Correspondence to: Diana G. Myles, Department of Molecular and Cellular Biology, University of California Davis, One Shields Avenue, Davis, CA 96516. Tel:(530) 752-1553 Fax:(530) 752-7522 E-mail:dgmyles{at}ucdavis.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous results, based on inhibition of fertilization by an anti6 integrin mAb (GoH3), suggest that the
6ß1 integrin on mouse eggs functions as the receptor for sperm (Almeida, E.A., A.P. Huovila, A.E. Sutherland, L.E. Stephens, P.G. Calarco, L.M. Shaw, A.M. Mercurio, A. Sonnenberg, P. Primakoff, D.G. Myles, and J.M. White. 1995. Cell. 81:10951104). Because the egg surface tetraspanin CD9 is essential for gamete fusion (Kaji, K., S. Oda, T. Shikano, T. Ohnuki, Y. Uematsu, J. Sakagami, N. Tada, S. Miyazaki, and A. Kudo. 2000. Nat. Genet. 24:279282; Le Naour, F., E. Rubinstein, C. Jasmin, M. Prenant, and C. Boucheix. 2000. Science. 287:319321; Miyado, K., G. Yamada, S. Yamada, H. Hasuwa, Y. Nakamura, F. Ryu, K. Suzuki, K. Kosai, K. Inoue, A. Ogura, M. Okabe, and E. Mekada. 2000. Science. 287:321324) and CD9 is known to associate with integrins, recent models of gamete fusion have posited that egg CD9 acts in association with
6ß1 in fusion (Chen, M.S., K.S. Tung, S.A. Coonrod, Y. Takahashi, D. Bigler, A. Chang, Y. Yamashita, P.W. Kincade, J.C. Herr, and J.M. White. 1999. Proc. Natl. Acad. Sci. USA. 96:1183011835; Kaji, K., S. Oda, T. Shikano, T. Ohnuki, Y. Uematsu, J. Sakagami, N. Tada, S. Miyazaki, and A. Kudo. 2000. Nat. Genet. 24:279282; Le Naour, F., E. Rubinstein, C. Jasmin, M. Prenant, and C. Boucheix. 2000. Science. 287:319321; Miyado, K., G. Yamada, S. Yamada, H. Hasuwa, Y. Nakamura, F. Ryu, K. Su- zuki, K. Kosai, K. Inoue, A. Ogura, M. Okabe, and E. Mekada. 2000. Science. 287:321324). Using eggs from cultured ovaries of mice lacking the
6 integrin subunit, we found that the fertilization rate, fertilization index, and sperm binding were not impaired compared with wild-type or heterozygous controls. Furthermore, a reexamination of antibody inhibition, using an assay that better simulates in vivo fertilization conditions, revealed no inhibition of fusion by the GoH3 mAb. We also found that an anti-CD9 mAb completely blocks sperm fusion with either wild-type eggs or eggs lacking
6ß1. Based on these results, we conclude that the
6ß1 integrin is not essential for spermegg fusion, and we suggest a new model in which CD9 acts by itself, or interacts with egg protein(s) other than
6ß1, to function in spermegg fusion.
Key Words: spermegg fusion, tetraspanin, membrane adhesion, oocyte, ovarian cultare
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Spermegg binding and fusion is critical to the initiation of development in many organisms, but identification of the molecules involved in the adhesion, fusion, and signaling is incomplete. In the present study, we examined the role of molecules on the egg surface that have been implicated in the process of gamete binding and fusion.
On the surface of the mammalian egg, two proteins, the integrin 6ß1 and the tetraspanin family member CD9, have been reported to act in spermegg binding and fusion. Integrins are transmembrane
ß heterodimers that play crucial roles in cellcell adhesion, cellextracellular matrix adhesion, and multiple signaling pathways (
6ß1 integrin was first proposed as the receptor for sperm on mouse eggs by
6ß1 had been found to be a laminin receptor whose adhesion activity was blocked by the anti-
6 mAb GoH3 (
6ß1 integrin in spermegg binding and fusion was called into question. All the binding and fusion assays of
6ß1 does have a role in spermegg binding and fusion.
