequently treated with 1% (wt/vol) trypsin at 378C prior to visualization using live confocal cell imaging. The merged panel demonstrates a clear area of intracellular co-localization (yellow) between LysoTrackerwGreen DND-26 (2 mM) and Rho-tat (5 mM). (C) Demembranation of spermatozoa does not decrease CPP accretion in intracellular structures. Rho-C105Y (5 mM)-treated spermatozoa were treated with 1% (wt/vol) trypsin and subsequently incubated in the presence and absence of 0.2% Triton X-100 for 10 min at 378C. Under both conditions, spermatozoa show a clear accumulation of Rho-C105Y in intracellular structures, whilst an enhancement in fluorescence intensity is evident in those spermatozoa treated with Triton-X100. Scale bars: Panel A, rhotat ¼ 5 mm and rho-penetratin ¼ 10 mm. Panel B ¼ 5 mm and Panel C ¼ 20 mm.

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Table II Differential subcellular localization of CPPs in bovine spermatozoa. Peptide

Subcellular compartment

.................................................................................................................................................................... Principal piece (tail)

Midpiece

Head (including equatorial regions and non-acrosomal regions)

Acrosome

Posterior ring and neck

............................................................................................................................................................................................. Penetratin

**

Tat

*

**

**

C105Y

**

***

***

MitP

*

***

*

iMitP

**

***

**

*

iMP

**

***

**

*

TP10

*

***

*

**

** *** ***

The degree of TAMRA-labelled CPP accumulation within subcellular compartments is indicated by (***) for heavy staining, (**) for medium staining and (*) for light staining.

Motility assays Media were prepared (either capacitating or non-capacitating) and peptides were added to individual volumes. Sperm cells were added to the media to give a total volume of 100 ml, a sperm cell concentration of 10 million/ml and peptide concentrations of 5 mM. Sperm cells in media only were used as control samples. All samples were kept at 378C throughout the experiments and under 6% CO2 when capacitating conditions were employed. 6 ml volumes of samples and controls were loaded into individual chambers of MicrocellTM (Vitrollife, San Diego, CA) 4 chamber slides (20 mm depth) which were pre-heated to 378C. This temperature was maintained while data were collected for 600 cells from each chamber. Each peptide was tested on samples from three individual donors. Motility data were collected using a Sperm Class Analyzer (Microptic S L, Barcelona, Spain) with SCAw v5.1 software.

Results Bovine spermatozoa lack endocytotic routes of internalization To confirm that bovine spermatozoa lacked the capacity for endocytotic uptake, isolated spermatozoa were treated with fluorescently labelled markers of endocytosis and analysed by live confocal cell imaging. Figure 1 clearly demonstrates that this differentiated haploid cell type is incapable of both clathrin-mediated endocytosis and macropinocytosis, whilst also being deficient in lysosomes. Somatic mammalian Swiss 3T3 cells were used as a comparative model of endocytotic competence (Fig. 1).

Chemically heterogeneous CPP readily translocate into bovine spermatozoa Live confocal cell imaging analysis clearly reveals the intracellular accumulation of selected CPP within bovine spermatozoa (Fig. 2A and B), whilst quantitative uptake analysis demonstrated a range of translocation efficacies amongst those CPPs tested (Fig. 3A). Moreover, live cell imaging demonstrated a distinct differential subcellular accumulation of rhodamine-labelled CPPs. Derived from the antennapedia

