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,
* Université Paris 13, UFR SMBH, Laboratoire de Biologie de la Reproduction, and AP-HP Hôpital Jean Verdier, Bondy, France;
INSERM U567, Paris, France; and
CNRS UMR8104, Institut Cochin, IFR Alfred Jost, France
1Correspondence: Université Paris 13, Laboratoire de Biologie de la Reproduction, UFR SMBH, 74, rue Marcel Cachin, 93017 Bobigny, France. E-mail: jean-philippe.wolf{at}jvr.ap-hop-paris.fr
ABSTRACT
Several families of molecules are implicated in the membrane fusion process between sperm and oocyte. Among these, CD9 tetraspanin, a membrane-organizing molecule, plays a crucial role, since the fertilizing ability of CD9–/– oocytes is dramatically impaired. CD9 controls alpha6-beta1 integrin relocation involved in the membrane reorganization that occurs on oocyte fertilization but is not expressed on sperm. We report here that, together with several other proteins, the CD9 tetraspanin is transferred from the oocyte to the fertilizing spermatozoa present in the perivitelline space before fertilization. Transfer of CD9 from oocyte to sperm from CD9–/– male mice still occurs. CD9 acquisition by sperm results from a transfer of membrane fragments from the plasma membrane of the oocyte, in a process similar to trogocytosis, the recently described mechanism of intercellular exchange of membrane patches. Acquisition of CD9 by the sperm may be crucial for the membrane reorganization in sperm required for fusion with the oocyte, a process that is similar to the role CD9 plays in oocyte membrane reorganization.—Barraud-Lange, V., Naud-Barriant, N., Bomsel, M., Wolf, J.-P., Ziyyat, A. Transfer of oocyte membrane fragments to fertilizing spermatozoa.
Key Words: trogocytosis • gamete fusion • tetraspanin • lipid transfer • protein transfer
FERTILIZATION RESULTS FROM A GAMETE FUSION process in which several families of molecules participate. Two are essential for membrane fusion: the spermatic Izumo protein (1)
and, on the oocyte surface, the CD9 tetraspanin, an organizer of multimolecular complexes (2)
. Indeed, the CD9 tetraspanin controls the relocation of 
6ß1 integrin into patches at the surface of the oocyte membrane, a process that is associated with fertilization (3)
. In the mouse, the CD9 tetraspanin knockout female has severely reduced fertility (4
5
6)
linked to specific failure of oocyte membrane fusion, whereas the CD9–/– male is normally fertile.
Sperm normally binds to and fuses with the microvillous region of the oocyte plasma membrane. A recent study (7)
showed that CD9 is enriched on this microvillous oocyte membrane region and is required for normal microvilli shape and distribution. Here we show that, in the mouse, before fusion, oocyte membrane fragments containing CD9 tetraspanin are transferred onto the sperm head, which do not otherwise express CD9.
TETRASPANIN CD9 APPEARS ON THE SURFACE OF THE SPERM HEAD UPON FERTILIZATION
To clarify the role of CD9 tetraspanin during fusion of mouse gametes, CD9 localization on oocytes fertilized in vitro was analyzed by immunofluorescence. When interacting with oocytes with an intact zona pellucida, only one spermatozoon enters the perivitelline space and interacts with the oocyte membrane due to a block to polyspermy. Surprisingly, the head of the spermatozoon present into the perivitelline space stained specifically with anti-CD9 antibody (4.1F12); (Fig. 1
B, D); (4)
. Since normal sperm do not express CD9 tetraspanin, these data suggest that a transfer to the sperm of oocyte membrane fragments, including the CD9 tetraspanin, which crosses the membrane four times, had occurred. To formally exclude that the contact with the oocyte could induce the expression of CD9 by sperm, sperm from CD9–/– males were used. When membrane fragment transfer was monitored using sperm from CD9–/– male mice, the CD9 tetraspanin was still detected on the sperm membrane after the membrane fragment exchange (data not shown).
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OOCYTES TRANSFER MEMBRANE FRAGMENTS TO SPERM
To investigate the hypothesis that membrane fragments are transferred from oocyte to fertilizing spermatozoon, zona-free oocyte membranes first were stained either with PKH26 (Red Fluorescent Cell Linker for General Cell Membrane Labeling) or PKH67 (green analog) and then were exposed to sperm. During in vitro fertilization of zona-free eggs, multiple spermatozoa are observed to swim around the oocytes. Some of the sperm bind to and fuse with oocytes, while others interact with the oocyte membrane then swim away, remaining free in the medium. In the absence of sperm, no dye transfer from one oocyte to another was detected (Fig. 2
A), while PKH26-stained spermatozoa bound to PKH67-stained oocytes (Fig. 2B
). Reciprocally, PKH67-stained spermatozoa bound to PKH26-stained oocytes (data not shown). Taken together, these data show that membrane transfer from oocyte to spermatozoa is specific. Forty-five minutes postinsemination, free spermatozoa were collected, washed with acidic Tyrode’s solution to remove any possible bound-cellular debris, and analyzed by immunofluorescence. Some sperm heads were stained at the equatorial region, with dots of PKH representing oocyte membrane fragments (Fig. 2C
). Additionally, this membrane transfer process was shown to be an active process and not due to passive diffusion of the dye, as incubation of sibling spermatozoa with the medium in which stained oocytes had been further incubated for 1 1/2 h remained PKH free.
