Involvement of Bicarbonate-Induced Radical Signaling in Oxysterol Formation and Sterol Depletion of Capacitating Mammalian Sperm During In Vitro Fertilization

Article (PDF Available)inBiology of Reproduction 88(1) · October 2012with28 Reads
DOI: 10.1095/biolreprod.112.101253 · Source: PubMed
Abstract
This study demonstrates for the first time that porcine and mouse sperm incubated in capacitation media supplemented with bicarbonate produce oxysterols. The production is dependent on a reactive oxygen species (ROS) signaling pathway that is activated by bicarbonate and can be inhibited or blocked by addition of vitamin E or vitamin A or induced in absence of bicarbonate with pro-oxidants. The oxysterol formation was required to initiate albumin dependent depletion of 30% of the total free sterol and >50% of the formed oxysterols. Incubation of bicarbonate treated sperm with oxysterol binding proteins (ORP-1 or -2) caused a reduction of >70% of the formed oxysterols in the sperm pellet but no free sterol depletion. Interestingly, both ORP and albumin treatments led to similar signs of sperm capacitation: hyper-activated motility, tyrosin phosphorylation, aggregation of flotillin in the apical ridge area of the sperm head. However, only albumin incubations led to high in vitro fertilization rates of the oocytes whereas the ORP-1 and -2 incubations did not. A pretreatment of sperm with vitamin E or A caused reduced in vitro fertilization rates with 47% and 100%, respectively. Artificial depletion of sterols mediated by methyl-beta cyclodextrin bypasses the bicarbonate ROS oxysterol signaling pathway but resulted only in low in vitro fertilization rates and oocyte degeneration. Thus bicarbonate induced ROS formation causes at the sperm surface oxysterol formation and a simultaneous activation of reverse sterol transport from the sperm surface which appears to be required for efficient oocyte fertilization.
BIOLOGY OF REPRODUCTION (2013) 88(1):21, 1–18
Published online before print 31 October 2012.
DOI 10.1095/biolreprod.112.101253
Involvement of Bicarbonate-Induced Radical Signaling in Oxysterol Formation and
Sterol Depletion of Capacitating Mammalian Sperm During In Vitro Fertilization
1
Arjan Boerke,
3
Jos F. Brouwers,
3
Vesa M. Olkkonen,
4
Chris H.A. van de Lest,
3
Edita Sostaric,
5
Eric J.
Schoevers,
6
J. Bernd Helms,
3
and Barend M. Gadella
2,3,6
3
Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The
Netherlands
4
Minerva Foundation Institute for Medical Research, Biomedicum Helsinki 2U, Helsinki, Finland
5
Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
6
Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
ABSTRACT
This study demonstrates for the first time that porcine and
mouse sperm incubated in capacitation media supplemented
with bicarbonate produce oxysterols. The production is depen-
dent on a reactive oxygen species (ROS) signaling pathway that
is activated by bicarbonate and can be inhibited or blocked by
addition of vitamin E or vitamin A or induced in absence of
bicarbonate with pro-oxidants. The oxysterol formation was
required to initiate albumin dependent depletion of 30% of the
total free sterol and .50% of the formed oxysterols. Incubation
of bicarbonate treated sperm with oxysterol-binding proteins
(ORP-1 or ORP-2) caused a reduction of .70% of the formed
oxysterols in the sperm pellet but no free sterol depletion.
Interestingly, both ORP and albumin treatments led to similar
signs of sperm capacitation: hyperactivated motility, tyrosin
phosphorylation, and aggregation of flotillin in the apical ridge
area of the sperm head. However, only albumin incubations led
to high in vitro fertilization rates of the oocytes, whereas the
ORP-1 and ORP-2 incubations did not. A pretreatment of sperm
with vitamin E or A caused reduced in vitro fertilization rates
with 47% and 100%, respectively. Artificial depletion of sterols
mediated by methyl-beta cyclodextrin bypasses the bicarbonate
ROS oxysterol signaling pathway but resulted only in low in vitro
fertilization rates and oocyte degeneration. Thus, bicarbonate-
induced ROS formation causes at the sperm surface oxysterol
formation and a simultaneous activation of reverse sterol
transport from the sperm surface, which appears to be required
for efficient oocyte fertilization.
acrosome reaction, in vitro fertilization (IVF), oxidative stress,
porcine/pig, sperm capacitation
INTRODUCTION
Before the sperm cell can enter the oocyte, it first needs to
be activated. This activation process has been described
extensively [1, 2]. One of the hallmarks of this activation
process (also termed capacitation) is the increased binding
affinity of the sperm cell to the zona pellucida (the extracellular
matrix) of the oocyte. The main components thought to be
responsible for sperm capacitation are bicarbonate and Ca
2
þ
levels. Both ions induce signaling cascades after elevated levels
inside the sperm cell [3]. Besides these two ions, albumin acts
in synergy by mediating efflux of sterols from the sperm’s
surface [4, 5]. In boar sperm (one of our model species in this
study), albumin is thought to selectively extract free sterols—
like cholesterol and desmosterol—from the sperm cells [5–7].
Albumin is rather specific for causing sterol depletion from the
sperm surface as phospholipids and glycolipid levels remain
unaltered [1, 2]. Cholesterol is preferentially removed from the
sperm surface despite of its high hydrophobicity when
compared to the other membrane bilayer preferring lipid
classes. Normally, albumin is involved in the transport of free
fatty acid in the circulation from donor to acceptor tissue [8].
One of the mechanisms that could play a role in sterol
depletion is the involvement of an active cholesterol transporter
in providing free cholesterol to the hydrophobic pocket of
albumin (as hypothesized previously [3]). Another option is
that sterols can be oxidized and that their oxidation products
(which are more hydrophilic) can be extracted by albumin
(observed in bovine sperm [4]) or can facilitate an oxysterol-
dependent scavenger-sensitive transport of free sterols to
albumin [9]. Inclusion of albumin to in vitro capacitation
media as well as the concomitant depletion of sterols by
albumin have been shown to be of fundamental importance to
achieve in vitro fertilization (IVF) [5].
Sterol Oxidation in Sperm
In bovine sperm, it has recently been demonstrated that
sperm capacitation leads to the formation of oxysterols, which
are preferentially extracted by albumin [4]. The formation of
oxysterols is believed to be initiated by reactive oxygen species
(ROS) as it can be induced by tert-butylhydroperoxide [4]. The
role of ROS in sperm physiology is ambivalent: On the one
hand, mild ROS formation is reported to be relevant for
oxidation-mediated sperm signaling events that are involved in
sperm capacitation [6, 7]. On the other hand, higher ROS
formation rates are reported to be damaging for the sperm cell
[10, 11].
Sterols and Lipid-Ordered Microdomain Formation on the
Sperm Cell
During in vitro capacitation, a lateral rearrangement of the
sperm surface sterols has been shown to be dependent on
bicarbonate, and this rearrangement preceded and was required
1
This work has been supported by the High Potential Program from
Utrecht University.
2
Correspondence: E-mail: b.m.gadella@uu.nl
Received: 11 April 2012.
First decision: 2 May 2012.
Accepted: 22 October 2012.
Ó 2013 by the Society for the Study of Reproduction, Inc.
This is an Open Access article, freely available through Biology of
Reproduction’s Authors’ Choice option.
eISSN: 1529-7268 http://www.biolreprod.org
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for albumin-mediated sterol depletion [3]. This lateral rear-
rangement coincides with higher membrane fluidity character-
istics of the remaining nonraft membrane area, in which lower
sterol levels are detected compared to the apical area of the
sperm head, membrane where raft aggregation takes place [2,
10, 12]. When albumin and bicarbonate together are used to
capacitate sperm, this causes a partial depletion of sterols
(.15% of total sterol and depletion was only from the nonraft
membrane area [2]). The treatment also leads to the
aggregation of lipid-ordered membrane microdomains at the
apical ridge area of the sperm surface in multiple species.
Several groups [13] independently established that the
detergent-resistant membrane fraction not only contains raft
markers such as caveolin, flotillin, gangliosides, sterols, and
sphingolipids (for reviews, see Gadella et al. [14] and Nixon
and Aitken [15]) but also is very highly enriched in cumulus-
and/or zona-binding proteins [16] and in proteins involved in
docking and priming of the sperm plasma membrane with the
outer acrosome membrane [17, 18]. These surface redistribu-
tions are, therefore, considered to be preparative steps for
interaction of sperm with the zona pellucida and/or cumulus
layer of the oocyte. This binding and induction of the acrosome
reaction enables the sperm to reach the oolemma, where
eventual fertilization can take place of the oocyte (for review,
see Tsai and Gadella [19]).
Sterol Oxidation and Sperm Capacitation
The relationship between capacitation of porcine sperm and
oxysterol formation was therefore investigated in the present
study with the rationale to see whether this formation could be
attributed to the depletion of sterols from the capacitating
sperm surface [20]. To this end, we studied whether incubation
of sperm with oxysterol-binding proteins could induce in vitro
capacitation of porcine sperm and possibly could result in
sperm capable of fertilizing oocytes in vitro in albumin-free
media. Sperm were, therefore, capacitated in the absence of
albumin but in the presence of two recombinant oxysterol-
binding protein-related proteins (ORP-1 and ORP-2) [21]. Both
ORPs have specific binding pockets for oxysterols and are able
to transport bound oxysterols from a donor membrane toward
specific acceptor membranes. The ORP-induced effects were
compared to methyl b cyclodextrin (MBCD) and to delipidated
bovine serum albumin (BSA)-mediated sterol extraction in
capacitating sperm. The role of bicarbonate-dependent oxy-
sterol formation and the subsequent (oxy)sterol depletion in the
induction of sperm capacitation in vitro, as well as the
modulatory role of pro- and antioxidants on these effects, are
discussed.
MATERIALS AND METHODS
Animal Experiments
The Institutional Animal Care and Use Committee of Utrecht University
approved this study.
Protein Expression of ORP-1 and ORP-2
Plasmid expression vectors for ORP-1 or ORP-2 were used for protein
production as previously described [21]. Briefly, glutathione S-transferase
fusion proteins of ORP-2 and ORP-1 were produced in E. coli BL21 and
purified over glutathione sepharose 4B (GE Healthcare, Amersham, Buck-
inghamshire, UK) columns as described by the manufacturer’s instructions.
Protein concentrations of purified ORP-1 and ORP-2 were determined by the
Bradford assay (Coomassie Plus, Pierce, Rockford, IL) according to the
manufacturer’s instruction. Purity of ORP-1 and ORP-2 protein preparations
was analyzed on sodium dodecyl sulfate polyacrylamide gels stained with
Coomassie brilliant blue [22].