homeodomain (Derossi et al., 1994), penetratin (5 mM) accumulated within the tail, midpiece, posterior ring and neck and the equatorial regions of the head (Fig. 2A(i)). HIV-derived tat (Vives et al., 1997) demonstrated a distinct accumulation within the head particularly the acrosomal region (Fig. 2A(ii)). Moreover, Fig. 4B indicates that when higher concentrations of LysoTrackerw (2 mM) were used as an acrosomal stain, a clear area of intracellular co-localization between LysoTrackerwGreen DND-26 (2 mM) and Rho-tat (5 mM) was observed. As an atypical CPP with minimal cationic charge and only recently identified, C105Y (Rhee and Davis, 2006) demonstrated a strong propensity for intracellular accumulation, giving the highest intracellular translocation efficacy of all peptide tested (7.29 + 0.70, Fig. 3A). Imaging analysis also showed intense staining of bovine spermatozoa (Fig. 2A(iii)). However, Figure 2A(iv) more clearly depicts the ultrastructural accumulation of C105Y. Whilst being absent from the interior head and acrosome, C105Y appears to markedly accumulate within the post-equatorial and equatorial subdomains of the head, the midpiece and posterior ring and neck. Isolated from Vespula lewisii, the tetradecapeptide MP has spawned many additional analogues, both endogenously derived and synthetically designed (Jones and Howl, 2006). One such analogue, the chargedelocalized and highly potent cytotoxin and secretagogue mitoparan (MitP) readily translocate plasma membranes of somatic cells to target mitochondria and enhances mitochondrial permeability with a subsequent induction of apoptotic events (Jones et al., 2008). We have also previously characterized a range of enantiomer-specific analogues of MP and MitP (Jones and Howl, 2012) and identified two further cell penetrant peptides, the highly efficient CPP inverso-MP (iMP) and a second mitochondrial localizing CPP inverso-MitP (iMitP). Figure 2B(i-iii) demonstrates that rho-MitP readily accumulated within the mitochondrial midpiece of bovine spermatozoa. Similarly, the wellcharacterized and inert CPP transportan 10 (TP10), a chimeric MP analogue (Soomets et al., 2000), demonstrated a propensity for mitochondrial colocalization in bovine spermatozoa (Fig. 2B(iv –v)). The enantiomeric analogues iMP and iMitP, though penetrant, showed a less distinct labelling of the midpiece and assumed a more generalized distribution throughout the spermatozoa tail, midpiece and head (Fig. 2B (vi –vii)). A summary of these data is provided in Table II.


porosity (0.24 cm2) and Cd is the concentration of the donor compartment at time 0.

10 A peptide mimetic of the first extracellular loop of the rat V1a vasopressin receptor (rV1aR102 – 113, Howl and Wheatley, 1996) was used as a control non-penetrant peptide. TAMRA-labelled rV1aR102 – 113 demonstrated a lack of propensity for cellular penetration into

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bovine spermatozoa (Fig. 2C), though a minor degree of binding was detected around the posterior ring and neck. Given its enhanced translocation efficacy into bovine spermatozoa, C105Y was further characterized by temporal analysis of intracellular


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uptake and compared with that of tat. Translocation kinetics were carried out using both spermatozoa and the somatic cell line Swiss 3T3 as a comparative model (Fig. 3B and C). Internalization of C105Y and tat occurred with first-order saturable kinetics (F ¼ Fmax x t/t0.5 + t, GraphPad Prism 5). Though the maximum uptake for C105Y (Fmax ¼ 10.24 and 9.05) greatly exceeded that of tat (Fmax ¼ 0.92 and 1.03) in both spermatozoa and Swiss 3T3, respectively, C105Y internalized into spermatozoa at a rate 10 times faster (t0.5 ¼ 0.70 min) than that for somatic Swiss 3T3 fibroblasts (t0.5 ¼ 7.02 min). The half-life for tat internalization (t0.5) was calculated at 2.64 min and 1.66 min for spermatozoa and Swiss 3T3, respectively. Due to the experimental protocol, including a centrifugation step, the first reliable time point that we could determine in these assays was at 5 min.

To establish that detected fluorescence was not attributable to surface-associated CPP, CPP (5 mM)-treated spermatozoa were incubated with high concentrations of exogenous trypsin that rapidly degrades polycationic peptides. Figure 4A indicates that CPP distributions were not influenced by exogenous trypsin treatment and confirms that these observed distributions are indeed intracellular. To further confirm that our fluorescently labelled CPPs were not merely surface associated, we extended our confocal investigations of polycationic CPP to include any observed co-localizations with established intracellular probes. Besides our reported mitochondrial localizing CPPs, MitP and TP10, which co-localized with MitotrackerTM-stained mitochondria in bovine spermatozoa (Fig. 2B), higher concentrations of LysoTrackerw were used as an acrosomal stain. Figure 4B demonstrates a clear intracellular co-localization between LysoTrackerwGreen DND-26 (2 mM) and Rho-tat (5 mM). Live confocal cell imaging analyses were similarly performed following exogenous trypsin treatment. To refine and extend these observations, Rho-C105Y (5 mM)treated spermatozoa were firstly incubated with high concentrations of exogenous trypsin followed by demembranation using 0.2% Triton X-100 as previously described (Ho et al., 2002). Live confocal cell imaging analyses confirmed that intracellular fluorescence did not decrease following demembranation (Fig. 4C) and clearly indicates the propensity of CPPs to accrete in intracellular structures of bovine spermatozoa. Corresponding quantitative measurements using

