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Sperm–egg interaction is a highly species-specific step during the fertilization process. The first steps consist of recognition between proteins on the sperm head and zona pellucida (ZP) glycoproteins, the acellular coat that protects the oocyte. We aimed to determine which sperm head proteins interact with ZP2, ZP3 and ZP4 in humans. Two approaches were combined to identify these proteins: immunoblotting human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far-Western blotting of human sperm proteins overlaid by each of the human recombinant ZP (hrZP) proteins. We used a proteomic approach with 2D electrophoretic separation of sperm protein revealed using either ASAs eluted from infertile patients or recombinant human ZP glycoproteins expressed in Chinese-hamster ovary (CHO) cells. Only spots highlighted by both methods were analysed by MALDI–MS/MS for identification. We identified proteins already described in human spermatozoa, but implicated in different metabolic pathways such as glycolytic enzymes [phosphokinase type 3 (PK3), enolase 1 (ENO1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase A (ALDOA) and triose phosphate isomerase (TPI)], detoxification enzymes [GST Mu (GSTM) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) 4], ion channels [voltage-dependent anion channel 2 (VDAC2)] or structural proteins (outer dense fibre 2). Several proteins were localized on the sperm head by indirect immunofluorescence, and their interaction with ZP proteins was confirmed by co-precipitation experiments. These results confirm the complexity of the sperm–ZP recognition process in humans with the implication of different proteins interacting with the main three ZP glycoproteins. The multiple roles of these proteins suggest that they are multifaceted or moonlighting proteins.

STUDY QUESTION Which human sperm proteins interact with zona pellucida (ZP) glycoproteins, ZPA/2, ZPB/4 and ZPC/3? SUMMARY ANSWER 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. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION 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. MATERIALS, SETTING, 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. MAIN RESULTS AND THE ROLE OF CHANCE 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. LIMITATIONS, REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the grant ReproRio (CNRS, INRA, INSERM and CEA) and the Societe d'Andrologie de Langue Francaise. TRIAL REGISTRATION NUMBER Not applicable.

Oocyte integrins have been described as essential for fertilization. But this concept has been challenged by deletion experiments. Recently, we have shown that sperm integrin alpha6beta1 plays a determinant role in mouse gamete interaction. In this study, we demonstrate the presence of alphavbeta3 integrin by Western blot and immunofluorescence on the sperm membrane. Oocytes and/or sperm preincubations with anti-alphav or anti-beta3 antibodies were performed before in vitro fertilization on cumulus-intact and zona-free egg assays. We observed inhibitory effects on the fusion process mostly by means of sperm function. An antibody directed against vitronectin inhibited gametes fusion, whereas the presence of exogenous vitronectin increased its efficiency. We suggest that vitronectin (on multimeric forms) can play a first nonspecific link corresponding to loosely bound spermatozoa to oocyte and that this link could be mediated by means of oocyte proteoglycans or integrins, and sperm alphavbeta3 integrin.

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.

In mammals, X- and Y-encoded genes are transcriptionally shut down during male meiosis, but the expression of many of them is (re)activated, after meiosis, in spermatids. Postmeiotic XY gene expression is timely regulated by active epigenetic marks, which are de novo incorporated in the sex chromatin of spermatids, and by repressive epigenetic marks inherited from meiosis; alteration in this process leads to male infertility. In the mouse, postmeiotic XY gene expression is known to depend on genetic information carried by the male specific region of the Y chromosome long arm (MSYq). The MSYq gene Sly has been shown to be a key regulator of postmeiotic sex chromosome gene expression and necessary for the maintenance/recruitment of repressive epigenetic marks on the sex chromatin, but studies suggest that another MSYq gene may be required. The best candidate to date is Ssty, an MSYq multicopy gene of unknown function. Here, we show that SSTY proteins are specifically expressed in round and elongating spermatids and colocalize with the postmeiotic sex chromatin. Moreover, SSTY proteins interact with SLY protein and its X-linked homolog SLX/SLXL1, and may be required for the localization of SLX/SLY proteins in the spermatid nucleus and sex chromatin. Our data suggest that SSTY is a second MSYq factor involved in the control of XY gene expression during sperm differentiation. As Slx/Slxl1 and Sly genes have been shown to be involved in the XY intragenomic conflict which affects the offspring sex-ratio, Ssty might constitute another actor of this conflict.

Antisperm antibodies eluted from the surface of spermatozoa obtained from infertile men recognised several common epitopes. We tested whether these epitopes were relevant to fertility by isolating the immunodominant 37/36 and 19/18 protein zones. These protein zones were cut out of preparative slab gels and electro-eluted. The isolated proteins, P36 and P18, were used for biochemical characterisation and to produce specific antibodies in rabbits. The specific reactivity of P36 and P18 with WGA and AAL lectins, respectively, indicated the presence of lactosaminyl structures with sialic acid termini in P36 and of fucosylated residues in P18. Isoelectric focusing showed that the two proteins consist of several polypeptides. Some of these polypeptides were recognised by both human and rabbit antibodies: the pl of these epitopes was around 5.5 for P36 and 8.3-10.3 for P18. Rabbit antibodies detected the corresponding proteins on the sperm heads of methanol-fixed and of live acrosome-reacted spermatozoa. Anti-P36 antibodies bound mainly to the equatorial segment. They reduced the binding and, consequently, the penetration of zona-free hamster oocytes by human spermatozoa. Anti-P18 antibodies gave more diffuse staining of the acrosomal and post-acrosomal regions and reduced sperm-oocyte penetration without a significant effect on sperm binding. These results suggest that P36 and P18 antigens located in different compartments of the sperm head may participate in the sperm-oolemma interaction. We are currently investigating the physiological role of these antigens by sequencing the proteins isolated from the gels.

Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol. Fil: Buschiazzo, Jorgelina. Universite Paris Descartes; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Ialy Radio, Come. Universite Paris Descartes; Francia Fil: Auer, Jana. Universite Paris Descartes; Francia Fil: Wolf, Jean Philippe. Universite Paris Descartes; Francia Fil: Serres, Catherine. Universite Paris Descartes; Francia Fil: Lefèvre, Brigitte. Universite Paris Descartes; Francia Fil: Ziyyat, Ahmed. Universite Paris Descartes; Francia

In order to identify the surface antigens of human spermatozoa recognized by the sera of immune infertile men, sperm membrane-specific antibodies were obtained from three serum samples exhibiting high titres of antibodies with different sperm-binding patterns. Serum antibodies were adsorbed onto normal motile spermatozoa and subsequently eluted from the sperm membrane. Using the immunoblotting technique under renaturating conditions, the sperm-eluted antibodies were tested against an electrophoretically fractionated sperm membrane preparation. A total of 15 protein bands ranging from 110 to 16 kDa were defined, but the immunoblot profiles differed quantitatively and qualitatively from one serum to another. Only two polypeptides were recognized by the three sperm membrane-specific antibody preparations; one of 90 kDa and another of 110 kDa. Blots were also used for the affinodetection of specific oligosaccharide side chains. Three lectins were tested (concanavalin A, Pisum sativum, and wheat germ agglutinin). Of the 15 protein zones recognized by the antibodies, 11 bound at least one lectin and should contain glycopeptides with oligosaccharides of four different types: N-linked biantennary complex type, N-linked fucosylated complex type, N-linked lactosaminyl complex type with terminal sialic acid and polysialyl type oligosaccharides. Further analysis of these glycoproteins will be pursued with sperm-associated antibodies eluted from the ejaculates of infertile men in order to define those with a potential role in the fertilization process.

Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis ofin vitroexchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists inCd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process.

Supplemental materials : www.landesbioscience.com/journals/epigenetics/article/21495; Genomic imprinting characterizes genes with a monoallelic expression, which is dependent on the parental origin of each allele. Approximately 150 imprinted genes are known to date, in humans and mice but, though computational searches have tried to extract intrinsic characteristics of these genes to identify new ones, the existing list is probably far from being comprehensive. We used a high-throughput strategy by diverting the classical use of genotyping microarrays to compare the genotypes of mRNA/cDNA vs. genomic DNA to identify new genes presenting monoallelic expression, starting from human placental material. After filtering of data, we obtained a list of 1,082 putative candidate monoallelic SNPs located in more than one hundred candidate genes. Among these, we found known imprinted genes, such as IPW, GRB10, INPP5F and ZNF597, which contribute to validate the approach. We also explored some likely candidates of our list and identified seven new imprinted genes, including ZFAT, ZFAT-AS1, GLIS3, NTM, MAGI2, ZC3H12Cand LIN28B, four of which encode zinc finger transcription factors. They are, however, not imprinted in the mouse placenta, except for Magi2. We analyzed in more details the ZFAT gene, which is paternally expressed in the placenta (as ZFAT-AS1, a non-coding antisense RNA) but biallelic in other tissues. The ZFAT protein is expressed in endothelial cells, as well as in syncytiotrophoblasts. The expression of this gene is, moreover, downregulated in placentas from complicated pregnancies. With this work we increase by about 10% the number of known imprinted genes in humans.

International audience; BACKGROUND: FOXL2 is a transcription factor essential for ovarian development and maintenance. It is mutated in the genetic condition called Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES) and in cases of isolated premature ovarian failure. We and others have previously shown that FOXL2 undergoes several post-translational modifications. METHODS AND PRINCIPAL FINDINGS: Here, using cells in culture, we show that interference with FOXL2 SUMOylation leads to a robust inhibition of its transactivation ability, which correlates with a decreased stability. Interestingly, FOXL2 SUMOylation promotes its transient recruitment to subnuclear structures that we demonstrate to be PML (Promyelocytic Leukemia) Nuclear Bodies. Since PML bodies are known to be sites where post-translational modifications of nuclear factors take place, we used tandem mass spectrometry to identify new post-translational modifications of FOXL2. Specifically, we detected four phosphorylated, one sulfated and three acetylated sites. CONCLUSIONS: By analogy with other transcription factors, we propose that PML Nuclear Bodies might transiently recruit FOXL2 to the vicinity of locally concentrated enzymes that could be involved in the post-translational maturation of FOXL2. FOXL2 acetylation, sulfation, phosphorylation as well as other modifications yet to be discovered might alter the transactivation capacity of FOXL2 and/or its stability, thus modulating its global intracellular activity.

