1 Section of Molecular and Cell Biology, University of California Davis,
One Shields Avenue, Davis, CA 95616, USA
2 Department of Cell and Human Anatomy, University of California Davis,
One Shields Avenue, Davis, CA 95616, USA
3 Department of Social and Environmental Medicine, Osaka University, Suita,
Osaka, Japan
* Author for correspondence (e-mail: pdprimakoff{at}ucdavis.edu)
Accepted 14 February 2003
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Summary |
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Key words: GPI-anchored protein, Sperm-egg fusion, Fertilization, Cre/loxP, Pig-a, Infertile
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Introduction |
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A number of surface proteins on sperm cells, including DE
(Cohen et al., 2000) and
equatorin (Toshimori et al.,
1998
), have been proposed to act in gamete fusion, but their role
in this process has not been thoroughly studied. CD9, a member of the
tetraspanin family of proteins present on eggs, has been shown to be essential
for sperm-egg fusion by gene knockout
(Kaji et al., 2000
;
Le Naour et al., 2000
;
Miyado et al., 2000
). The
exact role of CD9 on the oocyte plasma membrane is currently under
investigation. The large extracellular loop of CD9 seems to have functionally
significant cis interactions with other egg-surface proteins, and it seems to
have an active site in its large extracellular loop that acts in gamete fusion
(Zhu et al., 2002
).
Another group of egg surface proteins implicated in the gamete fusion
process is the lipid-linked, glycosylphosphatidylinositol-anchored proteins
(GPI-APs). Coonrod and coworkers reported that treatment of mouse eggs with
the enzyme PI-PLC (phosphatidyl inositol-specific phospholipase C) released
two GPI-APs (35-45 kDa and
70 kDa), and caused a reduction in
sperm-egg binding and a strong block of sperm-egg fusion
(Coonrod et al., 1999
). GPI-APs
are a functionally diverse group of proteins that includes adhesion molecules,
receptors, complement regulators, enzymes and signaling molecules
(Ferguson and Williams, 1988
;
Hooper, 1997
;
Low, 1989
). Aside from the
C-terminal lipid linkage another common characteristic they share is a high
degree of localization to cholesterol and sphingolipid-rich microdomains found
within the plasma membrane of every cell type examined
(Anderson and Jacobson, 2002
;
Brown and London, 2000
).
The finding that PI-PLC release of oocyte surface proteins blocks gamete
fusion could mean that one or both of these proteins (35-45 kDa and
70 kDa) has a role in sperm-egg fusion
(Coonrod et al., 1999
).
However, PI-PLC treatment could also have artifactual effects on membrane
fusion including: (1) the PI-PLC used was not tested by enzyme assay for
contaminating protease activity; (2) PI-PLC has a second substrate,
phosphatidylinositol, and its loss from the plasma membrane outer leaflet
might reduce membrane fusibility; (3) alternatively, PI-PLC-catalyzed
production of diacylglycerol (from phosphatidylinositol and GPI-APs) in the
outer leaflet of the plasma membrane could reduce fusibility; and (4) the
diacylglycerol produced in the outer leaflet might flip into the inner leaflet
and initiate signaling that would block gamete fusion. In addition, in vitro
experiments may show effects that can not be confirmed by in vivo tests using
gene deletion (Hynes, 1996
).
This has clearly been the case in fertilization research, where some of the
predicted roles of acrosin (Baba et al.,
1994
), Trp2 calcium channels
(Leypold et al., 2002
),
galactosyltransferase (Lu and Shur,
1997
) and the ADAMs and integrin
6ß1 mentioned above have failed to be
confirmed by in vivo knockout studies.
To determine whether GPI-APs have a role in fertilization in vivo we
created conditional, oocyte-specific GPI-AP-knockout mice. Pig-a,
phosphatidylinositol glycan class-A, encodes a subunit of an N-acetyl
glucosaminyl transferase that is involved in the first steps of GPI anchor
biosynthesis (Tiede et al.,
2000). Complete knockout of Pig-a is embryonic lethal
(Kawagoe et al., 1996
);
therefore the Cre/loxP recombination system was used to disrupt the
gene Pig-a so that GPI-AP delivery to the plasma membrane was
precluded in oocytes. Disruption of Pig-a exon 6, through flanking it
with direct loxP repeats
(Tarutani et al., 1997
),
occurred only in oocytes and not in other cells because Cre-recombinase was
expressed under control of the oocyte-specific promoter ZP3 (Shafi et al.,
2000). Transgenic mice carrying the ZP3-Cre gene and two copies of
the loxP-flanked Pig-a gene were considered to be
conditional knockouts. We have shown that egg GPI-APs are required for
sperm-egg fusion by use of the conditional knockout females.