Reduced efficacy of CPP-mediated protein delivery into bovine spermatozoa CPPs have proved to be efficient vectors for the intracellular delivery of the large protein cargoes into mammalian cells (Langel, 2011). Thus, biotinylation of the well-characterized CPPs tat and penetratin allowed for the assessment of CPP delivery of the large 66 kDa protein avidin into bovine spermatozoa. Figure 5A demonstrates that whilst avidin Texas Redw (TXR) appeared to accumulate around the equatorial and post-equatorial ridge subdomains of spermatozoa following delivery with biotinyl-tat and biotinyl-penetratin, avidin TXR alone also accumulated within these subdomains. These observations negate any successful CPP-mediated delivery of avidin into the intracellular milieu of bovine spermatozoa. To quantify the confocal observations in Fig. 5, translocation efficacies were quantitatively determined in bovine spermatozoa and Swiss 3T3 cells. The ratio of mean intracellular fluorescence (n ¼ 6) comparing delivery into Swiss 3T3 cells and bovine spermatozoa of rho-tat was calculated to be 3.95. Under identical experimental conditions, the corresponding ratio of biotinyl-tat-mediated protein delivery (avidin TXR) into Swiss 3T3 versus spermatozoa was 17.78. Thus, the efficiency of CPP-mediated protein delivery into spermatozoa compared with that of Swiss 3T3 is 22%. Synthesis of rho-biotinyl-TP10 and complex formation with avidin Alexa Fluorw 488 permitted the dual intracellular detection of both CPP and its protein cargo. As indicated in Fig. 5B, biotinyl-TP10 facilitated the intracellular delivery of avidin into Swiss 3T3 cells. Both rho-biotinyl-TP10 and avidin Alexa Fluorw 488 translocated fibroblast membranes to assume vesicular endocytotic distributions within the cytoplasm. In contrast, treatment of bovine spermatozoa with this dual-labelled biosensor further confirmed that CPP-mediated protein uptake was relatively inefficient. In these experiments we observed a consistent lack of intracellular fluorescence emitted by either of the two fluorophores.

Figure 5 CPP-mediated protein delivery into bovine spermatozoa. (A) Biotinyl-CPP constructs of tat and penetratin were synthesized and complexed with avidin Terxas Red (TXR) at a 3:1 molar ratio to assess the utility of CPP for the intracellular delivery of large protein cargoes such as avidin (see caption). Following 1 h incubation with bovine spermatozoa, live confocal cell imaging analysis demonstrated a distinct absence of CPP-mediated intracellular uptake of TXR-labelled avidin. Moreover, avidin TXR alone clearly demonstrated a degree of non-specific binding to equatorial and postequatorial ridge subdomains of live spermatozoa. Corresponding lower panels are presented here merged with images taken under differential interference contrast (DIC) so as to assist with visualization of subcellular distribution. (B) A TAMRA-labelled biotinylated construct of TP10 (rho-biotinyl-TP10) was synthesized and complexed with avidin Alexa Fluorw 488 at a 3:1 molar ratio to yield a dual-labelled biosensor of both CPP and protein uptake (see caption). Swiss 3T3 (upper panel) and bovine spermatozoa (lower panel) were treated for 1 h with the dual-labelled biosensor and viewed by confocal live cell imaging. Both rho-biotinyl-TP10 and avidin Alexa Fluorw 488 assume intracellular vesicular distributions within Swiss 3T3 cells, including liberated cargo (green fluorescence), liberated TP10 (red fluorescence) and colocalized moieties (yellow). In contrast, a distinct absence of both rho-biotinyl-TP10 and avidin Alexa Fluorw 488 is evident in bovine spermatozoa. Scale bars: Panel A, for biotinyl penetratin + avidin TXR, bars ¼ 10 mm, for biotinyl tat + avidin TXR, bars ¼ 20 mm and for avidin TXR bars ¼ 20 mm. Panel B, all bars ¼ 10 mm.