Sperm Incubations
Ejaculates were collected from boars with proven fertility at Varkens KI
Nederland (Deventer, The Netherlands), a commercial enterprise producing
insemination doses for pig artificial insemination for sow herds. Freshly
ejaculated sperm was filtered through gauze to remove gelatinous material and
subsequently diluted and washed in HEPES-buffered saline (HBS; 137 mM
NaCl, 2.5 mM KCl, 20 mM HEPES, pH 7.4). Mouse sperm was aspirated from
cauda epididymi of wild-type mice (strain B6129SF2/J; stock number 101045
Jackson Laboratories, Bar Harbor, ME). Next, sperm were washed through a
discontinuous Percoll (GE Healthcare, Diegem, Belgium) gradient of 70% (v/v)
and 35% (v/v) as described [3]. All solutions were made iso-osmotic (290–310
mOsm/kg) with HBS at 238C. Percoll layers were discarded and sperm pellets
resuspended at a final concentration of 100 million sperm cells/ml in Hepes-
buffered Tyrodes media (120 mM NaCl, 21.7 mM lactate, 20 mM Hepes, 5
mM glucose, 3.1 mM KCl, 2.0 mM CaCl
2
, 1.0 mM pyruvate, 0.4 mM MgSO
4
,
0.3 mM NaH
2
PO
4
; 300 mOsm/kg, pH 7.4; HBT condition hereafter referred to
as Bic) or supplemented with 15 mM NaHCO
3
, equilibrated with 5% CO
2
in
humidified atmosphere (hereafter referred to as
þ
Bic). Similarly, sperm were
incubated Bic or
þ
Bic media supplemented with 1) 0.3% w/v BSA (defatted
fraction V; Boehringer Mannheim, Almere, The Netherlands) or 2) with 4–16
lg/ml recombinant ORP-1 or ORP-2 or 3) with 0.5–10 mM MBCD. All
þ
Bic
conditions were incubated with open vials in 5% CO
2
atmosphere for 2 h at
38.58C. All Bic conditions were incubated in airtight vials for 2 h at 38.58Cin
a water bath. In some cases, sperm incubations were carried out in the presence
of either 0.5 mM vitamin E (alpha-tocopherol; Sigma-Aldrich, St. Louis, MO)
or 0.5 mM vitamin A (retinol; Sigma-Aldrich) or in the presence of a pro-
oxidant mix containing 0.2 mM FeSO
4
and 1 mM ascorbate or in the presence
of 30 lM 3-morpholinosydnonimine hydrochloride (SIN-1; Sigma Aldrich) for
the induced formation of peroxynitrite [20, 23].
Lipid Extraction and Mass Spectrometry
After incubation, lipids of 200 million sperm cells were extracted according
to the method of Bligh and Dyer (see Brouwers et al. [4]). Lipids were dried
under nitrogen and redissolved in chloroform/methanol (1/9; v/v), and a
fraction corresponding to ;1 million cells was subjected to reverse-phase
chromatography on a 150-mm 3 3-mm Kinetex 2.6-lm column (Phenomenex,
Torrance, CA), using isocratic elution (methanol/acetonitrile/2-propanol/
chloroform; 90/90/8.5/1.5; v/v/v/v). The column effluent was introduced into
an Atmospheric Pressure Chemical Ionization source of a 4000Qtrap mass
spectrometer (AB Sciex, Foster City, CA) operated under multiple reaction
monitoring mode. Eluting peaks were identified and quantified based on
comparison of retention time and product ion spectra with authentic standard,
as described previously [4]. Briefly, a dose-response curve was made of levels
of 50 fmole to 100 pmole of oxysterol standards and of cholesterol and
desmosterol that were injected into the same reverse-phase column elution. The
integrated detector response (linear to the entire concentration range) was used
to calculate the amounts of each individual (oxy)sterol species form sperm
extracts that were eluting from the reverse-phase column after injection (for
more experimental details, see Brouwers et al. [4]).
Western Blot Immunodetection of Tyrosine
Phosphorylation
After incubation, a total of 2 million sperm cells were resuspended in 25
ll of lithium dodecyl sulfate loading buffer (NuPAGE; Invitrogen, Carlsbad,
CA) in the presence of 0.1 M dithiothreitol and heated for 10 min at 958C.
Subsequently, solubilized proteins were loaded and separated on a 12%
polyacrylamide gel and run at 40 mA for 45 min. Subsequently, proteins
were blotted onto nitrocellulose paper (Protran BA85; Whatman, Dassel,
Germany) at 60 V for 1.5 h. In order to prevent aspecific binding of
antibodies used later in the blotting procedure, the nitrocellulose paper with
blotted proteins was first incubated in blocking buffer (Tris 25 mM, NaCl, pH
7.4, with 0.5% Tween, TBS-Tween 0.5%) for 10 min at room temperature.
Subsequently, the nitrocellulose paper with the blotted and blocked proteins
was incubated with 1% BSA TBS-Tween 0.05% for 1 h at room temperature.
After this step, the nitrocellulose paper, with the blotted proteins, was
incubated in 0.1% BSA TBS-Tween 0.05% supplemented with a mouse
monoclonal antibody raised against phosphotyrosine residues (PY20; Becton
Dickinson Transduction Laboratories, Franklin Lakes, NJ) at a final
concentration of 1 lg/ml overnight at 48C. The resulting nitrocellulose paper
with blotted proteins was washed six times with 10 min per washing step in
TBS. After this washing, the nitrocellulose was placed into TBS-Tween
0.05% containing 0.5 lg/ml monoclonal goat anti-mouse antibody conjugat-
ed with horseradish peroxidase (Nordic Immunology, Tilburg, The Nether-
BOERKE ET AL.
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lands) for 90 min at 48C. The resulting nitrocellulose paper with blotted and
immunolabeled proteins was washed as described before for removal of
unbound PY20 above. Protein bands indirectly immunolabeled with
horseradish peroxidase were visualized by chemiluminescence for 5 min
(enhanced chemiluminescence detection kit; Supersignal West Pico; Pierce,
Rockford, IL) and captured on a Molecular Imager Chemidoc XRS from Bio-
Rad Laboratories (Hercules, CA).
Immunofluoresence Detection of Tyrosine Phosphorylation
and of Flotillin
After incubation, 200 million sperm cells were subsequently spun down
at 600 3 g and resuspended in 1 ml and fixed in 2% paraformaldehyde in
PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na
2
HPO
4
, 1.47 mM KH
2
PO
4
,
pH 7.4) at room temperature for 10 min. The fixative was removed by three
washing steps and centrifugation (600 3 g) steps, and the final pellet was
resuspended with PBS at the original concentration. The washed sperm cells
were then smeared on Superfrost (Microm International, Walldorf,
Germany) glass slides and dried for 5 min at 378C and subsequently
placed in a 208C methanol bath for 1 min for full permeabilization of
membranes to ensure optimal intracellular immunolabeling. The immobi-
lized and permeabilized sperm cells were then incubated in blocking buffer
(PBS containing 1% BSA and 0.05% Tween20) for 1 h in a humid chamber
at 378C. After this step, cells were incubated in buffer containing 0.1% BSA
and 0.05% Tween20 and either supplemented with 5 lg/ml PY20 antibody
or 10 lg/ml mouse monoclonal anti-flotillin-1 (Becton Dickinson Trans-
duction Laboratories) for 1 h at 378C in a humidified chamber. Unbound
antibodies were removed by gently washing the Superfrost glasses with 3
ml of PBS containing 0.1% BSA and 0.05%Tween20. The resulting
specimen on Superfrost glass slides was subjected to 2 lg/ml monoclonal
rabbit anti-mouse antibody conjugated with Alexa-488 TM (Invitrogen) for
labeling PY20 or flotillin-1 for 2 h at 378C in a humidified chamber. After
immunolabeling, the nonbound antibody conjugates with Alexa-488 TM
were removed by three subsequent washing steps with PBS containing 0.1%
BSA and 0.05%Tween20 and finally rinsed with PBS and mounted in
Fluorsave (Calbiochem, San Diego, CA) and sealed airtight with nail polish.
Samples were examined with an Eclipse Ti microscope (Nikon, Tokyo,
Japan) equipped with a mercury lamp and appropriate filters at a minimum
magnification of 403.
Motility Assessment of Incubated Sperm
After sperm was incubated in HBT media, sperm motility was measured by
the SpermVision computer-assisted sperm analysis (CASA; Minitube,
Tiefenbach, Germany) system for 2 h. Sperm motility measurements were
performed in this system by using 20-lm-deep Leja-4 chambers (Leja Products,
Nieuw Vennep, The Netherlands). Standard instrument settings of SpermVision
Version 3.0 were used for the analysis of motility, as used by the artificial
insemination center (Varkens KI Nederland) [24]. For mouse sperm, the
percentage of cells with vigorous lateral head displacement movement was
counted.
In Vitro Fertilization
Ovaries were collected from the slaughterhouse material of adult sows
(VION, Groenlo, The Netherlands). The 3- to 6-mm follicles from individual
ovaries were aspirated to retrieve cumulus-oocyte complexes (COCs). The
COCs were individually selected based using criteria previously described [25].
Subsequently, the selected COCs were matured in vitro [25]. COCs were
collected in HEPES-buffered M199 (Gibco Laboratories, Grand Island, NY)
and washed in pre-equilibrated M199 supplemented with 2.5 mM NaHCO
3
,
0.1% (w/v) polyvinylpyrrolidone, 100 lM cysteamine, 75 lg/ml potassium
penicillin G, and 50 lg/ml streptomycin sulfate (oocyte maturation medium
[OMM]). The selected COCs were cultured for 22 h in humidified atmosphere
in air with 5% CO
2
at 388C in OMM supplemented with 0.05 IU/ml
recombinant human follicle-stimulating hormone (rhFSH; Organon, Oss, The
Netherlands). Subsequently, the COCs were transferred into OMM without
rhFSH for another 22 h. This second step is needed for the oocytes to reach the
final meiosis stage II of maturation (the stage they reach in vivo at ovulation).
