Veterinary Quarterly

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Dynamics in the membrane organization of

the mammalian sperm cell and functionality in
fertilization
B.M. Gadella , F.M. Flesch , L.M.G. van Golde & B. Colenbrander
To cite this article: B.M. Gadella , F.M. Flesch , L.M.G. van Golde & B. Colenbrander (1999)
Dynamics in the membrane organization of the mammalian sperm cell and functionality in
fertilization, Veterinary Quarterly, 21:4, 142-146, DOI: 10.1080/01652176.1999.9695009
To link to this article: http://dx.doi.org/10.1080/01652176.1999.9695009

Published online: 01 Nov 2011.

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DYNAMICS IN THE MEMBRANE ORGANIZATION OF THE

MAMMALIAN SPERM CELL AND FUNCTIONALITY IN
FERTILIZATION
B.M. Gadella1,2,3, F.M.Flesch2, L.M.G. van Golde2, B. Colenbrander1

SUMMARY
The capacitation process of sperm cells involves complex
changes in the composition and orientation of molecules
at the surface of the sperm cell. Here we focus on the lipid
architecture in the sperm plasma membrane and demonstrate that the sperm plasma membrane is not static but

Vet Quart 1999; 21: 142-6

Accepted for publication: July 15, 1999.

cell is important for fertilization. In this light it is interesting to

note that, indeed, the different discrete subdomains of the

sperm head plasma membrane are involved in separate gamete

organization in living cells and extremely rapid lipid

movements were observed. The orientation of lipids in
the sperm plasma membrane changed under capacitative treatments, was found to be sensitive for tempera-

interaction events (summarized in Figure 1, for reviews see

8,33). (i) The apical subdomain of the sperm head plasma
membrane is specifically involved in the sperm binding with
the zona pellucida. (ii) The then provoked acrosome reaction
is a multiple membrane fusion event between the plasma
membrane and the outer acrosomal membrane. The acrosomal
membrane fusions with the plasma membrane are confined to
the pre-equatorial and apical subdomains whereas the equatorial subdomain of the plasma membrane does not fuse (36).

ture and also changed upon binding of sperm cells to the

(iii) After zona penetration the sperm cell reaches the

is an extremely dynamic structure. Advanced fluoroscopic techniques enabled continuous monitoring of lipid

zona pellucida. The changes in membrane properties

coincided with an activation of protein kinases resulting

oolemma and fertilizes the oocyte. This is achieved after the

binding of the spermatozoon to the oolemma and the fusion of

in tyrosine phosphorylation of specific plasma membrane proteins. The detected membrane changes relate

the plasma membranes of the two gametes (fertilization

fusion). The remaining equatorial subdomain of the sperm

to intrinsic membrane properties such as fluidity, permeability, adhesiveness and fusibility. We think that these
results may provide a physiological basis for new assays,
able to discriminate between functional and non-physiological sperm cells.

plasma membrane after the acrosome reaction, is the exclusive

region of the sperm surface capable of interacting and fusing
with the oolemma.
Much attention has been paid to dynamic aspects in the organization of membrane proteins and the extracellular coating of

INTRODUCTION

The sperm plasma membrane is typically polarized into lateral domains (head, midpiece and tail) and into subdomains
within these regions (8,23,25,33,36). All these specialized

surface regions differ from each other in composition of

glycocalyx and membrane molecules. Lateral diffusion of
membrane molecules between the domains is blocked by
two trans membranous ring shaped diffusion barriers (the
annular and posterior rings). The mixing of membrane components between the subdomains of the three domains, however, is not hindered by ultra structural membrane diffusion
barriers (14).

In mammals fertilization is a highly speciali2ed process in

which the sperm cell and its surface faces a series of physiological and functional changes (8,36). The first characteristics of
the sperm membrane polarity appear during its development

in the testis. After its release to the seminiferous tubules the

sperm cell undergoes specific physiological processes that
modify the cell surface: maturation in the epididymis, mixing
of accessory fluids during ejaculation, capacitation in the female genital tract, gamete interaction (1,23,28). It is believed
that the highly regulated membrane organization of the sperm
Department Farm Animal Health, Division Male Reproduction, Faculty of
Veterinary Medicine, Utrecht University, the Netherlands.

2 Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine,

Utrecht University, the Netherlands.
3

Corresponding author; full address: Department of Farm Animal Health, Faculty of

Veterinary Medicine, Utrecht University, Yalelaan 7, 3584 CL Utrecht, the
Netherlands. Tel: +31-30-2535382 or +31-30-2535389. Fax: +31-30-2535492. Email: B.Gadella@vet.uu.nl.