Verification of CPP accretion in intracellular structures of bovine spermatozoa

fluorometric analysis of trypsin-treated spermatozoa pre-labelled with C105Y (5 mM) gave absorbance values of 33.2 + 3.8 in the absence and 40.0 + 3.0 in the presence of 0.2% Triton-X 100 [data are expressed as mean fluorescence (minus background) + s.e.m. of three independent experiments performed on each of two sperm samples (n ¼ 6)].

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CPP import is compatible with sperm viability and motility

Figure 6 (A) Correlation analysis between CPPs translocation efficacies in bovine spermatozoa (ordinate) and mammalian Swiss 3T3 cells (abscissa). (B) Correlation analysis between passive permeability across a phosphatidylcholine membrane (ordinate) and CPP translocation efficacy in bovine spermatozoa (abscissa).

Comparative CPP translocation efficacies Thus far we have established that the primary CPP import mechanism into spermatozoa is that of direct membrane translocation and is based on the following lines of evidence presented herein:

MTS conversion was used to assess whether CPP internalization had any detrimental effects on spermatozoa viability. Figure 7A clearly demonstrates that even up to application concentrations of 30 mM peptide, CPP import had a minimal influence on the viabilities of bovine spermatozoa. Figure 7B shows that CPP application at 5 mM to human sperm cells does not have a detrimental effect on cell motility over a 3 h period. In many cases, in both capacitating and non-capacitating conditions, addition of peptides resulted in increased motility. Motility data were obtained using a diverse range of CPPs, including designated inert CPPs, tat, C105Y and Cyt c 5 – 13 (Jones et al., 2010) and those with bioactive properties in somatic mammalian systems, the bioportides, nosangiotide, camptide and MitP (Howl et al., 2012).

Discussion (i) Bovine spermatozoa lack endocytotic routes of internalization as demonstrated by markers of endocytosis and an absence of lysosome formation (Fig. 1). (ii) TAMRA-labelled CPP do not assume a vesicular morphology following membrane translocation (Fig. 2).

Gross variation in the morphology of spermatozoa is a widely reported phenomenon (reviewed by Fawcett, 1970; Pesch and Bergmann, 2006), though there are many ultrastructural similarities between those of human (Pedersen, 1969) and bovine (Hancock, 1952) origins. Our findings that some CPPs differentially accumulate


These observations are also in accordance with previous reports that the sperm plasma membrane is incapable of active lipid recycling thus rendering sperm cells endocytosis incompetent (Gadella and Evans, 2011). Conversely, CPP import into somatic mammalian cell lines is clearly more complex and entails a combination of different mechanisms including various endocytotic routes in addition to direct membrane translocation. Thus, to refine our deductions, we sought to establish any concordance between CPP uptake into spermatozoa and somatic mammalian cells by comparing translocation efficacies of CPPs into bovine spermatozoa with those efficacies into Swiss 3T3 cells (Fig. 6A). Pearson correlation analysis (two-tailed, GraphPad Prism 5) confirmed there to be no correlation between CPP uptake into sperm and CPP uptake into Swiss 3T3 fibroblasts (P ¼ 0.4695 and R 2 ¼ 0.09032, whereby only 9.03% of the variance in Y could be explained by a variation in X ). To further characterize the translocation properties of CPPs across the spermatozoa plasma membrane, correlation analysis was employed to determine whether a relationship existed between CPP translocation into bovine spermatozoa and CPP translocation across an artificial lipid membrane (Fig. 6B). We have previously employed the parallel artificial membrane permeability (PAMPA) assay to assess passive permeability and direct membrane translocation of a variety of CPP across a pure phosphatidylcholine membrane (Jones and Howl, 2012). Interestingly, Pearson correlation analysis (two-tailed, GraphPad Prism 5) concluded there to be no significant relationship between CPP translocation efficacies in live bovine spermatozoa and assigned CPP passive permeability values (Papp) generated from the PAMPA assay (P ¼ 0.4233 and R 2 ¼ 0.1095, whereby only 10.95% of the variance in Y could be explained by a variation in X ). Intriguingly, C105Y with the highest translocation efficacy in live bovine spermatozoa was assigned a Papp of 0.0.