The fully matured oocytes were denuded (removal of cumulus cells) and
transferred into an IVF medium (113.1 NaCl, 3 mM KCl, 20 mM Tris, 11.0
mM glucose, 1.0 mM caffeine, 7.5 mM CaCl
2
, and 5.0 mM Na-pyruvate,
supplemented with either 0.1% [w/v] BSA, 4 lg/ml ORP-1, or 4 lg/ml ORP-
2). Oocytes were equilibrated in a humidified atmosphere in air with 5% CO
2
at
388C for at least 1 h before adding boar sperm. The sperm was washed through
Percoll as described above and diluted to a concentration of 10
5
cells/ml in the
same IVF medium as the oocytes. Next, depending on the amount of oocytes
(20–30) per group, 20–30 ll of this sperm suspension were added to the IVF
media that already contained the oocytes. The resulting IVF media contained a
total of 1000 sperm cells per oocyte. Subsequently, the oocytes and sperm cells
were incubated in a humidified atmosphere in air with 5% CO
2
at 388C for 24
h. Next, the oocytes were washed in PBS with 0.1% BSA and fixed in 4%
formaldehyde in PBS for a minimum of 1 h. After fixation, oocytes were
labeled with 1 lg/ml Sytox Green (Molecular Probes, Invitrogen, Leiden, The
Netherlands) in PBS for 5 min to label the chromatin. Subsequently, the
fertilization rate of the oocytes were scored, and for unfertilized oocytes, they
were scored for MII maturation phase rate; for the rate of cells that did not
develop further, the MI maturation stage or even the germinal vesicle stage
were scored as well as the rates of oocytes that were degenerated during this
IVF procedure.
IVF in the Presence of MBCD
To determine the fertilization rate of sperm submitted to 0.5–10 mM
MBCD, the standard IVF procedure was followed as described above, although
all IVF procedures were performed in the absence of BSA. A control IVF
experiment was performed on the same collection batch of follicles.
IVF in the Presence of Antioxidants
Washed sperm cells were pretreated for 30 min with 0.5 mM vitamin A or
0.5 mM vitamin E (each with a final ethanol concentration of 0.1 vol%). A
control sample was pretreated for 30 min with 0.1 vol% ethanol without
antioxidants. The pretreated sperm samples where then diluted 1:10 for further
IVF experiments and further processed as described above.
Determination of the Sperm-Zona Interaction at IVF
Conditions
After all IVF treatments, individual oocytes were, after extensive washing
of each individual oocyte, scored for the amount of sperm cells that had a firm
interaction with the surrounding zona pellucida. This was determined by using
the Sytox Green immunofluorescence and bright-field view on a Leica TCS
SP2 confocal system (Leica Microsystems, Wetzlar, Germany) equipped with a
488-nm laser using the laser power and acquisition settings at a submaximal
pixel value.
Measurement of Acrosome Integrity
The acrosome integrity of incubated sperm was measured after 30 and 120
min by flow cytometry. Sperm cells were stained directly from the different
incubations conditions diluted and supplemented with 1 lg/ml peanut
agglutinin conjugated to fluorescein isothiocyanate (PNA-FITC; EY Labora-
tory, San Mateo, CA) to distinguish acrosome-reacted cells and with the
membrane-impermeable vital stain propidium iodide (PI; final concentration 25
nM). After gentle homogenizing of the sperm suspension, sperm were analyzed
on a FACS Calibur flow cytometer equipped with a 100-mW argon laser
(Becton Dickinson, San Jose, CA). At the wavelength of 488 nm, sperm cells
were excited, and the FITC and PI emission intensity was detected in the
logarithmic mode of FL-1 (530/30-nm band-pass filter) and FL-3 (620-nm
long-pass filter). The forward and sideways scatter was detected in the linear
mode, and sperm-specific events were gated for further analysis. The resulting
two-dimensional dot plots represented 10 000 gated events and were made with
FL-1 data expressed on the x-axis and FL-3 data on the y-axis. The amount of
cells positively stained with one or both fluorescent dyes was scored using
quadrant analysis in Win MDI software (Version 2.8, J. Trotter, freeware). The
percentage of sperm that showed high intensities for the FL-1 channel were
regarded as acrosome reacted. Sperm samples were also stained for PNA-FITC
for microscopic discrimination of acrosome-intact and acrosome-reacted status.
Briefly, after fixation in 2% (w/v) paraformaldehyde for 15 min at room
temperature, sperm cells were spun down at 600 3 g and washed in PBS three
times. Subsequently, sperm cells were smeared on Superfrost glass slides and
dried at 378C. Next, sperm cells were incubated with 100 lg/ml PNA-FITC for
30 min at 378C in a humid chamber. After this, sperm cells were rinsed with
PBS and mounted in Fluorsave (Calbiochem) and sealed airtight with nail
polish. Samples were examined with a Nikon Eclipse Ti microscope equipped
with a mercury lamp and appropriate filters at a minimum magnification of
403. Sperm cells with no fluorescence at the acrosome region and with visible
apical ridges were considered acrosome intact, whereas sperm cells with PNA-
FITC-labeled acrosome regions and no visible apical ridge were considered
acrosome reacted.
OXYSTEROLS IN CAPACITATING MAMMALIAN SPERM
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RESULTS
Bicarbonate Induces an ROS Signaling-Dependent
Oxidation of Cholesterol That Can Be Blocked by Vitamin E
or Vitamin A
The possible oxidation effects of bicarbonate on free sterols
in porcine sperm cells by incubating Percoll-washed sperm in
the absence of sterol-depleting agents were examined.
Incubation of Percoll-washed sperm cells for 2 h in albumin-
free media that were supplemented with bicarbonate induced
the formation of oxysterols (Fig. 1). The levels of oxysterols
increased ;10-fold, from approximately 0.047 mole % of total
sterol (equivalent of 0.062 pmoles oxysterols per million sperm
in the absence of bicarbonate; BicBSA) to ;0.49 mole %
of total sterol (equaling 0.74 pmoles oxysterols per million
sperm in presence of bicarbonate;
þ
BicBSA). The formation
of all types of oxysterols was nearly completely inhibited when
0.5 mM vitamin E or 0.5 mM vitamin A was present during the
incubations (Fig. 1). On the other hand, when sperm were
incubated in the absence of bicarbonate but with added pro-
oxidants (FeSO
4
in the presence of ascorbate), similar
oxysterol formation was observed when compared to bicar-
bonate (Fig. 1), while SIN-1 (stimulates peroxynitrite forma-
tion) did not cause any oxysterol formation (Fig. 1). The
oxysterol species formed after bicarbonate incubation are
depicted in the mass spectrometry graphs (Fig. 2), clearly
depicting the formation of 7- and 25-hydroxycholesterols, 7-
and 22-ketocholesterols, and 5,6a-andb-epoxycholesterols.
Note that the intensity of ions detected as depicted in Figure 2
are corrected for (oxy)sterol-specific ion response curves as
described previously, and thus the peak height/signal is not
necessarily proportional to the amount of oxysterols present
[4]. The relative amounts of oxysterol species out of the total
amounts of oxysterols recovered from sperm are depicted in
Figure 3, and from these data the following findings were
derived: 1) In mouse and porcine sperm, the amount of
oxysterols formed in sperm incubated with bicarbonate or with
pro-oxidants (tested only for porcine sperm; Fig. 1) were
similar to the levels depicted in Figure 1 for
þ
BicBSA and
for BicBSA
þ
pro-oxidants (10-fold when compared to
BicBSA; Table 1). 2) In mouse and porcine sperm, the
presence of BSA did not change the amount of oxysterols
formed compared to the absence of BSA (Fig. 1 and Table 1).
3) The amount and composition of oxysterols formed in sperm
incubated with bicarbonate and vitamin E supplementation did
not differ from control cells (Figs. 1 and 3). 4) The relative
contribution of 7-ketocholesterol increased and 7-hydroxycho-
lesterols decreased in the presence of bicarbonate or after
inclusion of pro-oxidants (Fig. 3). 5) Interestingly, in the
presence of albumin (
þ
BSA
þ
Bic), a relative low proportion of
5,6a-epoxycholesterol and a much higher proportion of 5,6b-
epoxycholesterol and 7-ketocholesterol, as well as a more
pronounced decrease in 7-hydroxycholesterols, were detected
when compared to
þ
BicBSA. This probably relates to the
fact the oxysterols formed actively exchange between the
sperm membrane and albumin, which may affect molecular
interspecies conversions between the sperm surface and the
extracellular albumin (detected in both mouse and porcine
sperm; Table 2).
Vitamin E and A Inhibit the Induction of Hyperactivated
Sperm Motility
Bicarbonate and albumin have been described to synergis-
tically induce hyperactivated sperm motility [26], and we show
here that they also induce oxysterol formation, while vitamin E
and vitamin A inhibited the formation of oxysterols (Figs. 1
and 3). Thus, we tested whether sperm-hyperactivated sperm
motility could be inhibited by vitamin E and vitamin A. About
10% of control (BicBSA; Fig. 4A) sperm showed hyper-
activated sperm motility, whereas incubation for 2 h in the
presence of bicarbonate (
þ
BicBSA) induced signs of hyper-
activated sperm motility, as shown with CASA measurements
in ;20% of the sperm cells (Fig. 4A). Indeed, a synergistic
effect was seen when bicarbonate and albumin (
þ
Bic
þ
BSA)
were added. Under these conditions, 45% of the sperm showed
hyperactive motility (Fig. 4A). In the presence of vitamin E or
vitamin A, hyperactivation of motility by bicarbonate and by
the combination of bicarbonate and albumin was completely
abolished (Fig. 4A). In the presence of BSA, a higher
proportion of sperm cells showed signs of hyperactivated
sperm motility (45%; Fig. 4A). When sperm cells were
incubated in the presence of vitamin E, the induction of
hyperactivated sperm motility, in the presence of bicarbonate
alone, was completely inhibited. Likewise, inclusion of vitamin
E to the IVF-mimicking medium containing both BSA and
bicarbonate also showed a severe but not complete inhibition of
hyperactivated sperm motility (Fig. 4A). For the regulation of
hyperactivated sperm motility (on tyrosine phosphorylation),
similar results were found in mouse sperm, which could be
inhibited by vitamin E or with vitamin A (Table 3).
Vitamin E and A Reduce Sperm-Zona Binding and IVF
Since both vitamin E and vitamin A inhibited bicarbonate-
dependent oxysterol formation and hyperactivated motility
response to bicarbonate (in the presence or absence of
albumin), we tested whether the sperm-binding properties to
the zona pellucida and/or IVF rates were inhibited. We chose to
first entrap high levels (0.5 mM) of hydrophobic antioxidants
into the plasma membrane of sperm, followed by a 10-fold
FIG. 1. Oxysterol formation is induced by a bicarbonate-activated ROS-
dependent pathway. Bicarbonate-enriched medium (
þ
Bic) induces radical
formation, leads to oxidation of cholesterol, and can be blocked by
vitamin E or by vitamin A. Oxysterols were detected by mass spectrometry
(see Aitken et al. [20]). The relative amount of oxysterols as percentage of
cellular cholesterol is expressed (mean % 6 SEM, n ¼3; ejaculates were
obtained from individual boars). * indicates a significant induction of
oxysterol formation when compared to Bic (P , 0.05).
BOERKE ET AL.