Recently, another egg surface protein, CD9, has been discovered to play an essential role in spermegg fusion (
Tetraspanins are generally thought to work via lateral interactions with other cell-surface proteins such as CD4, CD8, CD19, CD21, major histocompatibility complexes I & II, integrins, and cytoplasmic signaling molecules (
The physical association of tetraspanins and integrins has been established in various cell types (6ß1 integrin in spermegg binding and fusion (
6 integrin, and are consistent with the conclusion that CD9 is essential for spermegg fusion, but acts either alone or through interactions with proteins other than
6ß1.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genotyping of 6 Knockout Mice
The generation of the total 6 integrin knockout mice (with a deletion of both A and B forms) has been described previously (
6 integrin subunit are born alive with severe skin blistering, reminiscent of epidermolysis bullosa (
6 intron upstream from the neomycin cassette insert, and the reverse primer 5'-TCAGAGCAGCCGATTGTC-3', which is located in the neomycin cassette (GIBCO BRL). These two primers produce an 820-bp band in homozygous and heterozygous mice. The wild-type allele was detected using the forward primer 5'-GTGATAACTCCAGCTTGTGTCAAG-3' and reverse primer 5'-CCTCTGCAGCGGGAGTGCTTC-3' (GIBCO BRL). These primers are within the region deleted in the mutant allele and give a product of 500 bp in wild-type mice. The PCR parameters were as follows: 3-min denaturation at 94°C, 34 cycles of amplification at 94°C (for 1 min), 55°C (for 1 min), 72°C (for 1 min), followed by 72°C (for 10 min) using a Robocycler (Stratagene).
Egg Isolation
Because pups died shortly after birth, it was necessary to isolate ovaries within 12 h after birth. Ovaries were dissected from newborn wild-type, heterozygous, and homozygous C57/Bl6 pups and placed into minimum essential medium (MEM; GIBCO BRL) at 37°C. They were prepared for grafting by removing the majority of the ovarian bursa. The ovaries were implanted under the kidney capsule of recipient females (
In Vitro SpermEgg Binding and Fusion Assay with Wild-type and Mutant Oocytes Isolated from Ovaries
Eggs, isolated as described, were treated with 10 µg/ml chymotrypsin in M199* + 0.3% BSA (Sigma Chemical Co.) for 3 min to loosen the zonae pellucidae. The zona pellucida was removed from the eggs because overnight culture can lead to modification (hardening) of the zona and, consequently, a reduction in fertilization rates. The treated eggs were transferred through three clean drops of medium using a narrow bore pipette to mechanically remove the loosened zonae. Zona-free eggs were preloaded with 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI;1 Polysciences Inc.) at 10 µg/ml in M199* + 0.3% BSA for 15 min at 37°C, 5% CO2. After DAPI treatment, the eggs were washed through three drops of clean M199* + 0.3% BSA. The eggs were allowed to recover for 3 h before insemination.
Sperm for the in vitro adhesion and fusion assay were isolated from the cauda epididymis and the vas deferens of 1012-wk-old male C57/Bl6 mice (Harlan Sprague Dawley, Inc.). Dissected cauda and vas deferens were placed into M199* + 3.0% BSA and were stripped of sperm. The sperm were allowed to dissociate for 15 min at 37°C in 5% CO2. Sperm were capacitated for 3 h in M199* + 3.0% BSA at a 1:10 dilution of the initial sperm suspension. This procedure results in a population of 6070% acrosome-reacted sperm (
Sperm and eggs were coincubated for 40 min at 37°C in 5% CO2. The eggs were scored for binding and fusion. Binding was scored under the light microscope at a magnification of 20. Fusion was scored by the fluorescent labeling of sperm nuclei by DAPI transfer from inside the preloaded eggs. The fertilization index (FI, the mean number of fused sperm per egg), and the fertilization rate (FR, the percentage of eggs fused with at least one sperm) were calculated. To test the effect of the anti-CD9 mAb KMC8.8 on the fertilization of mutant and wild-type eggs; zona-free eggs were preincubated for 30 min with 50 µg/ml KMC8.8 in M199* + 0.3% BSA. After the 30 min preincubation, sperm were added (final concentration 3 x 105 sperm/ml) to the drop containing the eggs and antibody. The gametes were coincubated for 40 min. Binding and fusion were assessed as described above. The control antibodies used were of the same species (rat) and isotype (IgG2a,) as KMC8.8.
Cumulus-intact Egg Assay for In Vitro Fertilization
To carry out in vitro fertilization assays with cumulus-intact eggs, 610-wk-old female ICR mice (Charles River) received pregnant mares' serum gonadotropin and 48 h later human chorionic gonadotropin. 14 h after hCG injection, cumulus masses containing eggs were isolated from the ampulla. The cumulus masses were washed through two 500-µl drops of M199* + 0.3% BSA, and then placed into a 50-µl drop of the same medium. Antibodies (500 µg/ml GoH3 or 50 µg/ml KMC8.8) were added to the drop with cumulus masses and preincubated for 45 min. After preincubation, sperm capacitated for 1.5 h at 37°C, 5% CO2 in M199* + 3.0% BSA were added to the cumulus masses at a final concentration of 15 x 106 sperm/ml. Gametes were allowed to coincubate overnight. Two cell embryos were counted to assess the FR. To determine if sperm penetrated the zona pellucida, the presence of sperm in the perivitelline space was scored using a Zeiss Axiophot microscope.