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in discrete structural compartments of bovine spermatozoa could be relevant to studies of human sperm ultrastructure (Pesch and Bergmann, 2006). Furthermore, and as recently reviewed (Langel, 2011; Verdurmen and Brock, 2011; Svensen et al., 2012) the utility of CPPs as cell- and tissue-selective pharmacokinetic modulators could be exploited to investigate fundamental processes of sperm physiology that include maturation, capacitation, hyperactivation and fertilization. The lipid composition of the plasma membrane surrounding highly polarized mammalian spermatozoa is also compartmentalized to ensure regional variations in glycoprotein and lipid composition that are related to physiological function (Wolf et al., 1986; Lenzi et al., 1996; James et al., 2004; Pesch and Bergmann, 2006). Indeed, the sperm plasma membrane is subject to intensive modification during transit within the epididymis (reviewed by Lenzi et al., 1996) and regional-specific surface domains in mature spermatozoa play

significant roles in processes such as acrosomal exocytosis and fusion to the ooycyte plasma membrane (reviewed by Gadella and Evans, 2011). Hence, prior to the studies reported herein it was uncertain whether common CPPs, so effective in other mammalian cell types, would readily translocate the sperm plasma membrane that is composed of a quite unique composition of lipids including a significant concentration of polyunsaturated fatty acids and plasmalogen (Lenzi et al., 1996). We were also aware that mammalian cells possess numerous endocytotic mechansims, all of which could contribute to the influx of CPPs (Duchardt et al., 2007; Langel, 2011; Madani et al., 2011). As reported herein, our cytochemical investigations confirmed that bovine spermatozoa lack lysosomes and are incapable of macropinocytosis or clathrin-mediated endocytosis, mechanisms that may facilitate CPP entry into mammalian cell types including Swiss 3T3 fibroblasts (Richard et al., 2003). Thus, we conclude that direct translocation across the plasma membrane is the primary mechanism


Figure 7 CPP import is compatible with sperm viability and motility. (A) Isolated bovine spermatozoa were treated with CPP for 1 h at the concentrations indicated. Cell viability was measured by MTS conversion and expressed as a percentage of those spermatozoa treated with vehicle alone (sEBSS). Data points are means + s.e.m. from three independent experiments performed on each of two sperm samples (n ¼ 6). (B) Motility data were collected from human sperm cells treated with six different CPPs. Each peptide was tested on samples from three individual donors. Data are shown as % rapid cells from treated samples relative to that of controls at six time points over 3 h (5, 15, 30, 60, 120 and 180 min) and expressed as the mean + s.e.m. MitP, nosangiotide and tat peptides were tested in non-capacitating buffer (i) and camptide, Cyt c 5 – 13 and C105Y were tested in capacitating media (ii). *rapid cells ¼ velocity (average path) ≥25 mms21 and straightness ≥80%.