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FIG. 2. Chromatograms of multiple reaction monitoring during the elution of (oxy-)sterols extracted from sperm cells. Oxysterols were identified based
on retention time and product spectra as annotated: triol, 3,5,6-trihydroxycholesterol; 7-OH, 7-hydroxycholesterol; 7-keto, 7-ketocholesterol; 22-keto,
22-ketocholesterol; 25-OH, 25-hydroxycholesterol; b-epoxy, 5,6b-epoxycholesterol; a-epoxy, 5,6a-epoxycholesterol. Each panel represents oxysterols
from 1 million sperm cells. The amount of desmosterol and cholesterol (note the discontinuous y-axis to accommodate visualization of the peak height of
these much more abundant sterol species) is depicted as well. The elution profile with m/z transitions intensities of 367.2/159.1 was used for oxysterols
and desmosterol (indicated as 367), the transition of 369.2/161.1 for cholesterol (indicated as 369), and the transition of 401.2/175.1 for ketosterols
(indicated as 401).
OXYSTEROLS IN CAPACITATING MAMMALIAN SPERM
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dilution in antioxidant-free IVF medium, and then perform the
IVF experiment for 24 h. In this way, only sperm and not the
oocyte had been in direct contact with high levels of membrane
antioxidants. The effective vitamin concentration during the
IVF incubation available for the oocytes was ,50 lM.
Pretreatment of sperm in 0.1% ethanol without antioxidants
and 1:10 dilution in IVF medium was used as internal control.
In all treatments, the oocytes did not show significant signs of
degeneration (in all cases ,10%). The pretreatment of sperm
for 30 min in 0.1% ethanol (either without antioxidants or with
either 0.5 mM vitamin A or 0.5 mM vitamin E) did not affect
sperm viability (for mouse, similar findings are presented in
Table 3). In fact, sperm that interacted with the zona pellucida
remained motile even after 24 h of IVF incubations. As
depicted in Figure 4B, a reduction of .60% in sperm binding
was noted after a pretreatment of sperm with 0.5 mM vitamin
A, and a 35% reduction in sperm binding was detected after a
pretreatment with 0.5 mM vitamin E. A considerable variety in
amount of sperm bound per oocyte was found for each
individual ejaculated producing boar tested (Fig. 4B). Remark-
ably, the amount of sperm bound to the zona pellucida of
unfertilized oocytes (M2), monospermic fertilized oocytes
(Mono), and polyspermic fertilized oocytes (Poly) was not
different (Fig. 4B). In line with the larger inhibitory effect of
vitamin A when compared to vitamin E on sperm-zona binding
were their respective effects on inhibition of IVF rates. When
sperm were pretreated for 30 min with 0.5 mM vitamin E prior
to IVF, a reduction of ; 50% in fertilization rates was
observed, while pretreatment with 0.5 mM vitamin A
completely blocked fertilization (Fig. 4C). Although the
vitamin E effect was significant (P , 0.05), the inhibitory
effect was highly variable between each of the 11 boars tested
FIG. 3. Relative molar contributions of cellular oxysterol species to the total oxysterol content in sperm cells after incubation in media supplemented and
centrifugation as indicated. Note the relative decrease of 7-OH and increase of 7-ketocholesterol under conditions with increased oxysterol formation.
The top panels refer to Bic and
þ
Bic, both in the absence of albumin. Conditions are marked a where control levels of oxysterol formation were
observed and ‘b’ where oxysterol levels were ;10-fold higher than under control conditions (data in Fig. 1). For the definitions of the oxysterol species
abbreviations used, see Figure 2 (numbers in graph depict sum of oxysterol species: mean 6 SD in pmol/10
6
cells; n ¼ 3).
BOERKE ET AL.
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(Fig. 4C). In line with the absence of sperm and oocyte
deterioration was the finding that there was no different
correlation between the ratio of monospermic and polyspermic
fertilized oocytes after vitamin E treatment. This shows that the
diluted vitamin E concentrations (0.05 mM) during the IVF
incubation were not affecting the oocyte’s receptivity for
sperm.
Effects of Oxysterol-Binding Proteins on Sperm Motility
Recombinant oxysterol-binding proteins ORP-1 and ORP-2
were produced and purified as described in Materials and
Methods (purity was checked in silver-stained gels; Fig. 5A,
lane 4). The effects of varying concentrations (4–16 lg/ml) of
ORP-1 and ORP-2 to sperm suspensions were examined. In the
presence of bicarbonate but in the absence of albumin (negative
control
þ
Bic–BSA), only 20% of sperm had hyperactivated
motility (Fig. 5B), while in the presence of albumin
(
þ
Bic
þ
BSA), about 45% of sperm showed hyperactivated
motility patterns (Fig. 5B). Both ORP-1 and ORP2 induced a
very strong hyperactivated motility in response to the sperm
with the highest effects in 55% of sperm already at the lowest
dose tested of 4 lg/ml (Fig. 5B).
Effects of Oxysterol-Binding Proteins on Tyrosine
Phosphorylation of Sperm Proteins
The hyperactivated motility pattern of capacitated sperm
relates to an increase in tyrosine phosphorylation of proteins in
the flagellum [27], and we examined whether tyrosine
phosphorylation occurred in incubated sperm samples. When
sperm were incubated in bicarbonate-enriched media
(
þ
BicBSA), only a weak protein tyrosine phosphorylation
increase as compared to control sperm was recovered (1.1-fold;
Fig. 5C). Inclusion of albumin (
þ
Bic
þ
BSA) caused an almost
4-fold increase in protein tyrosine phosphorylation (Fig. 5C).
The 4-lg/ml dose of ORP-1 or ORP-2 (eliciting maximal
stimulation in hyperactivated sperm motility; Fig. 5B) caused a
significant (;2-fold) but less prominent induction of protein
tyrosine phosphorylation compared to control sperm (Fig. 5C).
Interestingly, when compared to albumin, both ORPs induced
tyrosine phosphorylation in the sperm head of a larger
subpopulation of sperm cells (Fig. 5D), and this was
manifested at the apical ridge area and at the equatorial area
of the sperm head (Fig. 5D). Induction of tyrosine phosphor-
ylation was not observed when vitamin E or A was added to
bicarbonate-enriched media in hyper the presence or the
absence albumin or albumin (data not shown) for mouse
sperm (Table 3).
Effects of Oxysterol-Binding Proteins on the Aggregation of
Flotillin into the Apical Ridge Area
In a previous study, the depletion of sterols and the
aggregation of lipid raft marker proteins such as flotillin have
been demonstrated to result in the capacitation of porcine
sperm [2]. We now have extended this observation and studied
whether, as compared to sperm treated in bicarbonate-enriched
media, the albumin effect on sterol extraction and lipid raft
aggregation could be mimicked by incubating sperm in
bicarbonate-enriched media supplemented with recombinant
ORP-1 or ORP-2. Clearly, as reported previously [2, 12],
inclusion of BSA to the bicarbonate-enriched medium (
þ
Bic)
caused the aggregation of flotillin at the apical ridge area (for
topology, see Fig. 6, inset). In the absence of (oxy)sterol-
depleting agents, only ,20% of the sperm cells had this
flotillin distribution, and this proportion increased to 70% in
the presence of BSA (Fig. 6). When sperm were incubated in
bicarbonate-enriched media with either ORP-1 or ORP-2, this
also resulted in a significant increase of aggregation of flotillin
in the apical area of the sperm head, although 4 lg/ml ORP-2
was much more efficient (to a similar degree as BSA) when
compared to 4 lg/ml ORP-1 (Fig. 6). This phenomenon was
not observed when vitamin E or vitamin A was added to
bicarbonate-enriched media in either the presence or the
absence albumin (data not shown).
TABLE 2. The relative amount of cholesterol, desmosterol, and oxysterol
species extracted by albumin from in vitro-capacitating boar and mouse
sperm cells.
Component
Relative amount in supernatant
a
Boar semen Mouse semen
3,5,6-Trihydroxycholesterol 71 6 11% 74 6 17%
7-Hydroxycholesterol 59 6 14% 75 6 14%
7-Ketocholesterol 62 6 13% 80 6 16%
22-Ketocholesterol 55 6 10% Not detected
25-Hydroxycholesterol 56 6 13% 76 6 17%
5,6b-Epoxycholesterol 38 6 7% 53 6 13%
5,6a-Epoxycholesterol 35 6 7% 41 6 9%
Desmosterol 34 6 5% 38 6 5%
Cholesterol 38 6 5% 37 6 3%
a
The mean percentage 6 SD from the total of each component in semen
that is recovered in the supernatant after a 2-h in vitro-capacitating
incubation (
þ
Bic,
þ
BSA) is expressed (n ¼ 3).
TABLE 1. Formation and albumin-dependent extraction of oxysterols in boar and mouse sperm before and after in vitro capacitation.
a
Parameter
b
Incubation
c
0h 2h( albumin) 2 h (þ albumin)
Boar semen
Oxysterol/cholesterol (%) 0.05 6 0.02 0.82 6 0.16
d
0.94 6 0.20
d
Oxysterol in supernatant (%)46 286 3606 18
d
Mouse semen
Oxysterol/cholesterol (%) 0.14 6 0.08 1.03 6 0.29
d
1.51 6 0.60
d
Oxysterol in supernatant (%)86 3116 4726 12
d
a
Data obtained from four boars and four mice, using three individual ejaculates for each boar (n ¼4, r ¼3) or aspiration of two mouse epididymi (n ¼4, r
¼ 2).
b
The relative amount of total oxysterol formed from cholesterol (100%) is indicated, as is the distribution of the formed oxysterols (100% in semen), which
was recovered in the supernatant after centrifugation. The remaining proportion of the oxysterols were present in the sperm pellet. For partition of
molecular oxysterol species see after capacitation in presence of albumin see Table 2.
c
Values are mean 6 SD.
d
Data differed significantly from the 0-h condition. Similar data were reported for bovine sperm previously [4].
OXYSTEROLS IN CAPACITATING MAMMALIAN SPERM
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FIG. 4. Effects of the bicarbonate/ROS pathway on hyperactivated motility, zona binding, and IVF. A) Vitamin E and vitamin A inhibit bicarbonate- and
albumin-induced hyperactivated sperm motility. Bicarbonate in the absence (
þ
BicBSA) and especially in the presence (
þ
Bic
þ
BSA) of albumin induces
hyperactivated sperm motility. In the presence of vitamin E or vitamin A, no signs of hyperactivated sperm motility were seen in the absence of albumin
(
þ
BicBSA
þ
VitE), and in the presence of albumin (
þ
Bic
þ
BSA
þ
VitE;
þ
Bic
þ
BSA
þ
VitA), the levels of hyperactivated sperm motility were comparable to
control sperm that were incubated in the absence of bicarbonate and albumin (BicBSA). The data represent mean 6 SEM, n ¼ 3. * indicates a
BOERKE ET AL.