1 42

Figure 1. Schematic representation of the sequence of interactions between the male and female gamete leading to fertilization.
(/)Sperm binding to the zona pellucida with its apical plasma membrane,

(2) the sperm acrosome reaction, a multiple fusion event of the outer
acrosomal membrane with apical and pre-equatorial plasma membrane,
(3)the penetration of the sperm cell through the zona pellucida; note that
the equatorial membrane remains intact, (4) sperm binding to and fusion
with the oolemma (fertilization); both are exclusive events for the equato-

rial plasma membrane, (5) activation of the oocyte by specific sperm

cytosolic factors, (6) secretion of cortical granules (cortical reaction)
causing a definitive poly-spermy block.

THE VETERINARY QUARTERLY, VOL 2 1 , N o 4 , OCTOBER, 1 9 9 9

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the sperm plasma mem-

(19,29,31), but lipids also

CHANGES IN THE TRANSVERSE DISTRIBUTION OF

PHOSPHOLIPIDS ACROSS THE SPERM PLASMA
MEMBRANE BILAYER
Vesicles containing nitrobenzoxadiazolyl-aminohexanoyl
(NBD) acylated phospholipid analogues were incubated with
spermatozoa, and the rate of fluorescence incorporation was
monitored directly by flow cytometry. The membrane-impermeable reducing agent, dithionite, was used to destroy NBD
fluorescence in the outer leaflet of the bilayer in order to allow quantitation of the proportion of incorporated label that

function as second messen-

had been translocated across the bilayer (16,21). NBD-

gers (30). We have devel-

phosphatidylserine and to a lesser extent NBD-phosphatidylethanolamine were translocated rapidly and essentially corn-

brane, both in their relation

to the fertility processes
(4,8,36). However, changes
in membrane lipids are also
important: not only does the
lipid topology, metabolism
and composition change
during the sperm's life-time

oped advanced fluoroscopic

techniques for continuous

monitoring of the lipid

organization in living sperm

cells(3,12,13,16,21,22).

100

Both the lateral polarity and

the transbilayer topology of

lipids in the sperm plasma

membrane as well as fluidity

a) -a 80
N

and the fusogenicity of this

membrane were followed

Figure 2. Lateral distribution of fluorescent glycolipids after their incorporation into the plasma mem-

brane on boar sperm cells with

intact acrosomes
membranes.

and

plasma

(A) The distribution of SGalCer(C12-

Furthermore induction of

cr) 7-0

protein tyrosine phosphorylation (a hallmark of sperm

capacitation) was followed
in complete cells and in iso-

lated sperm plasma mem-

brane of a freshly ejaculated sperm

cell. (B-D) Subsequent stages of migration of SGalCer(C12-LRh) during

the changes we detected in

capacitation in vitro in a Tyrode's

based medium containing 2 mM
CaCl2 for a period of (13) 30 minutes, (C) 90 minutes, (D) 4 hours.
The distribution of the C12-LRh lipid
label intensity was measured in situ

on the sperm surface with an epifluorescence microscope (Rhodamine filter setting) connected to a
CCD camera and an image analyser
[13]. Cells were stained with

Hoechst 33258 and a peanut agglutinin-FITC conjugate in order to

El 60

during in vitro capacitation

and control incubations.

fluorescent analogue of
seminolipid) in the plasma memLRh) (a

0
)

40

a.-

co

*3

20

branes. This paper describes

10

20

30

40

50

60

10

20

30

40

50

60

the sperm plasma membrane under conditions that

induced capacitation and/ or
acrosome reaction in vitro
and discusses the physiological and clinical implications.

LATERAL REDISTRIBUTION OF LIPIDS IN THE

SPERM HEAD PLASMA

membrane and the acrosome reMEMBRANE
spectively. The length of the sperm
Acyl-labeled fluorescent
head is approximately 8 mm.
lipid analogues were transferred into the sperm plasma membrane. The labeled spermatozoa were incubated under various conditions, and latassess the intactness of the plasma

50

Cu

a)

30

.c

20

10

eral distribution of the incorporated lipid label was

followed in situ by analyzing digitized images obtained by

fluorescence microscopy (12,13). Figure 2 demonstrates
the lateral topology of a fluorescent analogue of the sperm
specific glycolipid (seminolipid) on boar spermatozoa. The
fluorescent glycolipids selectively incorporated in the api-

cal subdomain of the sperm head plasma membrane of

freshly ejaculated sperm cells (Figure 2A). However, the
fluorescent glycolipids redistributed towards the equatorial
subdomain during incubation with a bicarbonate and calcium enriched medium at 38C (a capacitative treatment;
Figure 2B-D). This demonstrates the possibility of membrane redistribution processes during capacitation over the
sperm head subdomains involved in binding to the zona
pellucida.