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et al., 2005). We have very recently proposed (Jones and Howl, 2012) that two very different phenomena may account for the differential intracellular accumulation of monomeric CPPs. Firstly, the translocation of CPPs into sperm cells may be influenced by the distinct lipid and glycoprotein composition of membrane surface domains (Phelps et al., 1990; Wolf et al., 1990; James et al., 2004). The sperm plasma membrane is compartmentalized to enable fusion events and other physiological functions (Gadella and Evans, 2011). Moreover, the plasma membrane surrounding the sperm head contains diffusion barriers that may restrict the lateral diffusion of proteins and lipid rafts thus sequestering molecular complexes within distinct domains such as the equatorial segment (James et al., 2004) that can be rearranged during capacitation (Gadella et al., 2008). One possibility, therefore, is that individual CPPs preferentially interact with lipid domains within the sperm plasma membrane and so translocate at these sites with varying degrees of efficacy. Even modest changes to the sequence of polybasic arginine-rich peptides can markedly change their interaction with biological membranes (Walrant et al., 2011). It is noteworthy that the membrane potential (Vm) of uncapacitated bovine sperm ( 230 mV; Zeng et al., 1995) would provide a reduced electrochemical driving force for cationic peptide entry (Rothbard et al., 2005) compared with a majority of eukaryotic cells (Vm 275 mV). However, hyperpolarization (Vm 260 mV) during capacitation, a consequence of increased K+ current (Navarro et al., 2007), would increase the electrochemical driving force to exacerbate the unidirectional rate of CPP uptake. A second factor that influences the observed intracellular distribution of fluorescent CPPs is their specific binding to intracellular protein targets (Jones et al., 2008; Jones and Howl, 2012). This is best exemplified by the observed intracellular distribution of mitoparan, a cell-penetrant bioportide (Jones et al., 2008; Howl et al., 2012) that is selectively sequestered by the mitochondria of both sperm and other eukaryotic cell types. Unfortunately, our efforts to more precisely analyse and define these dynamic processes, using time lapse photography, have been hampered by the rapid translocation kinetics of CPPs into sperm. Our results indicate that the utility of CPPs as pharmacokinetic modulators could be adapted to deliver bioactive cargoes to specific compartments of mammalian sperm, thus providing a strategy to investigate and modulate those physiological processes essential for fertilization. Biological investigations also provided overwhelming evidence that CPPs have no discernible influence upon the viability of spermatozoa. Furthermore, both capacitated and non-capacitated human spermatozoa maintained motility when exposed to a range of chemically diverse CPPs and bioportides. Thus, it should be possible to design mimetic peptides that can modulate the various calcium stores and signal transduction pathways that are so fundamental to sperm physiology (Abou-haila and Tulsiani, 2009; Costello et al., 2009). CPP technologies might also be exploited to load sperm with bioactive agents (e.g. siRNA) that, following fertilization, are active in later events critical to the development of the blastocyst (Boerke et al., 2007). There is a caveat, however, as our results indicate that the delivery of larger proteins such as avidin (68 kDa) into spermatozoa using a variety of common CPPs is a relatively ineffective process most likely a consequence of the lack of the endocytotic machinery required for the trafficking of larger CPP-protein constructs (Raä¨gel et al., 2009; Mishra et al., 2011). Nevertheless, the smaller proteins Rab3A and GST, both 25 kDa, are delivered into sperm


by which cationic CPPs gain entry into the intracellular compartments of spermatozoa. This conclusion is further evidenced by a lack of correlation when comparing the uptake efficacies of CPPs for sperm and Swiss 3T3 cells. Our kinetic investigations indicate that the direct translocation process is rapid, saturable and, in terms of efficacy, CPP sequence dependent. The same investigations also identified the C105Y peptide (Rhee and Davis, 2006) as the most efficient delivery vector for sperm studies. Intriguingly, with a Papp of 0.0, C105Y did not translocate across a pure phosphatidylcholine membrane. Whilst phosphatidylcholine is a major component of eukaryotic cell membranes and the PAMPA assay is a reliable tool for the measurement of passive permeability, the variations in lipid composition and the highly compartmentalized features of the sperm plasma membrane, cannot readily be replicated in such a system. Clearly though, the unique lipid composition of the mammalian spermatozoa membrane favours the incorporation of the CPP C105Y, which is not a phosphatidylcholine-dependent phenomenon. There is in fact a bulk of published evidence to support the contention that CPPs may directly interact with anionic lipid membranes or their components. Such interactions may facilitate non-canonical routes of internalization via mechanisms such as dynamic localized membrane perturbations (Hirose et al., 2012) promoting inverted micelles or pore formation (Baumga¨rtner et al., 2007; Alves et al., 2008; Ter-Avetisyan et al., 2009; Yesylevskyy et al., 2009; Alves et al., 2011; Mishra et al., 2011). The formation of membrane-soluble ion-pair complexes is an alternative explanation for the efficient uptake of some guanidium-rich transporters (Rothbard et al., 2005). Clearly, distinguishing direct membrane translocation from other process such as adsorptive-mediated endocytosis (Drin et al., 2003) and the localized formation of ‘particle-like’ multivesicular structures (Hirose et al., 2012) is very difficult indeed in a majority of eukaryotic cells that possess a multitude of endocytotic mechanisms (Duchardt et al., 2007; Gump and Dowdy, 2007; Langel, 2011; Madani et al., 2011). Thus, in this regard the spermatozoon represents a convenient and quite unique cell type in which to study the direct membrane translocation of CPPs in the absence of endocytotic events. Moreover, the observations presented herein, that CPPs but not CPP-protein (avidin) cargoes were readily internalized by sperm, further indicates that large-sized cargos may require endocytotic uptake pathways. One of the most fascinating aspects of this study was the consistent observations that different rhodamine-conjugated CPPs preferentially accumulated in different sub-cellular compartments of bovine spermatozoa. We deliberately selected a chemically diverse range of CPPs for this study, including D-amino acid analogues of mastoparan and mitoparan (iMP and iMitP; Jones and Howl, 2012), since the substitution of D-amino acids into certain CPPs can lead to a stereochemicaldependent reduction in uptake efficiency in selected cell types (Verdurmen et al., 2011). Moreover, retroinverso transformation of CPPs may also promote a dramatic increase in cytotoxicity via the destabilization of both plasma and mitochondrial membranes (Holm et al., 2011). When observing intracellular distribution we were also particularly wary of the reported changes of sperm morphology resulting from dying cells, exposure to low temperatures or fixation (Hancock, 1952; James et al., 2004; Pesch and Bergmann, 2006) and so restricted our observations to viable cells harvested by the swim-up method and maintained at 378C in a physiologically relevant buffer containing bicarbonate and bovine serum albumin (Bedu-Addo