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Materials and Methods |
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Generation of conditional knockout female mice
Pig-aflox females carrying the eGFP-GPI
transgene (Pig-a f/f:eGFP-GPI) were mated with ZP3-Cre males to generate
ZP3-Cre:Pig-a f/y:eGFP-GPI pups. These male mice were mated with Pig-a
f/f:eGFP-GPI females to generate a conditional knockout female with the
genotype ZP3-Cre:Pig-a f/f:eGFP-GPI.
In vivo fertility test
Conditional knockout or wild-type C57BL/6 females, 2 to 6 months old, were
individually housed for 21 days with a wild-type C57BL/6 male (Charles River)
aged eight to 10 weeks. Females were then separated from the males and allowed
to rest for 21 days, the average gestation period in mouse. A female was
considered fertile if she gave birth to pups. Litter size was determined by
counting pups.
In vivo fertilization assay
Wild-type C57BL/6 and conditional knockout females were superovulated with
10 IU pregnant mares' serum gonadotropin (PMSG, Sigma) followed 46 to 50 hours
later with 10 IU human chorionic gonadotropin (hCG, Sigma). Immediately
following hCG injection a wild-type C57BL/6 male was introduced into the cage
with a single female. 40 hours later the females were euthanased and the
oviducts were removed. The oviducts were placed in supplemented M199, which is
M199 medium (GIBCO BRL) supplemented with 3.5 mM sodium pyruvate, 100 IU/ml
Penicillin, 100 ug/ml Streptomycin and containing 0.4% BSA (Fraction V, fatty
acid free, Sigma). To release unfertilized oocytes and/or two cell embryos the
oviducts were minced with dissecting scissors. Two-cell embryos and
unfertilized oocytes were transferred through three 500 µl drops of medium
then viewed by light microscopy at 20x magnification to score the number
of two-cell embryos and unfertilized oocytes.
In vitro fertilization assay
Egg collection
Wild-type C57BL/6 and conditional knockout females were superovulated as
described above. 13 to 14 hours post-hCG injection cumulus masses were
retrieved from the oviducts of euthanized females and placed in supplemented
M199 medium + 0.4% BSA. The supplemented medium was pre-equilibrated overnight
under light mineral oil (Fisher) in a humidified incubator with 5%
CO2 at 37°C. Cumulus cells were removed from oocytes by
treatment in 300 µg/ml hyaluronidase Type I-S (Sigma) for 5 minutes
followed by washing through three 500 µl drops of medium. To remove the
zona, oocytes carrying a first polar body were transferred to a 100 µl
medium drop containing 30 µg/ml -chymotrypsin (Sigma) and incubated
for 3 minutes. Oocytes were transferred to a fresh medium drop and then passed
several times through a narrow bore (
80 µm), hand-pulled Pasteur
pipette to release eggs from the partially digested zona. Zona-free oocytes
were washed through two 100 µl drops of medium and placed in a fresh drop
to recover for 3 hours. Recovered oocytes were loaded with 4',6'
Diamidino-2-phenylindole dihydrochloride (DAPI) to label DNA by incubating the
cells for 12 minutes at 100 µg/ml, 37°C, 5% CO2 followed by
washing through three drops.
Sperm isolation
Sperm were isolated from 10- to 12-week-old ICR males (Charles River). The
cauda epididymis and vas deferens were removed from euthanased males and
placed in a 500 µl drop of supplemented M199 + 3% BSA under light mineral
oil. Sperm were squeezed from the vas deferens, and the epididymis was cut in
several places and placed for 15 minutes in a humidified CO2
chamber to allow sperm to swim out. Tissue fragments were removed, and the
sperm diluted 10-fold into a fresh 500 µl drop to a concentration of
1-5x106 sperm/ml and capacitated for 3 hours at 37°C, 5%
CO2.