143

Min of incubation with vesicles

Figure 3. Time dependent transfer of C6N8D-labelled phospholipids across
the lipid bilayer of the boar sperm plasma membrane.
Suspensions of washed boar spermatozoa were pre-incubated in variations
of a Hepes-buffered Tyrode's-based medium before being mixed with vesicles loaded with (A) C6NBD-acylated phosphatidylserine (NBD-PtdSer), (B)
C6NBD-acylated phosphatidylcholine (C6NBDPtdCho). At intervals during

further incubation at 38C samples were counter stained with propidium

iodide (a DNA stain which is excluded from living cells) and subjected to flow
cytometric analysis before and after brief treatment with dithionite, in order
to detect the amount of C6NBD-acylated phospholipid incorporated and the
relative amount of internalized in the live sperm cells (for further details see

16). (0) preincubation and lipid labelling in a non-capacitative Tyrode's

Medium; () 15 mM bicarbonate/5 % CO2 included in theTyrode's Medium
during pre-incubation and labelling (capacitative treatment).

THE VETERINARY QUARTERLY, VOL 21, No 4 , OCTOBER, 1999

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101

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210
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FL3-Height

FL3-Height

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Figure 4. Flow cytometric dot plots of merocyanin stained boar sperm populations.
Sperm cells were incubated in control (A) or in capacitation (B) medium for 2 hours. The region indicated with L depicts living cells with low merocyanin
fluorescence, whereas region H depicts living cells with high merocyanin fluorescence (FL 3 Height). Cells in region D are stained positive for Yo-Pro and

therefore considered to be deteriorated (Yo-Pro is a membrane impermeant DNA stain (F 1Height). For further details see (22) Each panel depicts
10.000 cells.

pletely; however, NBD-phosphatidylcholine was only slowly

translocated (to a limit of 12%). Figure 3 depicts that capaci-

tative treatment caused a significant slowing of the rate of

NBD-phosphatidylserine translocation (Figure 3A) and an
increase in the limit of translocated NBD-phosphatidylcholine (to 30%, Figure 3B). This demonstrates that capacitation
results in transbilayer redistributions of phospholipids. The
observed decrease in lipid asymmetry is believed to be functional in the preparation for the acrosome reaction (21).
INCREASE IN FLUIDITY OF THE SPERM PLASMA
MEMBRANE

Sperm cells were loaded with merocyanin 540 to detect

tion status of control and capacitated protein samples was

analyzed by immunoblotting using antibodies raised against
phosphorylated tyrosine residues (Figure 5). Tyrosine
phosphorylation of sperm plasma membrane proteins was
observed after capacitation but not before (Figure 5 lanes 2
and 4). Protein tyrosine phosphorylation was observed in
complete sperm samples under both control as well as capa-

citated condition, although the phosphorylation pattern

differed somewhat (Figure 5 lanes 1 and 3).

changes in the lipid architecture of the plasma membrane according to Harrison et al. (22). The cells were counterstained

INCREASE IN PROTEIN TYROSINE PHOSPHORYLATION AT THE SPERM HEAD PLASMA MEMBRANE

The sperm head plasma membrane was isolated from control
incubated and in vitro capacitated cells according to Flesch
et al. (9) and purity was checked with marker lectins and
marker enzymes. The proteins from pure plasma membrane

preparations and from complete sperm cell preparations

were separated by SDS-PAGE. The tyrosine phosphoryla-

1 44

kDa
103

77

with Yo-Pro and the MC540 fluorescence of the living

sperm cells was monitored in a flow cytometer. Under control conditions only 2-5 % of the living cells were brightly
fluorescent for MC540 (Figure 4A) whereas 30-55 % of the
living cells acquired bright fluorescent staining within ca. 5
min (see Figure 4B). The observed increase in MC540 staining indicates that the packing of plasma membrane phospholipids, becomes disordered (34) in capacitating sperm cells.
Most likely this is due to an increase in membrane fluidity
which has been demonstrated in capacitating sperm cells
with the fluorescence recovery after photo bleaching technique (32,35).

111111101110

111101,

1.31111

48
34
28
21

Figure 5. Immunodetection of tyrosine phosphbrylation in boar sperm proteins

Solubilized protein samples were loaded and separated by SDS-PAGE and
after blotting on polyvinylidene fluoride membranes phosphotyrosine residues (PY) were detected by PY-20 (monoclonal anti PY) conjungated to

horseradish peroxidase. Lane 1 Control sperm cells, lane 2 control

plasma membranes, lane 3 capacitated sperm cells, lane 4 capacitated
plasma membranes. Capacitation was performed for a 2 h period.