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Effects of Oxysterol-Binding Proteins and Antioxidants on
the Generation of Zona Affinity of Sperm and IVF
In vitro capacitation leads to the generation of hyper-
activated sperm motility but also to the generation of affinity
for the zona pellucida. In the absence of bicarbonate (Bic), no
sperm bound to the zona pellucida (data not shown). Only a
minimal amount of sperm cells showed binding to the zona
pellucida (0.8 sperm cells per zona pellucida on average) when
incubated in bicarbonate-enriched medium in the absence of
(oxy)sterol-interacting proteins (BSA; Fig. 7A). When
incubated in the presence of BSA, an average of eight sperm
cells were binding to each zona pellucida (
þ
BSA; Fig. 7A).
Incubation of sperm in bicarbonate-enriched media (
þ
Bic)
with ORP-1 also induced sperm zona binding (two to three
sperm cells per zona pellucida), whereas ORP-2 failed to
induce this compared to bicarbonate-enriched medium without
(oxy)sterol-interacting proteins (Fig. 7A). The fertilization rates
of sperm cells incubated under these four conditions were also
followed. IVF in the presence of bicarbonate and albumin
(
þ
BSA) led to a fertilization rate of 60% (Fig. 7B) where
omission of (oxy)sterol-binding proteins (BSA) led to
fertilization rates of only 2.4%. Despite the positive effects of
ORPs on signs of sperm capacitation, they led to only marginal
increases of fertilization rates: 3.5% and 4.1% for a 4-lg/ml
dose of ORP-1 and ORP-2, respectively. Figure 7C depicts the
localization and presence of sperm when interacting with the
oocyte-zona complex under IVF conditions. In the absence of
(oxy)sterol-interacting proteins, almost no interacting sperm
cells were detected. In the presence of ORP-1 or –ORP-2, some
sperm cells were present at the zona pellucida and showed
some signs of zona penetration, indicating that these cells not
only interact with the zona pellucida but also had induced the
acrosome reaction, which is a step required for zona
penetration (Fig. 7C). However, a deeper penetration as shown
for BSA was only rarely found under ORP-1 or ORP-2
incubations.
Effects of Oxysterol-Binding Proteins on the Induction of
the Acrosome Reaction
The observation that fertilization rates were low in the
presence of ORPs in bicarbonate-enriched media (in contrast to
BSA) but that sperm incubated with ORPs showed some zona-
binding affinity and some zona penetration ability led us to
investigate whether ORPs were inducing premature acrosome
reactions. Incubation of sperm in bicarbonate-depleted medium
or in bicarbonate-enriched medium (in either the absence or the
presence of BSA) did not induce the acrosome reaction (the
amount of acrosome-reacted cells that stained positive for
fluorescent-conjugated peanut agglutinin; PNA-FITC, positive
cells ;10%; Fig. 8). Note that BSA had a stabilizing effect on
the acrosome integrity, which has been explained in a previous
study showing that this condition causes stabilization of
docked SNARE complexes [18]. In contrast, ORP-1 and
ORP-2 caused premature acrosome reactions in 20%–30% of
the sperm cells (Fig. 8, B and C).
Albumin, Oxysterol-Binding Proteins, and Methyl b-
Cyclodextrin Efficiently Reduce Oxysterol Levels in
Bicarbonate Stimulated Sperm
Experiments were designed in which sperm cells were
treated with bicarbonate (
þ
Bic) in the absence and in the
presence of agents that can reduce sterols and/or oxysterols in
sperm. The sterol reducing agents albumin, MBCD, or the
recombinant oxysterol-binding proteins ORP1 and ORP2 were
tested. All agents caused a reduction of 50% or more of the
oxysterols formed when compared to the amount of oxysterols
formed under bicarbonate alone (Fig. 9A). Interestingly, both
ORPs at 4 lg/ml caused a very reproducible and more
pronounced reduction in oxysterols levels than albumin or
MBCD (a reduction of 70%; Fig 9A).
TABLE 3. Effects of bicarbonate and membrane antioxidants during in vitro capacitation of mouse sperm.
a
Parameter BicþBSA þBicþBSA þBicþBSAþVitE þBicþBSAþVitA
Hyperactivated motility
b
4 6 3% 48 6 14%
d
14 6 5%
e
6 6 4%
e
PY20 positive in tail
bc
3 6 2% 52 6 19%
d
12 6 8%
e
7 6 2%
e
Membrane intact
b
93 6 4% 78 6 12% 92 6 5% 89 6 6%
a
Mean percentage of sperm cells 6 SD with indicated properties after a 2-h incubation (n ¼ 3, r ¼ 2).
b
For all samples, the overall amount of motile sperm cells was .75%.
c
Note that PY20 labeling in some sperm cells was present in the cytoplasmic droplets and/or in the postacrosomal head region. Although the percentage
of such aberrant PY20 labeling patterns was ,5%, it was invariable between samples (not induced by bicarbonate or inhibited by hydrophobic
antioxidants).
d
Indicates a significant difference induced by bicarbonate (
þ
Bic
þ
BSA vs. –Bic
þ
BSA).
e
Indicates a significant inhibition of the bicarbonate-induced effects by inclusion of hydrophobic antioxidants.
3
significant increase of effects compared to BicBSA; *** indicates a significant lower effect compared to
þ
Bic
þ
BSA and a significant effect compared to
BicBSA; # indicates a significant lower amount of hyperactive motile sperm when compared to BicBSA (in all predictions, P , 0.05). B) Sperm
binding to oocytes after a pretreatment of 30 min in the IVF medium containing 0.1% ethanol (control) or in 0.5 mM of vitamin A (VitA) or vitamin E (VitE).
The individual black squares indicate the amount of sperm that bound to the zona pellucida for each of the boar ejaculates tested (eight different boars
indicated by A-H on the x-axis). The mean amount of sperm bound to unfertilized oocytes (M2), monospermic fertilized oocytes (Mono), and polyspermic
fertilized oocytes (Poly) is indicated in the top panels. For control and VitE, eight individual boars were scored for VitA, three boars were scored, and no
fertilized oocytes were detected. C) Vitamin A blocked and vitamin E significantly reduced (P , 0.05) IVF rates. The relative reduction compared to
control IVF rates is the expressed normalized fertilization rate (NFR). The fertilization rate (in %, y-axis) and the ratio of polyspermic fertilization/total
fertilization (in %, x-axis) are indicated for ejaculate obtained from 11 individual artificially inseminated boars. For each IVF treatment and each individual
boar, .30 oocytes were used.
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FIG. 5. The effects of oxysterol-binding proteins on sperm capacitation. B) Purification of recombinant oxysterol-binding proteins ORP-1 and ORP-2. A)
Silver-stained SDS-PAGE cells loaded with ORP preparations. Lane 1: protein supernatant isolated after ultracentrifugation of transfected E. coli
suspensions; lane 2: supernatant after an additional bead treatment with glutathione sepharose; lane 3: the empty wash fraction of glutathione sepharose
column; and lane 4: the eluted proteins with 10 mM glutathione, which released the ORP-GSH fusion constructs (see also Suchanek et al. [21]). B)
Inclusion of albumin (
þ
Bic
þ
BSA) and 4–16 lg/ml ORP-2 (
þ
Bic
þ
ORP-2) and for lower doses of 4 lg/ml and, to a lesser extent, 8 lg/ml ORP-1
BOERKE ET AL.
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Desmosterol and Cholesterol Are Depleted by Albumin but
Not by Oxysterol-Binding Proteins in Bicarbonate-
Stimulated Sperm
Sperm stimulated with bicarbonate in the presence of
albumin (
þ
Bic
þ
BSA) showed a 63% decrease of cholesterol
and almost a 34% decrease in desmosterol when compared to
the negative control (no bicarbonate; Supplemental Figure S1,
available online at www.biolreprod.org), which was recovered
in the albumin-containing supernatant after centrifugation (Fig.
8B and Table 2). The depletion of these sterols could be
inhibited by vitamin E or vitamin A (Fig. 8B). Independent of
bicarbonate inclusion, the MBCD also caused a significant (but
lower) depletion of cholesterol (27% and 19%) and desmos-
terol (27% and 33%) in the absence and the presence of
bicarbonate, respectively, in line with previous studies (Fig. 9B
and Supplemental Data [2, 3]). Desmosterol is a precursor
sterol in the cholesterol biosynthetic pathway present in all
samples in a molar ratio of 1.5% relative to cholesterol. Note
that the intensity of ions detected as expressed in Figure 2 are
detected with varying levels of sensitivity because of the
different ionization efficiencies in the APCI process. They
were corrected for a response curve made for each individual
oxysterol species and sterol species detected as described
previously [4]. Interestingly, the addition of pro-oxidants in the
presence of albumin and bicarbonate (i.e., under oxysterol-
forming conditions; Fig. 1) resulted not only in a modest
reduction the level of cholesterol (13%) but also a pronounced
reduction of desmosterol (43%) when compared to the
Bic
þ
BSA control (Fig. 9B and Supplemental Figure S1).
Moreover, the two oxysterol-binding proteins ORP-1 and
ORP-2, although very efficient in reducing oxysterol levels in
sperm cells (Fig. 9A), did not cause significant reductions in
the levels of cholesterol or desmosterol (Fig. 9B). The
incubations with bicarbonate and BSA caused the depletion
of oxysterols cholesterol and desmosterol from the sperm
surface into the medium (for the partition of each sterol
component in in vitro-capacitated mouse and boar sperm, see
Tables 1 and 2).
MBCD-Mediated Sterol Depletion Causes Tyrosine
Phosphorylation and Hyperactivated Motility in a Different
Fashion Than Recombinant ORPs or BSA
In addition to the effects of recombinant ORP-1 and ORP-2
on sperm capacitation, we compared the effects of a much less
specific sterol depletor on sperm capacitation (i.e., MBCD).
Figure 9 shows that MBCD has the capacity to deplete the
bicarbonate-dependent production of oxysterols. However,
MBCD is a potent depletor of free sterols in both the absence
and the presence of bicarbonate (Fig. 9; [28–30]). In fact, in the
dose range of 0.5–10 mM, MBCD caused an increase in
tyrosine phosphorylation in both the absence (Bic) and the
presence (
þ
Bic) of bicarbonate (Fig. 10A), with highest
stimulation to similar levels as that of BSA (Fig. 4C; note
that BSA had this effect only in the presence of bicarbonate). In
the absence of bicarbonate, only the specific concentration of 2
mM MBCD led to the induction of hyperactivated sperm
motility (Fig. 10B), whereas in the presence of bicarbonate,
MBCD did not attenuate the bicarbonate induction of hyper-
activated sperm motility and significantly inhibited this
response at higher dosages (Fig. 9B).