Co-incubation of gametes
Capacitated sperm were diluted 10-fold to a final concentration of
1-5x105 sperm/ml in a 100 µl drop of supplemented M199 +
0.4%BSA along with DAPI-loaded, zona-free oocytes. Gametes were co-incubated
for 40 minutes at 37°C, 5% CO2. Sperm-egg complexes were washed
through one 100 µl medium drop and loaded onto a microscope slide to score
for the fertilization rate (FR; percentage of eggs fused with at least one
sperm), fertilization index (FI; total number of sperm fused per total number
of eggs) and sperm bound per egg. The transfer of DAPI from pre-loaded eggs to
sperm was used to score sperm-egg fusion.
Indirect immunofluorescence
Zona-free oocytes from wild-type C57BL/6, from Pig-a f/f:eGFP-GPI, and from
conditional knockout ZP3-Cre:Pig-a f/f:eGFP-GPI females were collected as
described above. To assess surface expression of GPI-APs, oocytes were probed
with a rabbit polyclonal anti-GFP antibody (Abcam Limited, ab290) to detect
the presence of eGFP-GPI. Eggs were washed through four 100 µl drops of PBS
(137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM
KH2PO4, pH 7.4) + 3% BSA (PBS/BSA), transferred to 100
µg/ml rabbit anti-GFP in PBS/BSA and incubated at room temperature for 1
hour. Eggs were washed through three 100 µl drops of PBS/BSA, transferred
to 20 µg/ml Alexa Fluor® 568 goat anti-rabbit secondary antibody
(Molecular Probes) and incubated for 30 minutes at room temperature. Eggs were
washed through three drops of PBS/BSA and placed under a raised coverslip on a
microscope slide and viewed by laser scanning confocal microscopy (model LSM
410, Carl Zeiss). The 568 laser line was used to excite the Alexa Fluor®
-568-conjugated secondary antibody.
Essentially the same protocol was used to test a set of antibodies to known mammalian GPI-APs (anti-CD24, -CD48 and -Qa-2 from Pharmingen, San Diego, CA; anti-CD55, GPC3, uPAR, Gas1 antibodies from Santa Cruz Biotechnology, Santa Cruz, CA) on zona-free oocytes. In the case of CD55 the first antibody was 100 µg/ml polyclonal goat anti-mouse CD55 (Santa Cruz Biotechnology) and the secondary antibody was 20 µg/ml Alexa Fluor® 488-donkey anti-goat (Molecular Probes). PBS + 1% polyvinyl alcohol (PBS/PVA) was used as the buffer for antibody incubation and all washes. As a control for primary antibody binding, wild-type oocytes were incubated with an irrelevant Goat IgG (Zymed) at 100 µg/ml. To determine if oocyte CD55 was PI-PLC sensitive, wild-type oocytes were treated with 1 U/ml PI-PLC (kind gift of Martin Low, Columbia University) in supplemented M199 + 0.4% BSA for 30 minutes, 37°C, then washed through three 100 µl droplets of medium prior to antibody labeling.
Western blotting of PI-PLC-treated eggs
Zona-intact oocytes were retrieved as described above and incubated with or
without 1 U/ml PI-PLC in PBS. The oocytes were incubated for 30 minutes at
room temperature, after which cells were removed, washed through four 100
µl drops of PBS/PVA, then transferred to an Eppendorf tube with SDS sample
buffer (2% SDS, 125 mM Tris-HCl, pH 6.8, 20% glycerol and 0.2% bromophenol
blue). The samples were heated, separated on 10% SDS-PAGE and transferred to
PVDF for western blotting. The PVDF membrane was blocked for 1 hour at room
temperature in TBST (50 mM Tris-HCl pH 7.3, 500 mM NaCl, + 0.5% Tween 20) + 5%
non-fat milk. The membrane was probed for 1 hour at room temperature in the
same blocking solution + 40 ng/ml goat anti-mouse CD55. HRP-conjugated donkey
anti-goat (Santa Cruz Biotechnology) at 2 ng/ml was incubated with the
membrane in blocking solution for 1 hour. The membrane was washed three times
for 10 minutes in TBST at room temperature then developed in Pierce's
WestFEMTO chemiluminescent reagent following the manufacturer's
instructions.
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Results |
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Appropriate matings allowed us to obtain conditional knockout female mice
designated ZP3-Cre:Pig-a f/f:eGFP-GPI (Fig.