THE VETERINARY QUARTERLY, VOL 21 , No 4 , OCTOBER, 1999

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INCREASE IN FUSOGENICITY AT THE EQUATORIAL

SUBDOMAIN OF THE SPERM HEAD PLASMA

MEMBRANE

If lipid vesicles containing N-rhodamine, and N-NBDheadgroup labeled phosphatidylethanolamine are illuminated so as to excite NBD (470 nm), little NBD fluorescence
(530 nm) but considerable rhodamine fluorescence (585 nm)

is observed; the rhodamine has been excited by absorbing

the emission energy from the NBD (a process known as fluorescence resonance energy transfer; FRET). When the vesicles fuse with cell membranes, the dilution of the two lipid
probes diminishes this FRET greatly and consequently NBD
fluorescence increases while the rhodamine fluorescence decreases. By monitoring FRET, it was found that fusion of the

fluorescent vesicles with sperm cells only took place after

the acrosome reaction (3,14). Fusion appears to be restricted
to the equatorial subdomain of the sperm head (Figure 6). In
this way we visualized the unique function of the equatorial
segment of the sperm head plasma membrane, described in
literature as the exclusive site of the sperm cell that fuses
with the oolemma during fertilization.
DISCUSSION

The current knowledge on the molecular organization of the

sperm plasma membrane and its relevance in sperm-egg interactions is very limited. However, the molecular organization of this organelle plays an important role in the physiology of the sperm cell. Changes in the molecular architecture
relate to other transitions of the sperm cell that enhance the
acrosome reaction such as changes in the intracellular ion

brane is an important event

for the sperm cell during

sperm-egg interactions: it
enables the sperm cell to
fuse at that site with the
oolemma (fertilization fusion) (8). Gaining insight
in mechanisms that regulate and modify the

(sub)domain structure of
the sperm plasma membrane, and thereby allo-

;if

wing transport, binding

and response to the oocyte,

as well as unraveling the

basic requirements for
sperm-egg fusion is of both
cell biological and clinical
interest.

The polarized membrane

structure, the involvement
in recognition and fusion
processes, and the occurrence of exocytosis, make
the

spermatozoon to a
valuable model system. It

should be noted that the

fine tuned surface lipid or-

ganization of the sperm

Figure 6. Fusion of PS liposomes with

intact porcine sperm cell after induc-

tion of the acrosome reaction on the

composition (especially Ca2+) (1,5,11), the activation of

(phospholipase dependent) signalling pathways (7,30) and

cell is sensitive for lipoproteins (15,19,29), peroxida-

membrane potential (4,18), the activation of protein kinases

(17,24,26). This article overviews the development and results of novel techniques used to study changes in the plasma
membrane in living sperm cells; this work offers new tools to
study the mechanism of sperm-oocyte interactions.

tion (2), and phospholipa-

Capacitation is defined as a preparative step; the spermatozoon must undergo a priming process before it can efficiently bind to the zona pellucida and respond appropriately
after this binding with the initiation of the acrosome reaction
(20,36). Although the physiological importance of capacitation has been acknowledged extensively in literature, the
molecular level of the processes occurring during this step
are ill defined up till now. With the techniques employed in
this paper we detected that the lateral and bilayer organization of sperm plasma membrane lipids alters upon capacita-

to manipulate the sperm

tion in vitro prior to the acrosome reaction. These lipid

ACKNOWLEDGEMENTS

changes may facilitate the induction of the multiple membrane fusions during the acrosome reaction and/or enhance

B.M. Gadella is a post-doc fellow granted by the Royal Dutch Academy of

Arts and Sciences (KNAW). F.M. Flesch is a PhD student financed by the
Graduate School of Animal Sciences (GSAH).

the sperm affinity for binding to the zona pellucida in the apical region of the sperm head surface (which primes the signalling cascade leading to the acrosome reaction;
17,24,26,27). In fact under these conditions we found that at
the plasma membrane level proteins became tyrosine phosphorylated. Preliminary results indicate that these proteins are

actually involved in sperm binding to the zona pellucida.

Therefore, upon capacitation sperm cells may generate high
affinity for the zona pellucida by activating endogenous tyrosine kinases. Later, after the initiation of the acrosome re-

ses (30) as well as for

changes in the buffer composition and the temperature (6). Future experiments should be designed
surface in such way that the

sperm cells obtain optimal

fertilizing ability. Applied
research on this topic may

145

capacitated for 4 hrs and incubated

with porcine zonae pellucidae according to Fazeli et al. [10]. at 370C.

After washing these preparations,

vesicles containing N-Rh-PE and NNBD-PE were added and allowed to
fuse with the sperm cells. The zona

bound spermatozoa were counter

stained with the vital stain Hoechst
33258 and only the living cells (i.e.
those that excluded this stain) were
analyzed. The length of the sperm
head is approximately 8 mm. For details about fluorescence microscopy
see Figure 2.

result in novel assays for

the prediction of the capacity of (sub)fertile sperm samples
to fertilize, important for the decision whether or not to apply
ICSI or rely on clinical IVF.

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