FIG. 6. The bicarbonate/oxysterol pathway induces protein tyrosine
phosphorylation and aggregation of lipid-ordered domains. Incubations
with specific depletion of oxysterols result in aggregation of flotillin-1 into
the apical ridge area of the sperm head. Incubation in the presence of
albumin (
þ
Bic
þ
BSA) or 4 lg/ml ORP2 (
þ
BIC
þ
ORP2) induced immuno-
labeling of flotillin-1 in the apical ridge area of the sperm head when
compared to sperm cells incubated in the absence of sterol-depleting
agents (
þ
BicBSA). Only 4 lg/ml ORP-1 (
þ
Bic
þ
ORP-1) slightly induced
the apical ridge of this relocation of flotillin-1. The data represent mean 6
SEM, n ¼ 3. * indicates a significant increase of effects compared to
þ
BicBSA; *** indicates a significantly lower effect compared to
þ
Bic
þ
BSA and a significant effect compared to
þ
BicBSA (in all
comparisons, P , 0.05).
3
(
þ
Bic
þ
ORP-1) induced hyperactivated sperm motility. The highest dose of 16 lg/ml ORP-1 (
þ
Bic
þ
ORP-1) showed no increase of hyperactivated sperm
motility and was comparable to the condition without added sterol-depleting agents (
þ
BicBSA). C) Western blot analysis revealed that tyrosine
phosphorylation is increased 3.5-fold in the bicarbonate and BSA condition (
þ
Bic
þ
BSA) when compared to the absence of sterol-depleting agents
(
þ
BicBSA). Inclusion of 4 lg/ml ORP-1 (
þ
Bic
þ
ORP-1) or 4 lg/ml ORP-2 (
þ
Bic
þ
ORP-2) also induced tyrosine phosphorylation albeit to a smaller
population of incubated sperm cells. D) Besides tail labeling (causing the hyperactivated motility), PY20 labeling of tyrosine phosphorylated proteins was
found on the apical ridge area (left arrow) and at the equatorial area (right arrow) of the sperm head. C) Clearly, the inclusion of albumin (
þ
Bic
þ
BSA), 4
lg/ml ORP-1 (
þ
Bic
þ
ORP-1), or 4 lg/ml ORP-2 (
þ
Bic
þ
ORP-2) to bicarbonate-enriched media induced, in increasing order, more PY20 labeling on both
of the sperm head areas when compared to medium without sterol-depleting agents (
þ
BicBSA). The data represent mean 6 SEM, n ¼ 3. * indicates a
significant increase of effects compared to –BicBSA; ** indicates a significant additional increase of effect when compared to
þ
Bic
þ
BSA; *** indicates a
significant lower effect compared to
þ
Bic
þ
BSA and a significant effect compared to –BicBSA; (in all predictions, P , 0.05).
OXYSTEROLS IN CAPACITATING MAMMALIAN SPERM
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MBCD Treatment Induces Low IVF Rates and Oocyte
Degeneration
Analogous to the IVF experiments in bicarbonate-enriched
media with BSA or with recombinant ORPs, we tested the effect
of MBCD on fertilization rates. The fertilization rates after
treatment of sperm with 0.5–10 mM MBCD compared to those
of BSA (for BSA .60%, see Fig. 7B) were dramatically
reduced (Fig. 10C). However, in contrast to ORP-1 and ORP-2
incubations, the 2-mM-MBCD condition led to much lower
fertilization rates (30%). At lower and higher levels of MBCD
(0.5 and 10 mM, respectively), even lower fertilization rates
were observed when compared to sperm treated with 0mM
MBCD (Fig. 10C) or with recombinant ORPs (Fig. 7B). A
dose-dependent degeneration of oocytes was observed when
IVF was performed in the presence of 0.5–10 mM MBCD. In
FIG. 7. The bicarbonate/oxysterol/BSA pathway generates zona affinity and IVF, but replacement of BSA by ORPs is insufficient. A) Sperm cells
incubated in bicarbonate-enriched medium with albumin (
þ
Bic
þ
BSA) showed affinity for the zona pellucida when compared to bicarbonate-enriched
medium without sterol-depleting agents, where essentially no zona binding was observed (
þ
BicBSA); 4 lg/ml ORP-1 (
þ
Bic
þ
ORP-1) but not 4 lg/ml
ORP-2 (
þ
Bic
þ
ORP-2) caused a smaller but significant increase in zona affinity than
þ
Bic
þ
BSA when compared to
þ
BicBSA. B) Only the
þ
Bic
þ
BSA
condition gave rise to efficient fertilization rates, while
þ
Bic
þ
ORP-1 or
þ
Bic
þ
ORP-2 did not have positive effects when compared to
þ
BicBSA. For each
experiment and each incubation type, at least 20 oocytes were used. The data represent mean 6 SEM, n ¼3. C) Representative microscopic photographs
of sperm-zona interactions under the incubations provided in B. Green fluorescence indicated labeling of DNA and are depicted as merged with the
corresponding bright-field images obtained. The dashed lines indicate the border of the zona pellucida. For
þ
BicBSA, no sperm interaction with the
zona pellucida was noted. In the presence of albumin (
þ
Bic
þ
BSA), full penetration through the zona pellucida was noted, while interaction with the zona
pellucida (
þ
BSA
þ
ORP-1) or slight penetration (
þ
Bic
þ
ORP-2) was noted when albumin was replaced with recombinant ORP.
BOERKE ET AL.
12 Article 21
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the presence of BSA, these rates were ,10% (Fig. 7B), whereas
2 and 10 mM MBCD caused unacceptably high levels of oocyte
degeneration (of 30% and 100%, respectively; Fig. 10C).
DISCUSSION
Induction and Prevention of Oxysterol Formation
In this article, the formation of oxysterols is described in
sperm under in vitro-capacitating conditions. Oxysterol
formation was dependent on bicarbonate ions that must have
induced the formation of radical oxygen species [31] because
oxysterol formation was essentially blocked when mouse or
porcine sperm cells were treated in bicarbonate-enriched media
in the presence of vitamin E or vitamin A. In a previous article,
we have shown from bovine sperm that oxysterol formation
can be induced by incubating sperm in the presence of pro-
oxidants like tert-butylhydroxide [4]. Here we showed that
oxysterols can be formed in the absence of bicarbonate under
certain pro-oxidant conditions (FeSO
4
þ
ascorbate) but not by
the peroxynitrite forming agent SIN-1. Both bicarbonate and
the FeSO
4
þ
ascorbate incubations induced especially the
production of 7-ketocholesterol and 5,6b-epoxycholesterol.
Oxysterol Formation and Interaction with Oxysterol-
Binding Proteins Induces Various Characteristics of Sperm
Capacitation
When sperm are capacitated in vitro in the presence of
bicarbonate and BSA, they show signs of hyperactivated sperm
motility [27], increased levels of tyrosine phosphorylation [27],
surface changes (including the already noted removal of sterols
[32]), redistribution and aggregation of lipid microdomains
[12], and higher affinity for the zona pellucida [11, 16]. These
changes together result in the sperm becoming competent to
fertilize the oocyte during IVF incubations [32]. In the present
work, in which BSA was replaced for either recombinant ORP-
1 or ORP-2, a number of capacitation responses, were detected,
including 1) the induction of hyperactivated sperm motility, 2)
increased tyrosine phosphorylation, 3) aggregation of lipid-
ordered microdomain membrane markers at the apical ridge
area of the sperm head, and 4) to some extent higher affinity for
the zona pellucida. In fact, ORP treatment led to some limited
penetration activity of sperm cells through the zona pellucida,
but this response was limited when compared to BSA. All these
responses were present only when sperm were incubated in
bicarbonate-rich media (
þ
Bic), and the responses were
inhibited in presence of vitamin E or blocked by vitamin A.
Thus, an essential part of sperm capacitation is due to the
formation of oxysterols and putatively to the interaction of
these formed oxysterols with proteins involved in sterol
depletion at the sperm surface [8, 19].
Depletion of Oxysterols, Desmosterol, and Cholesterol
Sperm capacitation performed in the presence of the sterol-
depleting molecule BSA (routinely done in IVF [11]) resulted
in an efficient reduction of the majority of oxysterols (50%)
from the sperm surface into the albumin-containing fraction not
associated with the sperm surface (recovered in the supernatant
after centrifugation). An even more severe reduction of
oxysterols were detected when sperm were incubated with
recombinant oxysterol-binding proteins (ORP-1 and ORP-2; in
both cases, a reduction of .70% of the oxysterols took place),
which are known to bind oxysterols [11, 33]. Importantly,
ORPs are soluble proteins enhancing exchange of sterols from
donor to acceptor membranes over the cytosol in a variety of
cells. After isolating these two recombinant proteins from
Escherichia coli, we used them as tools for the specific
interaction of oxysterols in sperm suspensions under various in
vitro capacitation conditions. We must stress here that this
extracellular type of action is not mimicking eventually
endogenous ORPs under physiological conditions where they
(if at all present in sperm) are active intracellularly. The effects
of ORPs, similar to BSA, were seen only in bicarbonate-
enriched media but not in the absence of bicarbonate. When
sperm were incubated with Bic
þ
ORPs, no depletion of
cholesterol or desmosterol was monitored, while Bic
þ
BSA
caused a depletion of .30% cholesterol and desmosterol.
Interestingly, ORP treatment caused only a minor depletion
(,10%) of the oxysterols in the supernatant after centrifuging
sperm. This implies that ORP (in contrast to BSA) does not
deplete oxysterols efficiently from sperm but scavenges
oxysterols (by binding to the sperm surface?) during the
induction of their production at the sperm surface by
bicarbonate. It is likely that the bicarbonate-induced capacita-
tion responses are not inhibited (instead, it was even promoted)
by ORP treatment, as intracellular ROS formation and ROS
signaling is not affected. It seems that the concomitant high
levels of oxysterols in sperm treated with bicarbonate alone can
be compensated by albumin (depletion) or by ORP (likely
surface binding and scavenging). Moreover, oxysterol forma-
tion was inhibited by pretreating sperm with vitamin A; this
caused a complete block of IVF, and vitamin E had only a
partial inhibiting effect. Both membrane antioxidants also
inhibited sperm zona binding but not the integrity of sperm or
oocytes. These findings indicate that lowering ROS and ROS-
dependent signaling processes at the sperm surface prior to an
IVF experiment blocks oxysterol formation and sperm
capacitation and reduces sperm-zona-binding and IVF rates.