Indirect Immunofluorescence with Zona-free Eggs
Zona-free eggs were prepared and allowed to recover as described above. After the recovery period, eggs were incubated with either 50 µg/ml KMC8.8 (PharMingen) or 100 µg/ml GoH3 (PharMingen or Immunotech) for 45 min at 37°C, 5% CO2. The medium used for the primary antibody incubation was M199* + 0.3% BSA. The eggs were transferred through two 100-µl wash drops containing PBS + 0.1% polyvinyl alcohol (PVA; Sigma Chemical Co.), fixed for 12 min using 4% paraformaldehyde in PBS + 0.1% PVA, and transferred through two wash drops containing M199* + 0.3% BSA. An Oregon greenTMconjugated goat antirat secondary antibody (Molecular Probes) was used to determine the localization of the primary antibody binding. Staining was visualized using a laser scanning confocal microscope (model LSM 410; Carl Zeiss). The control antibodies used were of the same species and isotype as the primary antibody being tested, unless otherwise noted.
Indirect Immunofluorescence with Cumulus-intact Eggs
Cumulus masses were collected as mentioned above except using M199* + 0.1% PVA. Cumulus masses were washed through three 500-µl drops of fresh medium. Primary antibody staining with either GoH3 (500 µg/ml), KMC8.8 (50 µg/ml), or irrelevant IgG was carried out in 50-µl drops of the same medium for 45 min, followed by washing the cumulus masses through three 500-µl drops of medium. Secondary antibody staining was done with an Oregon greenTMconjugated goat antirat antibody in a 500-µl drop for 45 min, and cumulus masses were washed through three 500-µl drops. Cumulus-intact eggs were viewed using an LSM 410 confocal microscope.
Calcium Imaging
Zona-free eggs were mixed with 25 µg/ml Oregon greenTM-BAPTA AM (Molecular Probes) for 1 h at 37°C, 5% CO2. Once loaded, the eggs were washed through three drops of M199* + 0.3% BSA. Eggs were inseminated on the microscope stage using an open perfusion microincubator (Medical Systems Corp.) to keep the fertilization drop at 37°C. Calcium oscillations were observed using an LSM 410 confocal microscope. Spermegg fusion was assessed by DAPI transfer. These techniques are described in detail in
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Eggs from Mice Lacking the 6 Gene Are Morphologically Normal and Do Not Have the
6 Integrin Subunit on the Egg Surface
Mice lacking the 6 gene die shortly after birth. Following previous work of
6 +/+), heterozygous (
6 +/-), or homozygous (
6 -/-) females at birth, culture these ovaries for 34 wk, and recover fertilizable eggs (see Materials and Methods). Eggs from wild-type (
6 +/+) or heterozygous (
6 +/-) ovaries were equivalent to each other in fertilization assays, were used interchangeably, and are referred to as control eggs or
6+ eggs. Eggs from homozygous, mutant (
6-/-) ovaries are termed
6- eggs. Previous expression studies of the
6 integrin subunit in the developing embryo indicated a potential role in ovarian development (Fröjdman et al., 1995;
6 (-/-) females, we observed normal egg morphology. Cultured
6+ and
6- eggs are morphologically similar (Fig 1A and Fig B). The number of eggs/female collected from either cultured control (wild-type or heterozygous) ovaries or cultured
6 knockout ovaries were 43 ± 13 and 66 ± 18, respectively (mean ± SEM, n = 4). The percentages of cultured control eggs or cultured
6- eggs that produced first polar bodies after the overnight culture were 75 ± 5% and 76 ± 4%, respectively (mean ± SEM, n = 4). These data indicate that the integrin
6ß1 does not have a required role in ovary development or oogenesis because apparently normal eggs, capable of fertilization (see below), can be isolated from
6 (-/-) pups via our culture method.
|
As expected, eggs isolated from 6 (-/-) ovaries do not bind the anti-
6 mAb GoH3 (Fig 2 B).