1). These females carry the ZP3-Cre transgene, two
Pig-aflox alleles (Pig-a f/f) and an internal control
transgene encoding a GPI-anchored eGFP
(Kondoh et al., 1999). To test
fertility, 10 conditional knockout females were mated for 21 days with a
wild-type C57BL/6 male (one female housed with one male) and then rested
another 21 days during which time no visible pregnancies or births were
observed (Table 1). All 10
wild-type control females became pregnant and gave birth to an average of
6.2±2.5 pups within 23 days of males being introduced.
|
|
Normal numbers of oocytes lacking surface expression of GPI-APs are
retrieved from conditional knockout mice
One possible explanation for the infertility found in conditional knockout
mice is that the absence of GPI-APs from the surface of developing oocytes
prevents maturation of oocytes. To determine whether mature oocytes could be
retrieved, wild-type and conditional knockout mice were superovulated with
serum gonadotropins, and zona-intact oocytes were retrieved from the ampullae
of treated females as described in Materials and Methods. The number of
oocytes and percentage of mature oocytes having a first polar body from
conditional knockouts and wild-type animals were essentially the same
(Table 2).
|
To determine the extent to which GPI-AP expression was affected, oocytes were retrieved from knockout females carrying the eGFP-GPI transgene and analyzed by confocal microscopy to detect expression of eGFP-GPI on the egg plasma membrane. eGFP-GPI, detected with a GFP-specific antibody, was present on the surface of wild-type oocytes but was absent from the surface of conditional knockout oocytes (Fig. 2), which indicates successful Pig-a disruption.
|
Conditional GPI-AP-knockout females do not produce fertilized eggs
when mated
Although they produce normal numbers of superovulated, mature oocytes,
conditional GPI-AP-knockout females never became visibly pregnant. To test if
fertilization was occurring in vivo we mated gonadotropin-treated females and
scored the number of two-cell embryos as a measure of fertilization. A male
was introduced into the cage with a single superovulated female immediately
post-hCG injection as described in Materials and Methods and left for 40
additional hours. Females were subsequently euthanized, then cells
(unfertilized eggs or two-cell embryos) were retrieved from the oviducts of
mated wild-type and conditional knockout females. From three wild-type females
83% of the 52 healthy cells retrieved were two-cell embryos. However, from a
total of five conditional knockouts 74 healthy, unfragmented cells were
retrieved, and among these an average of only 1.3% were two-cell embryos
(Fig. 3). In addition, multiple
sperm were detected in the perivitelline space of several
Pig-a/ eggs. This indicates that there was
no block to multiple sperm traversing the zona and entering the perivitelline
space, a block that does occur if a sperm fuses with the egg.
|
Wild-type sperm do not fuse with GPI-AP/
eggs
Since unfertilized eggs with perivitelline sperm were observed and two-cell
embryos were almost absent, it appears that sperm-egg fusion is defective in
conditional knockout females when females are mated. To test for a fusion
defect in GPI-AP-knockout eggs, zona-free oocytes obtained from conditional
knockout and wild-type females were inseminated with wild-type sperm for 40
minutes. The fertilization rate was about eight-fold lower and the
fertilization index about nine-fold lower in GPI-AP/
eggs compared to the wildtype (Fig.
4). Sperm binding to the knockout eggs was not significantly
different from the control (3.9±1.8 and 2.4±1.9 wil type versus
knockout; P=0.09). These findings indicate that when GPI-APs are not
expressed on the surface of mouse eggs, the eggs are defective in fusing with
sperm.
|
Identification of GPI-anchored CD55 on mouse eggs
PI-PLC treatment of surface-biotinylated mouse eggs releases an 35-45
kDa and a 70 kDa protein detectable with avidin
(Coonrod et al., 1999
). We
sought to identify these bands or other egg-surface GPI-APs initially by using
commercially available antibodies to known mammalian GPI-APs. One of the
commercial antibodies identified an egg-surface GPI-AP as CD55 in indirect
immunofluorescence and western blot experiments. CD55 is a complement
regulatory protein also known as decay accelerating factor, DAF. Zona-free
wild-type mouse oocytes were stained with polyclonal goat anti-CD55
(Fig. 5A). Pretreatment of eggs
with PI-PLC, which releases GPI-APs from the cell surface, diminished staining
(Fig. 5B), indicating that
oocyte CD55 is PI-PLC sensitive. By western blotting, a single
70 kDa
protein band was recognized in mouse eggs
(Fig. 6). PI-PLC sensitivity
was also demonstrated in this blot by the decrease in staining intensity of
the
70 kDa band when eggs were pre-treated with PI-PLC. Unfortunately,
when supernatants from PI-PLC-treated eggs were analyzed, the anti-CD55
antibody was unable to detect protein, suggesting that the epitopes are less
available or altered after CD55 release.