Jones et al.


Authors’ roles S.J.: study design, peptide synthesis, confocal microscopy, biochemical assays, data interpretation, wrote manuscript, M.L.: peptide synthesis, confocal microscopy, data interpretation, J.S.: peptide synthesis, manuscript correction, S.O.: study design, motility assays, data interpretation, manuscript correction, C.B.: study design, data interpretation, manuscript correction, S.P.: study design, data interpretation, manuscript correction, D.M.C.: peptide synthesis, manuscript correc¨ .L.: study design, data interpretation, manuscript correction, tion, U J.H.: study design, peptide synthesis, data interpretation, wrote manuscript.

Funding We are pleased to acknowledge financial support from the following sources: The Wellcome Trust, TENOVUS (Scotland), University of Dundee, Medical Research Council, NHS Tayside and Scottish Enterprise and the Research Institute in Healthcare Science, University of Wolverhampton.

Conflict of interest None declared.

References Abou-haila A, Tulsiani DRP. Signal transduction pathways that regulate sperm capacitation and the acrosome reaction. Arch Biochem Biophys 2009;485:72 – 81.

Bedu-Addo K, Lefie`vre L, Mosely FLC, Barratt CLR, Publicover SJ. Bicarbonate and bovine serum albumin reversibly ‘switch’ capacitation-induced events in human spermatozoa. Mol Hum Reprod 2005;11:683 – 691. Boerke A, Dielman SJ, Gadella BM. A possible role for sperm RNA in early embryo development. Theriogenology 2007;68S:S147– S155. Castro-Gonza´lez D, Alvarez M, Muro J, Esteso MC, de Paz P, Anel L, Martıńez-Pastor F. The acidic probe LysoSensor is not useful for acrosome evaluation of cryopreserved ram spermatozoa. Reprod Domest Anim 2010;45:363– 367. Costello S, Michelangeli F, Nash K, Lefievre L, Morris J, Machado-Oliveira G, Barratt C, Kirman-Brown J, Publicover S. Ca2+-stores in sperm: their identities and functions. Reproduction 2009;138:425 – 437. Derossi D, Joliot AH, Chassaing G, Prochiantz A. The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem 1994;269:10444 – 10450. Drin G, Cottin S, Blanc E, Rees AR, Temsamani J. Studies on the internalization mechanisms of cationic cell-penetrating peptides. J Biol Chem 2003;278:31192 – 31201. Duchardt F, Fotin-Mleczek M, Schwarz H, Fischer R, Brock R. A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 2007;8:848 – 866. Fardilha M, Esteves SLC, Korrodi-Grego´rio L, Pelech S, da Cruz e Silva OAB, da Cruz e Silva E. Protein phosphatase 1 complexes modulate sperm motility and present novel targets for male infertility. Mol Hum Reprod 2001;17:466 – 477. Fawcett DW. A comparative view of sperm ultrastructure. Biol Reprod 1970;2:90 – 127. Gadella BM, Evans JP. Membrane fusions during mammalian fertilization. Adv Exp Med Biol 2011;713:65 – 80. Gadella BM, Tsai PS, Boerke A, Brewis IA. Sperm head membrane reorganisation during capacitation. Int J Dev Biol 2008;52:473 – 480. Garavito RM, Ferguson-Miller S. Detergents as tools in membrane biochemistry. J Biol Chem 2001;276:32403– 32406. Gump JM, Dowdy SF. TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med 2007;13:443– 448. Guzick DS, Overstreet JW, Factor-Litvak