Identification of sperm head proteins involved in zona pellucida binding.
OBJECTIVE
Which human sperm proteins interact with zona pellucida (ZP) glycoproteins, ZPA/2, ZPB/4 and ZPC/3?


CONCLUSIONS
Co-precipitation experiments with recombinant human ZP (rhZP) coated beads demonstrated interactions with various proteins, including glutathione S-transferase M3 (GSTM) with ZPB/4 and voltage-dependent anion channel 2 (VDAC2) with ZPA/2 and ZPC/3.


BACKGROUND
Regarding sperm-ZP binding, several target spot/proteins have been detected in several species, but not all have been characterized. The limit of these studies was that a mixture of the different ZP glycoproteins was used and did not allow the identification of the specific ZP glycoprotein (ZPA/2, ZPC/3 or ZPB/4) involved in the interaction with the sperm proteins.


METHODS
To identify the human sperm proteins interacting with the oocyte ZP, we combined two approaches: immunoblot of human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far western blot of human sperm proteins overlayd by each of the rhZP proteins.


METHODS
We used rhZP expressed in Chinese hamster ovary (CHO) cells and ASA eluted from infertile patients undergoing IVF failure. Sperm proteins separated by two-dimensional (2D) electrophoresis recognized by both sperm-eluted ASAs from infertile patients and rhZP were identified by mass spectrometry (MALDI-MS/MS). Some of these proteins were further validated by co-precipitation experiments with rhZP and functional zona binding tests.


RESULTS
We identified proteins that are glycolytic enzymes such as pyruvate kinase 3, enolase 1, glyceraldehyde-3-phosphate dehydrogenase, aldolase A, triosephosphate isomerase, detoxification enzymes such as GSTM or phospholipid hydroperoxide glutathione peroxidase, ion channels such as VDAC2 and structural proteins such as outer dense fibre 2. Several of the proteins were localized on the sperm head. However, these proteins have also been described to exert other functions in the flagellum. Co-precipitation experiments with rhZP-coated beads confirmed the direct interaction of GSTM with ZP4 and of VDAC2 with ZP2 and ZP3.


CONCLUSIONS
We used recombinant ZP in place of native ZP. Thus, the post-translational modifications of the proteins, such as glycosylations, can be different and can influence their function. However, CHO cell-expressed rhZP are functional, e.g. can bind human spermatozoa and induce the acrosome reaction. Moreover, the identification of relevant proteins was limited by the need for sufficient amounts of proteins on the preparative 2D-gel to be subsequently analysed in MALDI-TOF MS/MS.


CONCLUSIONS
Our results bring new insights on the ability of sperm proteins to exert several functions depending on their sub-cellular localization, either the head or flagellum. Their multiple roles suggest that these sperm proteins are multifaceted or moonlighting proteins.


BACKGROUND
This work was supported by the grant ReproRio (CNRS, INRA, INSERM and CEA) and the Société d'Andrologie de Langue Française.


BACKGROUND
Not applicable.
AP-HP, Laboratoire de génétique moléculaire, Hôpital Antoine Béclère, Clamart 92141, France.
F M
FM
Petit
C
C
Serres
F
F
Bourgeon
C
C
Pineau
J
J
Auer
D000818:Animals; D026481:Blotting, Far-Western; D016466:CHO Cells; D006224:Cricetinae; D004527:Egg Proteins / Q000378:metabolism / Q000502:physiology*; D005260:Female; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013077:Sperm Head / Q000378:metabolism*; D013084:Sperm-Ovum Interactions / Q000502:physiology*; D050996:Voltage-Dependent Anion Channel 2 / Q000737:chemistry / Q000378:metabolism; D015044:Zona Pellucida / Q000737:chemistry*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C494765:VDAC2 protein, human; D050996:Voltage-Dependent Anion Channel 2; C000606515:ZP2 protein, human; C000606517:ZP3 protein, human; C082833:ZP4 protein, human; D000072216:Zona Pellucida Glycoproteins; C483009:GSTM3 protein, human; D005982:Glutathione Transferase