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Discussion |
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The in vivo data suggest that the female infertility observed is the result of a block to fertilization at the level of sperm-egg fusion. Multiple sperm enter the perivitelline space of Pig-a/ eggs without causing a fusion-induced block to polyspermy. The in vitro fertilization data confirm that the defect is at the level of gamete fusion because wild-type sperm fuse poorly with Pig-a/ eggs.
A number of interpretations can be considered to explain why eggs lacking
GPI-APs are defective in fusion. One possibility is that eggs may require
their full complement of GPI-APs to maintain the correct membrane organization
to be fusion-competent. Our data along with the known lack of an effect of the
CD55 knockout on fertility indicate that this possibility is not the case. The
GPI-AP CD55 was identified on mouse eggs. However, in previous studies
CD55-knockout mice have been generated, and they do not show altered fertility
(Sun et al., 1999). Therefore,
even though the CD55-knockout female mice lack the full complement of
GPI-anchored proteins, they have normal fertility.
A second possibility is that at least some GPI-anchored proteins must be
present to support the interaction of proteins in organized plasma membrane
lipid domains (e.g. rafts). In some cell types when all GPI-APs are absent the
cells still contain rafts, indicating that GPI-APs are not indispensable
structural elements of these microdomains
(Abrami et al., 2001). However,
the absence of all the egg GPI-APs may alter the normal lipid domain
composition and may change the repertoire of protein-protein interactions in
lipid domains. The lipid domain is an important functional unit for cell
signaling molecules, for cell activation, adhesion and viral infectivity. For
example, in T cell receptor (TCR)-mediated signaling and activation, the
function of the TCR and the complex of proteins associated with it is
dependent on lipid domain integrity and the presence of GPI-APs
(Montixi et al., 1998
;
Romagnoli and Bron, 1997
).
Disruption of lipid domains by cholesterol depletion or lack of GPI-APs in T
cells results in decreased signaling. T cells lacking GPI-APs have decreased
phosphorylation downstream of TCR activation probably because src family
kinases found in lipid raft fractions have decreased activity in the absence
of GPI-APs (Romagnoli and Bron,
1997
). Lipid rafts may also serve as platforms for viral entry
into some cell types (Dimitrov,
2000
), and disruption of these domains prevents entry of a variety
of pathogens (Campbell et al.,
2001
). Thus, the absence of egg GPI-APs from lipid domains might
block egg signaling or egg functions that normally result in membrane fusion
through steps about which nothing is currently known.
A final explanation is that a single egg GPI-AP has a specific, required
function in sperm-egg fusion. Biotinylation of the egg surface followed by
PI-PLC treatment and detection with avidin reagents revealed GPI-APs of
70 kDa and
35-45 kDa (Coonrod et
al., 1999
). We found that CD55 is a GPI-AP on the egg surface,
which has a Mr
70 kDa and may be the biotinylated 70
kDa protein. However, CD55 apparently does not have a required function in
fertilization as CD55-knockout mice have normal fertility
(Sun et al., 1999
). To
evaluate a possible specific function in gamete fusion for a single egg
GPI-AP, it will be necessary to characterize and identify the remaining
35-45 kDa GPI-AP or other so far undetected GPI-APs that may be of lower
abundance and/or poorly biotinylated.
Since egg GPI-APs and CD9 are now the two known gene products whose
ablation blocks sperm-egg fusion, it is worth considering if and how they may
interact. Tetraspanins in general and CD9 in particular are not concentrated
in the kinds of lipid domains that are enriched for GPI-APs
(Claas et al., 2001). Although
CD9 is known to form associations with many other plasma membrane proteins
(e.g. Ig superfamily, membrane-anchored growth factors and integrins), none of
the known CD9 partners has a GPI anchor. These preliminary ideas suggest the
GPI-APs and CD9 act in distinct segments of a fusion pathway in which
additional components remain to be revealed.
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Acknowledgments |
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