The difference in degree of effect between vitamin A and
vitamin E may lie in the intrinsic properties of both
hydrophobic antioxidants and the way we pretreated sperm
prior to IVF. It is possible that vitamin A either resides better in
the sperm surface during the IVF procedure (more hydropho-
bic) or elicits its antioxidant properties more efficiently when
compared to vitamin E (vitamin A is a stronger antioxidant).
Note that the pretreated sperm was diluted 10 times and used
for 24 h in the IVF experiment. Under such conditions, back
exchange of the antioxidants to albumin may occur. The
decrease of IVF rates by vitamin E has been reported for
bovine sperm [34, 35]. Moreover, inseminating oocytes with
hydrogen peroxide-pretreated sperm has been shown to
increase fertilization, cleavage, and blastocyst rates [36]. Taken
together, we believe that these data show that depletion of
sperm surface lipid peroxides (or, in the absence of
bicarbonate, the lack of formation of sperm surface lipid
peroxides) causes a reduction in IVF rates. The formation of
lipid peroxides under bicarbonate allows reverse transport of
(oxy)sterols to BSA and high IVF rates, while excessive lipid
peroxidation is detrimental to the sperm cell and thus to
fertilization [10].
Oxysterol Reduction Alone Is Insufficient for Obtaining IVF
Success
Interestingly, unlike albumin, ORPs were not able to reduce
cholesterol or desmosterol, which fits to their higher specific
affinity for oxysterols when compared to albumin. This lack of
reducing levels of free sterols at the sperm surface likely relates
to the fact that incubations of BSA did result in 60%
fertilization rates after IVF, whereas ORP incubations largely
failed to result in fertilization. Both ORP and BSA incubations
OXYSTEROLS IN CAPACITATING MAMMALIAN SPERM
13 Article 21
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FIG. 8. Sperm incubations with ORPs induce the acrosome reaction. A) Merged phase-contrast image and PNA-FITC labeling of an acrosome-intact
sperm (left) and an acrosome-reacted sperm cell (right). B) The relative amount of acrosome-reacted sperm cells in the absence (
þ
BicBSA) and in the
presence of sterol-depleting agents (
þ
Bic
þ
ORP-1;
þ
Bic
þ
ORP-2 or
þ
Bic
þ
BSA, respectively). The doses of ORP were 4 lg/ml. The data represent mean 6
SEM, n ¼ 3. Fluorescence intensity histograms of the PNA-FITC labeling of sperm incubated in the presence of (C) albumin (
þ
Bic
þ
BSA) and in the
presence of (D) ORP-1 (
þ
Bic
þ
ORP-1) are included to show the shift in the amount of sperm cells with their acrosome reaction induced by ORP-1
BOERKE ET AL.
14 Article 21
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induced in vitro sperm capacitation, although ORP also
induced preliminary acrosome reactions, while BSA-treated
cells remained acrosome intact. Therefore, it is possible that the
concentration and length of ORP treatments need to be refined
and that the currently used circumstances were simply too
rough for the sperm to become fertilization competent. Support
for this possibility is that the lowest dose of ORP-1 or ORP-2
caused already maximal stimulation of hyperactivated motility,
and higher doses were partly inhibiting this effect. The
previously mentioned possibility that ORPs act to inhibit
oxysterol formation by scavenging oxysterols may at higher
doses become a too efficient process and thus will inhibit ROS-
dependent capacitation responses. Presently, we are studying
how extracellular added ORPs on the sperm surface cause the
cellular responses that in part run parallel to bicarbonate/BSA-
induced capacitation. The oxysterol-binding protein family
contains .10 proteins, and their role in cell physiology has
recently been reviewed [37]. Of course, another possibility for
the discrepancies between BSA and ORPs may lie in that BSA
has other (oxysterol-independent) effects on sperm [38].
MBCD Causes a Partly Bicarbonate-Independent Sterol
Depletion from Sperm
Besides BSA and ORP, another molecule with affinity for
sterols, MBCD, has been exploited either to elicit sperm
capacitation [30] or to achieve BSA-independent IVF [39]. The
latter can be useful in developing methods to treat sperm under
fully pathogen-free declared materials. Because only a part of
the BSA effects can be obtained by ORP replacement, the
effects of MBCD were tested. As reported in the literature,
MBCD is indeed capable of inducing hyperactivated sperm
motility [40, 41] and tyrosine phosphorylation in the sperm tail
[27, 40] and causes a depletion of cholesterol, desmosterol
[28], and oxysterols (this study). However, MBCD does work
in a different fashion when compared to BSA and ORP-1 and
ORP-2, as it elicits these effects independently from bicarbon-
ate and does not stop extracting sterols in the presence of
vitamin E [28, this study]. Moreover, MBCD fails to induce
aggregation of flotillin and tyrosin phosphorylation in the
sperm head [28] and extracts sterols from both lipid rafts and
from the nonraft membranes, whereas BSA extracts sterols
only from the nonraft domain [2]. In the presence of
bicarbonate, higher levels of MBCD inhibit sperm motility
(this study) and also cause sperm membrane deterioration and
premature acrosome reactions [29]. Despite all these differ-
ences to BSA, 2-mM-MBCD incubation led to IVF rates of
30%. However, already at this concentration, MBCD caused a
nonacceptable level of oocyte degeneration (.30%) that was
observed for all oocytes at 10 mM MBCD.
In conclusion, the bicarbonate-dependent formation of ROS
leads to the production of oxysterols. It is likely that the
subsequent interactions of the formed oxysterols with oxy-
sterol-binding proteins (ORPs or albumin) and substantial
depletion of oxysterols enable sperm capacitation and related
specific sperm surface changes [13]. We postulate that that
these subtle changes in sperm sterols (only less than 1% of
sterols are converted to oxysterols) may activate a sterol
transporter protein. In this respect, it is possible that a protein
like the CD36 (a multifunctional protein homologous to the
class B scavenger receptor SR-B1) acts as a lipid sensor [42] to
FIG. 9. Reduction of cellular cholesterol and desmosterol levels and
cellular oxysterol levels after incubation with sterol-interacting proteins.
A) Oxysterol-binding proteins ORP-1 and ORP-2 are more effective in
reducing oxysterols from bicarbonate-activated sperm than albumin and
MBCD. The total amount of oxysterols indicated is relative to the amount
of oxysterols formed in the absence of oxysterol-interacting molecules
(BicBSA ¼ 100% conform; Fig. 1). B) The cellular levels of cholesterol
and desmosterol (total amount of both sterols indicated here as data for
desmosterol and cholesterol separately; Supplemental Figure S1) are
reduced by albumin (BSA) in a bicarbonate-specific manner that can be
blocked by vitamin E (vitE). MBCD lowers both free sterols to a lesser
extent but in a bicarbonate-independent manner. The ORP-1 and ORP-2
treatments did not lower the cellular levels of cholesterol or desmosterol.
In all cases, the molar ratio of cholesterol to desmosterol remained
approximately 10:1. Nevertheless, a slightly higher proportion of
desmosterol was depleted by albumin and MBCD when compared to
cholesterol (Supplemental Figure S1). Mean values 6 SEM are indicated,
n ¼ 3. * indicates a significant effect when compared to
þ
BicBSA; **
indicates a more pronounced effect when compared to
þ
Bic
þ
BSA (in all
comparisons, P , 0.05). For distribution of molecular oxysterol species,
cholesterol and desmosterol in the cell pellet and the supernatant of boar
and mouse sperm specimen after treatment see Table 2.
3
compared to albumin. The left population is acrosome intact, and the right population is acrosome reacted, as depicted with the line connector arrows to
A. * indicates a significant increase of effects compared to BicBSA (in all comparisons, P , 0.05).
OXYSTEROLS IN CAPACITATING MAMMALIAN SPERM
15 Article 21
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oxysterols. In fact, a recent report on bull sperm has indicated
that CD36 is a biomarker for male fertility [43]. SR-B1 itself (a
translation product from the SARB-1 gene) has also been
implied in regulating reverse cholesterol transport to high-
density lipoproteins in other tissues [44] and could be activated
by surface rearrangements typical for capacitating sperm as
postulated previously [1]. Other candidates for a reverse sterol
transporter potentially active at the sperm surface belong to the
ATP cassette transporters; ABCA1, ABCA7, ABCA17, and
ABCG1 are all detected in sperm [45, 46], and both ABCA1
and ABG1 have in fact been shown to transport oxysterols out
of cells [9].
Our results provide evidence that only with a cholesterol/
desmosterol-accepting protein in the sperm incubation buffer
do sperm became capable of fertilizing the oocyte. ORP-1 and
ORP-2 turned out to be too specific for reducing oxysterol
FIG. 10. Effects of MBCD-mediated sterol depletion on sperm activation and IVF. A) In the absence of bicarbonate (Bic), MBCD induced PY20 labeling
in the concentration range of 0.5–10 mM MBCD used and with maximal effect at 2 mM. In the presence of bicarbonate (
þ
Bic), a similar maximal
response was seen at 0.5 mM, while the higher doses of MBCD were still but less effective. B) MBCD only induced hyperactivated sperm motility at a 2-
mM dose in the absence of bicarbonate (Bic) but had no effects at lower or higher doses. In the presence of bicarbonate, only a marginal increase of
hyperactivated sperm motility was noted at lower doses of MBCD (0.5 or 2 mM), whereas the highest dose of 10 mM MBCD had an adverse effect and
inhibited hyperactivated sperm motility. C) MBCD-mediated sterol depletion inhibits IVF and induces oocyte degeneration. Increasing amounts of MBCD
in albumin-free bicarbonate-enriched incubation media did not result in high IVF rates. Only at the dose of 2 mM MBCD did ;30 percent of the oocytes
become fertilized, which is a relatively poor figure when compared to IVF rates obtained with albumin (Fig. 7). A dose-dependent degeneration of porcine
oocytes was noted when IVF was performed in bicarbonate-enriched media with inclusion of MBCD. The data represent mean 6 SEM, n ¼ 3, per
experiment, and for C in each incubation type, at least 20 oocytes were used. * indicates a significant effect when compared to 0 mM MBCD (P , 0.05).
BOERKE ET AL.
16 Article 21
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levels but failing to deplete cholesterol/desmosterol, which
could render the treated sperm unfertile under IVF conditions.
The sperm surface interaction of ORP also induced some
premature acrosome reactions. Under bicarbonate conditions,
BSA was capable of extracting cholesterol desmosterol and
oxysterols, thus allowing the depletion of a large amount of
total free sterols, an effect apparently required for fertilization.