6+ eggs, used as controls, showed the typical pattern of GoH3 binding to the microvillus region and no binding over the metaphase plate (Fig 2 A).
|
Eggs Lacking the 6 Integrin Subunit Are Fully Functional in SpermEgg Binding and Fusion
We directly tested the requirement for 6ß1 in spermegg plasma membrane binding and fusion by testing the ability of mature eggs lacking the
6 subunit to bind to and fuse with sperm. Immature eggs were collected from transplanted ovaries of wild-type and
6 (-/-) mice, the granulosa cells were removed to induce oocyte maturation, and oocytes were cultured overnight to obtain metaphase II eggs for in vitro fertilization assays. After culturing, the zona pellucida was removed by chymotrypsin, and the eggs were used for in vitro fusion assays. The following three parameters in these assays were measured: (1) FR, the percent of eggs fused with at least one sperm; (2) FI, the total number of fused sperm/total number of eggs; and (3) the mean number of sperm bound to the equator of the egg. There was no reduction in the number of sperm bound or fused with eggs lacking
6 compared with wild-type eggs (Fig 3). In seven experiments, using a total of 284 eggs, the mean FR for the cultured control eggs was 56% ± 8% (FR ± SEM). The fertilization rate for the
6- eggs, in parallel experiments, was 59 ± 9%. The FI of
6- and
6+ eggs were equivalent (Fig 3 B). In seven experiments, using a total of 230 eggs, the mean FI for both the cultured control eggs and the cultured
6- eggs was 0.67 ± 0.09 and 0.70 ± 0.1 (FI ± SEM). The mean number of sperm bound to the egg equator was 5.3 ± 2.8 sperm/egg with cultured control eggs and 7.8 ± 2.1 sperm/egg with
6- eggs. The increased level of sperm binding to
6- eggs in this assay is not significantly different than control eggs.
|
In addition to the in vitro assays carried out using standard procedures, in some experiments we directly observed the interactions of sperm and egg binding and fusion using a microscope equipped with Hoffman modulation optics. The characteristics of sperm binding were similar in assays using 6- eggs and control eggs. Sperm were bound via both their tip and lateral head regions, presumably corresponding to the inner acrosomal and equatorial regions of the sperm membrane (data not shown).
Sperm Can Initiate Calcium Oscillations in Eggs Lacking the 6 Integrin Subunit
Even though 6ß1 was not required for spermegg binding or fusion, we considered the possibility that
6ß1 was a required component in spermegg signaling. A hallmark of early signaling in the egg is the series of transient [Ca2+]i oscillations that occur directly after sperm binding/fusion. It has been suggested that integrins may participate in initiation and/or propagation of the calcium signal during egg activation (
6 integrin subunit.
6+ and
6- eggs were loaded with a calcium-sensitive dye, Oregon greenTM BAPTA-AM, and fertilized under conditions where Ca2+ oscillations could be observed. We observed no difference in the calcium oscillations of
6- eggs as compared with
6+ eggs. Calcium oscillations in the
6- eggs showed typical time of onset, amplitude, and frequency (Fig 4;
|
The Anti-6 mAb GoH3 Has No Effect on Sperm Fusion with Cumulus-intact Eggs In Vitro
Our results with the 6- eggs led us to reexamine the interpretations of previous GoH3 inhibition studies (
|
To determine if this assay could accurately measure antibody inhibition, we used the anti-CD9 antibody KMC8.8 as a control. Using cumulus-intact wild-type eggs in the same assay, we measured a 95% inhibition of fertilization in the presence of 50 µg/ml KMC8.8 antibody. The average fertilization rate for control eggs was 58% ± 12%, but for KMC8.8-treated eggs, the fertilization rate was reduced to 3% ± 1% (Fig 5 B, n = 4). Neither the GoH3 nor KMC8.8 antibodies inhibited sperm passage through the cumulus or zona as sperm were observed in the perivitelline space of antibody-treated unfertilized eggs. Diffusion of GoH3 or KMC8.8 to the egg plasma membrane was apparently not impeded in cumulus-intact eggs as shown by indirect immunofluorescence staining of the egg plasma membrane by both antibodies (Fig 5C and Fig D).
6ß1 Is Not Required for the Function of CD9 during SpermEgg Binding and Fusion
Previous studies have reported that CD9 has a role in spermegg fusion (6ß1. To directly test the model that during spermegg binding and fusion CD9 functions via associations with
6ß1, we tested the effects of the anti-CD9 antibody, KMC8.8, on cultured
6+ and
6- eggs. Sperm fusion with both
6+ and
6- eggs was completely inhibited in the presence of 50 µg/ml KMC8.8 (Fig 6). No apparent decrease in sperm binding was observed in the presence of either the control IgG or the KMC8.8 antibodies (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of morphologically normal eggs from ovaries of 6 (-/-) knockout pups allowed us to measure if eggs lacking the
6ß1 integrin bind and fuse with sperm. The fertilization rate and index both demonstrate that the
6ß1 integrin is not essential for spermegg binding and fusion. Furthermore, the inhibition of sperm fusion with
6- eggs in the presence of an anti-CD9 antibody shows that CD9 acts alone or in association with proteins other than
6ß1 to function in gamete binding and fusion.