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QUANTIFICATION OF SPERM HEAD MEMBRANE CAPTURE
Membrane transfer from oocyte to sperm head was next quantified by flow cytometry. A mean of 10% of the recovered sperm had captured PKH-labeled oocyte membrane fragments (Fig. 3
A). To assess a functional relationship between this membrane fragment transfer and oocyte fertilizing capability, we evaluated whether already fertilized eggs were still able to provide membrane fragments to sperm heads. Zona-free oocytes were first exposed to sperm for 45 min and washed, and the same oocytes, containing from 1 to 6 sperm heads per egg, were stained with PKH and re-exposed to fresh sperm. A very limited number of sperm were observed to fuse during this second insemination of zona-free oocytes. A significant reduction in gamete fusion ability paralleled a dramatic reduction in transfer of oocyte membrane fragments to sperm (from 10 to 3%), suggesting a direct link between these two phenomena (Fig. 3C
).
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HYPOTHESIS: MEMBRANE TRANSFER FROM EGG TO SPERM PARTICIPATES IN GAMETE FUSION
The transfer of such membrane fragments between gametes has not previously been reported but could correspond to the presence of small membrane vesicles covering the sperm head of fertilizing spermatozoa present into the perivitelline space, that was previously reported at the ultrastructural level (8)
. The transfer is also reminiscent of trogocytosis, a phenomenon involved in lymphocyte activation, in which lymphocytes actively capture plasma membrane fragments of antigen-presenting cells with which they establish a tight contact, forming an immune synapse (9)
. We previously described oocyte membrane modifications upon fertilization, with the appearance of surface patches of alpha6beta1 integrin. These membrane modifications were dependent on CD9 tetraspanin (3)
. Recently, we have found that the alpha6beta1 integrin is also expressed on the sperm membrane (our unpublished observations) and is transferred from oocyte to fertilizing spermatozoon, together with CD81, another tetraspanin. Alpha6beta1 consequently increases on the surface of the fertilizing sperm. However, since the CD9 tetraspanin is the only well-characterized molecule controlling gamete fusion that is not expressed by sperm, one can speculate that before fusion, sperm have to reorganize their membrane-binding machinery into complexes, as is the case for oocytes (3)
. Such reorganization of the sperm membrane would require the presence and activity of a membrane "organizer," such as the CD9 tetraspanin. Accordingly, the transfer of egg membrane fragments to sperm that we describe here would allow the fertilizing sperm to acquire membrane "organizer" molecules, including the CD9 tetraspanin that sperm does not express. Alternatively, CD9 tetraspanin on sperm membranes could interact with other proteins within the egg membrane. Accordingly, it has been demonstrated that constructs including the large extracellular loop of CD9 significantly inhibit gamete fusion only when incubated with oocytes, but not when incubated with sperm (10)
. Such constructs would compete with the sperm CD9 tetraspanin at a yet unknown oocyte "receptor."
As a model, we suggest that sperm CD9 tetraspanin interacts, in a nonexclusive manner, with other sperm molecules, such as Izumo, to form multimolecular complexes like that recently described on the oocyte (3)
or even participates in a ligand-receptor interaction with oocyte surface proteins. Actually, Izumo belongs to the immunoglobulin superfamily (IgSF) as do EWI-2 and EWI-F, which are proteins directly associated with the CD9 tetraspanin (11
12
13)
. These two IgSF molecules may serve as linkers between surface proteins and ezrin, radixin, and moesin (ERM proteins), which in turn bind to the actin core of microvilli (14)
. Finally, the transfer of membrane fragments between gametes is likely to be essential to gamete fusion, as fertilization significantly reduces the ability of oocytes to transfer membrane fragments to sperm.
ACKNOWLEDGMENTS
We thank C. Boucheix and E. Rubinstein (INSERM U602) for providing anti-CD9 antibody, CD9 knock out mice and help for confocal microscopy analysis. We thank S. Chambris (Université Paris 13) for technical assistance, P. Bourdoncle for confocal microscopy analysis assistance (Institut Cochin), S. Line and L. Gattegno for flow cytometry analysis (Université Paris 13), and B. Weksler (Cornell University) for English editing of the manuscript.
Received for publication February 6, 2007. Accepted for publication May 3, 2007.
REFERENCES
6ß1, which are involved in human and mouse gamete fusion. J. Cell Sci. 119,416-424This article has been cited by other articles:
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  K. Miyado, K. Yoshida, K. Yamagata, K. Sakakibara, M. Okabe, X. Wang, K. Miyamoto, H. Akutsu, T. Kondo, Y. Takahashi, et al. The fusing ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice PNAS, September 2, 2008; 105(35): 12921 - 12926. [Abstract] [Full Text] [PDF]
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