Blocking oxysterol formation in the sperm membrane with
hydrophobic antioxidants reduced sperm motility tail tyrosine
phosphorylation and IVF rates and, to a lesser extent, inhibited
sperm-zona binding. This may indicate that ROS formation and
peroxidation of sterols (and possibly other lipids) in the sperm
membrane are required for proper zona penetration rather than
the recognition of this structure. The bicarbonate- and ROS-
independent sterol depletion by MBCD also induces some
aspects of sperm capacitation but fails to generate good IVF
results because of the adverse effects on oocyte physiology. It
is likely that more subtle treatments with ORPs or MBCD
could result in better IVF results. The balance between the rate
of oxysterol formation and of oxysterol depletion are probably
determining how the sperm are going through the time window
of sperm capacitation b to reach the state of deterioration (see
the model in Fig. 11; see also Aitken [31]). Thus, this study has
shed new light on the mechanisms of how oxysterols are
formed in the sperm and that this process precedes free sterol
depletion from the sperm surface during IVF. Apparently, a
bicarbonate-induced ROS-dependent membrane process is
required to activate reverse (oxy)sterol transport from the
capacitating sperm surface. From this study it is not clear
whether the formation of oxysterols itself is involved in the
activation of a reverse sterol transporter or whether they are just
molecular signs of ROS emerging in the sperm surface and
indirectly linked to the depletion of (oxy)sterols by albumin.
Future research should focus on the exact conditions that can
be used to optimize IVF with sterol-depleting agents. More
important, experiments should be planned to unravel the
mechanism of reverse sterol transport from capacitating sperm
as well as determining the involvement of oxysterols in this
sterol transport, which is crucial for IVF.
ACKNOWLEDGMENT
Dr. R.L. Serrano has helped with the transfection experiments and
purification of recombinant ORP-1 and ORP-2 proteins. Dr. P.S. Tsai has
helped with IVF and sperm incubation experiments.
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BOERKE ET AL.
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    • "Importantly, in vitro capacitation of sperm in the presence of albumin did not result in cholesterol depletion in the DRM fraction (in contrast to MBCD treatment; van Gestel et al. 2005b). Nevertheless, MBCD treatment allows cholesterol depletion in sperm and this does result in increased zona binding of stallion sperm (Bromfield and Nixon 2013a, b) and to some extent in vitro fertilization of porcine oocytes (Boerke et al. 2013). Note that sperm capacitation is a process essential for sperm to become competent to fertilize the oocyte (Gadella et al. 2008; Aitken and Nixon 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Lipid rafts are micro-domains of ordered lipids (L o phase) in biological membranes. The L o phase of cellular membranes can be isolated from disordered lipids (L d phase) after treatment with 1 % Triton X-100 at 4 °C in which the L o phase forms the detergent-resistant membrane (DRM) fraction. The lipid composition of DRM derived from Madin-Darby canine kidney (MDCK) cells, McArdle cells and por-cine sperm is compared with that of the whole cell. Remarkably, the unsaturation and chain length degree of aliphatic chains attached to phospholipids is virtually the same between DRM and whole cells. Cholesterol and sphingomyelin were enriched in DRMs but to a cell-specific molar ratio. Sulfatides (sphingolipids from MDCK cells) were enriched in the DRM while a seminolipid (an alkylacylglycerolipid from sperm) was depleted from the DRM. Treatment with<5 mM methyl-ß-cy-clodextrin (MBCD) caused cholesterol removal from the DRM without affecting the composition and amount of the phospholipid while higher levels disrupted the DRM. The substantial amount of (poly)unsaturated phospholipids in DRMs as well as a low stoichiometric amount of cholesterol suggest that lipid rafts in biological membranes are more fluid and dynamic than previously anticipated. Using negative staining, ultrastructural features of DRM were monitored and in all three cell types the DRMs appeared as multi-lamellar vesicular structures with a similar morphology. The detergent resistance is a result of protein–cholesterol and sphingolipid interactions allowing a relatively passive attraction of phospholipids to maintain the L o phase. For this special issue, the relevance of our findings is discussed in a sperm physiological context.
    Full-text · Article · Sep 2015
    • "ROS are well recognized as key regulators of mammalian sperm capacitation , yet a fine balance exists between a 'beneficial' presence of ROS to promote cholesterol efflux (Boerke et al., 2013), cAMP production (Zhang and Zheng, 1996; Ickowicz et al., 2012 ), protein tyrosine phosphorylation ( Leclerc et al., 1997) and the acrosome reaction (de Lamirande et al., 1998) and the 'detrimental' presence of excess ROS that can lead otherwise viable cells down an intrinsic apoptotic-like pathway (Aitken et al., 2012b). The present study suggests that levels of oxidative stress that might be encountered in vivo (Uchida, 2003; Aitken et al., 2012b ) have relatively little effect on events associated with early capacitation, such as membrane fluidity, raft redistribution and protein tyrosine phosphorylation. "
    [Show abstract] [Hide abstract] ABSTRACT: STUDY QUESTION How does oxidative stress impact upon human sperm-egg interaction and in particular the formation of zona pellucida-receptor complexes on the sperm surface? SUMMARY ANSWER Oxidative stress during human sperm capacitation resulted in the chemical alkylation of the molecular chaperone heat shock protein A2 (HSPA2), a concomitant reduction in surface expression of the zona pellucida-receptor arylsulphatase A (ARSA) and a severe loss of zona pellucida binding ability. WHAT IS KNOWN ALREADY An inability to bind to the zona pellucida is commonly encountered in the defective spermatozoa generated by male infertility patients; however, the underlying mechanisms remain unresolved. Recent studies have revealed that zona pellucida binding is mediated by molecular chaperones, particularly HSPA2, that facilitate the formation of multimeric zona pellucida-receptor complexes on the surface of mammalian spermatozoa during capacitation. STUDY DESIGN, SIZE, DURATION Spermatozoa were collected from healthy normozoospermic donors (n = 15). Low levels of oxidative stress were induced in populations of non-capacitated spermatozoa by a 1 h treatment with 4-hydroxynonenal (4HNE) or hydrogen peroxide (H2O2) and then these insults were removed and cells were capacitated for 3 h. PARTICIPANTS/MATERIALS, SETTING, METHODS Motility, membrane fluidity, protein tyrosine phosphorylation and lipid raft distribution were evaluated after sperm capacitation to determine the impact of oxidative stress on this process. The surface expression of ARSA and sperm adhesion molecule 1 (SPAM1) was observed using fluorescence microscopy, and the ability of treated cells to interact with homologous human zonae pellucidae was assessed through gamete co-incubation. Proximity ligation was used to evaluate the state of the HSPA2-laden zona pellucida-receptor complex and an immunoprecipitation approach was taken to establish the chemical alkylation of HSPA2 by the cytotoxic lipid aldehyde 4HNE. The validity of these findings was then tested through treatment of oxidatively stressed cells with the nucleophile penicillamine in order to scavenge lipid aldehydes and limit their ability to interact with HSPA2. All experiments were performed on samples pooled from two or more donors per replicate, with a minimum of three replicates. MAIN RESULTS AND THE ROLE OF CHANCE The oxidative treatments employed in this study did not influence sperm motility or capacitation-associated changes in membrane fluidity, tyrosine phosphorylation and lipid raft redistribution. However, they did significantly impair zona pellucida binding compared with the capacitated control (P < 0.01). The reduction in zona pellucida binding was associated with the impaired surface expression (P < 0.02) of a zona pellucida-receptor complex comprising HSPA2, SPAM1 and ARSA. Proximity ligation and immunoprecipitation assays demonstrated that impaired zona pellucida binding was, in turn, associated with the chemical alkylation of HSPA2 with 4HNE and the concomitant disruption of this zona pellucida-receptor complex. The use of penicillamine enabled a partial recovery of ARSA surface expression and zona pellucida adherence in H2O2-treated cells. These data suggest that the ability of low levels of oxidative stress to disrupt sperm function is mediated by the production of lipid aldehydes as a consequence of lipid peroxidation and their adduction to the molecular chaperone HSPA2 that is responsible for co-ordinating the assembly of functional zona pellucida-receptor complexes during sperm capacitation. LIMITATIONS, REASONS FOR CAUTION While these results extend only to one particular zona pellucida-receptor complex, we postulate that oxidative stress may more broadly impact upon sperm surface architecture. In this light, further study is required to assess the impact of oxidative stress on additional HSPA2-laden protein complexes. WIDER IMPLICATIONS OF THE FINDINGS These findings link low levels of oxidative stress to a severe loss of sperm function. In doing so, this work suggests a potential cause of male infertility pertaining to a loss of zona pellucida recognition ability and will contribute to the more accurate diagnosis and treatment of such conditions. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Health and Medical Research Council. Grant # APP1046346. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER N/A. © 2015 The Author 2015. 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] /* */
    Article · Sep 2015
    • "Oxysterol formation also takes place under in vitro capacitation conditions in porcine and mouse sperm [13]. Indeed, in both mammalian species, sperm capacitation led to the formation of oxysterols and the depletion of free sterols [13] . The formation of oxysterols was bicarbonatedependent and could also be induced by prooxidants in absence of bicarbonate or blocked by hydrophobic antioxidants in presence of bicarbonate. "
    [Show abstract] [Hide abstract] ABSTRACT: The fusion of a sperm with an oocyte to form new life is a highly regulated event. The activation-also termed capacitation-of the sperm cell is one of the key preparative steps required for this process. Ejaculated sperm has to make a journey through the female uterus and oviduct before it can approach the oocyte. The oocyte at that moment also has become prepared to facilitate monospermic fertilization and block immediately thereafter the chance for polyspermic fertilization. Interestingly, ejaculated sperm is not properly capacitated and consequently is not yet able to fertilize the oocyte. During the capacitation process, the formation of competent lipid-protein domains on the sperm head enables sperm-cumulus and zona pellucida interactions. This sperm binding allows the onset for a cascade reaction ultimately resulting in oocyte-sperm fusion. Many different lipids and proteins from the sperm surface are involved in this process. Sperm surface processing already starts when sperm are liberated from the seminiferous tubules and is followed by epididymal maturation where the sperm cell surface is modified and loaded with proteins to ensure it is prepared for its fertilization task. Although cauda epididymal sperm can fertilize the oocyte IVF, they are coated with so-called decapacitation factors during ejaculation. The seminal plasma-induced stabilization of the sperm surface permits the sperm transit through the cervix and uterus but prevents sperm capacitation and thus inhibits fertilization. For IVF purposes, sperm are washed out of seminal plasma and activated to get rid of decapacitation factors. Only after capacitation, the sperm can fertilize the oocyte. In recent years, IVF has become a widely used tool to achieve successful fertilization in both the veterinary field and human medicine. Although IVF procedures are very successful, scientific knowledge is still far from complete when identifying all the molecular players and processes during the first stages the fusion of two gametes into a new life. A concise overview in the current understanding of the process of capacitation and the sperm surface changes is provided. The gaps in knowledge of these prefertilization processes are critically discussed. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Aug 2015
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