Earlier evidence suggested the 6ß1 integrin, on the egg surface, played a crucial role in sperm binding leading to fusion (
6 integrin mAb GoH3 at 50400 µg/ml inhibited spermegg binding. Furthermore, spermegg fusion could be inhibited, presumably by blocking all sperm binding and by incubating eggs with the GoH3 antibody at a concentration of 400 µg/ml.
In light of our current data, we asked why do the 6 knockout results not agree with previous findings implicating
6, specifically GoH3 inhibition (
6ß1 during fertilization used a protease (chymotrypsin) treatment to prepare the zona-free eggs for the in vitro fertilization assays (
Because fertilization could be inhibited in assays using chymotrypsin-treated eggs, but not acid-treated or zona-intact eggs, we chose to test the ability of the GoH3 mAb to inhibit fusion using cumulus-intact eggs. The assay with cumulus-intact eggs is the closest experimental system to in vivo fertilization as it avoids the use of both crude preparations of hyaluronidase to remove the cumulus cells and chymotrypsin or acid to remove the zona. Data from the assay with cumulus-intact eggs demonstrate that GoH3 has no effect on fertilization under conditions where neither the cumulus cell layer nor the zona pellucida are disrupted. Experiments demonstrating the inhibitory effect of the anti-CD9 antibody in the assay with cumulus-intact eggs show that the assay can detect antibody inhibition, and that the zona does not act as a barrier to antibody diffusion to the plasma membrane. Furthermore, immunofluorescent detection of GoH3 and CD9 on the plasma membrane of cumulus-intact eggs demonstrates that these antibodies can reach the plasma membrane.
Normal sperm fusion with eggs lacking the 6 integrin subunit could mean that another integrin or receptor can substitute for the
6ß1 integrin in knockout eggs. However, our results with cumulus-intact eggs and previous results with zona-intact eggs (
6 integrin subunit are not impaired in spermegg binding or fusion, we suggest that
6ß1 is not required for spermegg binding and fusion.
What other egg surface protein might act as a receptor for sperm? One possibility is a different ß1 integrin. An anti-ß1 integrin polyclonal antibody moderately inhibited spermegg binding ( integrin subunits,
2,
3,
5, and
V, known to pair with ß1, have been reported to be present on the egg surface (
A new and exciting development is the discovery of a role for the egg surface protein CD9. Anti-CD9 antibodies were reported to inhibit binding and fusion (6 knockout eggs.
A Major Question to Be Answered Is How Does CD9 Function in the Fusion Process
In other systems, CD9 does not appear to function as a receptor itself, but has associations with several other cell-surface molecules (6ß1 integrin in other systems (
6ß1 has been suggested in models for spermegg binding leading to fusion (
6ß1 would affect
6ß1's ability to bind its sperm surface ligand (
6ß1 transduces signals to CD9 to initiate/promote fusion (
6ß1 to function in spermegg binding or fusion.
![]() |
Footnotes |
---|
1 Abbreviations used in this paper: DAPI, 4',6'-diamidino-2-phenylindole dihydrochloride; FI, fertilization index; FR, fertilization rate.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The authors would like to thank Drs. John Eppig (Jackson Laboratory, Bar Harbor, ME), Gerald Kidder (Department of Physiology, The University of Manitoba, Winnipeg, Manitoba, Canada), and Allen Hall (University of California Davis Medical Center, Davis, CA) for advice on surgical techniques. Thanks go to David Griffith for help with genotyping and colony maintenance, and Drs. Chunghee Cho (Laboratory of Reproductive and Developmental Toxicology, NIEHS, NIH, Chapel Hill, NC) and Jean-Emmanuel Faure (Ecole Normale Superieure de Lyon CNRS - INRA - ENS Lyon - University of Lyon, France) are gratefully acknowledged for advice on laboratory techniques. We also thank Jennifer Alfieri and Kathryn Kreimborg for critically reading the manuscript.
This work was supported by grants from the National Institutes of Health (HD16580 and HD29125).
Submitted: 10 April 2000
Revised: 9 May 2000
Accepted: 10 May 2000
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|