P, Brazil CK, Nakajima ST, Coutifaris C, Carson SA, Cisneros P, Steinkampf MP, Hill JA et al. Sperm morphology, motility, and concentration in fertile and infertile men. N Eng J Med 2001;345:1388 – 1393. Hancock JL. The morphology of bull spermatozoa. J Exp Biol 1952; 29:445– 453. Hirose H, Takeuchi T, Osakada H, Pujals S, Katayama S, Nakase I, Kobayashi S, Haraguchi T, Futaki S. Transient focal membrane deformation induced by arginine-rich peptides leads to their direct penetration into cells. Mol Ther 2012;20:984– 993. Ho H-C, Granish KA, Suarez SS. Hyperactivated motility of bull sperm is triggered at the axoneme by Ca2+ and not cAMP. Dev Biol 2002; 250:208– 217. ¨. Holm T, Johansson H, Lundberg P, Pooga M, Lindgren M, Langel U Studying the uptake of cell-penetrating peptides. Nat Protocols 2006; 1:1001 – 1005. ¨. Holm T, Raä¨gel H, El Andaloussi S, Hein M, Maë M, Pooga M, Langel U Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity. Biochim Biophys Acta 2011;1808:1544 – 1551. Howl J, Wheatley M. Molecular recognition of peptide and non-peptide liagnds by the extracellular domains of neurohypophysial hormone receptors. Biochem J 1996;317:577– 582. ¨ stenson C-G, Howl J, Matou-Nasri S, West DC, Farquhar M, Slaninova´ J, O ¨ stlund P, Kumar S, Langel U ¨ et al. Bioportide: an emergent Zorko M, O concept of bioactive cell penetrating peptide. Cell Mol Life Sci 2012; 69:2951– 2966.


when fused to a polyarginine CPP (RRRQRRKRRRQ) at the C terminus (Lopez et al., 2007) and the translocation of exogenous Rab3A is sufficient to trigger acrosomal exocytosis. Thus, we predict that CPP technologies can be employed to effectively deliver a range of smaller-sized bioactive moieties, peptides, proteins, oligonucleotides and biochemicals, to modulate sperm physiology and function in the absence of cytotoxicity or a generalized detrimental influence upon motility. Specifically, cell-penetrant agents could be employed on immobilized individual sperm prior to their intracytoplasmic injection into oocytes to assist fertilization (Palermo et al., 1992; Montag et al., 2000) or to improve general sperm motility, an obvious index of male fertility (Guzick et al., 2001). Moreover, intrinsically bioactive CPPs or bioportides (Howl et al., 2012) will overcome the limitations associated with the delivery of larger bioactive agents that usually enter cells by an endocytotic route. It is feasible, therefore, to consider K+ channels (Navarro et al., 2007), Ca2+ stores (Costello et al., 2009), protein phosphatases (Fardilha et al., 2001) and a unique isoform (a4) of Na,K-ATPase (Woo et al., 2000) as intracellular targets for cell-penetrant agents capable of modulating sperm motility. The fact that CPPs translocate into a majority of viable sperm in any sample is an obvious advantage to this approach. In summary, our findings demonstrate that structurally diverse CPPs will readily translocate into spermatozoa, which are endocytosisincompetent cells and endorse the potential of these pharmacokinetic modulators to deliver diagnostic and therapeutic agents to spermatozoa.

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