A clinically significant proportion of couples experience difficulty in conceiving a child. In about half of these cases male infertility is the cause and often genetic factors are involved. Despite advances in clinical diagnostics ∼50% of male infertility cases remain idiopathic. Based on this, further analysis of infertile males is required to identify new genetic factors involved in male infertility. This review focuses on cation channel of sperm (CATSPER)-related male infertility. It is based on PubMed literature searches using the keywords 'CATSPER', 'male infertility', 'male contraception', 'immunocontraception' and 'pharmacologic contraception' (publication dates from January 1979 to December 2009). Previously, contiguous gene deletions including the CATSPER2 gene implicated the sperm-specific CATSPER channel in syndromic male infertility (SMI). Recently, we identified insertion mutations of the CATSPER1 gene in families with recessively inherited nonsyndromic male infertility (NSMI). The CATSPER channel therefore represents a novel human male fertility factor. In this review we summarize the genetic and clinical data showing the role of CATSPER mutation in human forms of NSMI and SMI. In addition, we discuss clinical management and therapeutic options for these patients. Finally, we describe how the CATSPER channel could be used as a target for development of a male contraceptive.

Little is known about the molecular impact of in vivo exposure to endocrine disruptors (EDs) on sperm structures and functions. We recently reported that the lifelong exposure of rats to the antiandrogenic compound vinclozolin results in low epididymal weight, changes in sperm kinematic parameters, and immature sperm chromatin condensation, together with the impairment of several fertility end points. These results led us to focus specifically on possible molecular abnormalities in sperm. Sperm samples were recovered from the frozen epididymides of rats exposed during the previous study. The proteins present in the samples from six exposed and six control rats were analyzed in pairs, by two-dimensional fluorescence difference gel electrophoresis, to investigate possible exposure-induced changes to sperm protein profiles. Twelve proteins, from the 380 matched spots observed in at least five gels, were present in larger or smaller amounts after vinclozolin exposure. These proteins were identified by mass spectrometry, and several are known to play a crucial role in the sperm fertilizing ability, among which, two mitochondrial enzymes, malate dehydrogenase 2 and aldehyde dehydrogenase (both of which were present in smaller amounts after treatment) and A-kinase anchor protein 4 (larger amounts of precursor after treatment). Finally, Ingenuity Pathway Analysis revealed highly significant interactions between proteins over- and underexpressed after treatment. This is the first study to show an association between in vivo exposure to an ED and changes to the sperm protein profile. These modifications may be at least partly responsible for the reproductive abnormalities and impaired fertility recently reported in this rat model of vinclozolin exposure.

In order to identify new protein markers modified in placental diseases, high-throughput analysis of proteins in the plasma of pregnant women was carried out for normal and pathological pregnancies (Preeclampsia and/or Intra-Uterine Growth Restriction) using iTRAQ technology. We could identify 166 proteins that were modified (p0.05) and the technique used allowed the detection of previously undetected factors, such as various members of the SERPINA clade. The modifications of two proteins (C reactive protein and antichymotrypsin, SERPINA3) were validated on individual samples. Complement and coagulation cascades proteins were significantly enriched among modified protein clusters in the case of intra-uterine growth restriction (p2.6.10(-11)). Several proteins were specifically enriched in isolated preeclampsia and depleted when preeclampsia was complicated by intra-uterine growth restriction. These findings suggest that the growth restricted foeto-placental unit is able to moderate some changes in maternal plasma composition. Overall, the use of iTRAQ technology, for the first time on this subject, enabled us to provide a new list of proteins modified in placental diseases, among which proteins expressed at a low level that were not accessible by other methods.

FOXL2 is a forkhead transcription factor, essential for ovarian function, whose mutations are responsible for the blepharophimosis syndrome, characterized by craniofacial defects, often associated with premature ovarian failure. Here, we show that cell stress upregulates FOXL2 expression in an ovarian granulosa cell model. Increased FOXL2 transcription might be mediated at least partly by self-activation. Moreover, using 2D-western blot, we show that the response of FOXL2 to stress correlates with a dramatic remodeling of its post-translational modification profile. Upon oxidative stress, we observe an increased recruitment of FOXL2 to several stress-response promoters, notably that of the mitochondrial manganese superoxide dismutase (MnSOD). Using several reporter systems, we show that FOXL2 transactivation is enhanced in this context. Models predict that gene upregulation in response to a signal should eventually be counterbalanced to restore the initial steady state. In line with this, we find that FOXL2 activity is repressed by the SIRT1 deacetylase. Interestingly, we demonstrate that SIRT1 transcription is, in turn, directly upregulated by FOXL2, which closes a negative-feedback loop. The regulatory relationship between FOXL2 and SIRT1 prompted us the test action of nicotinamide, an inhibitor of sirtuins, on FoxL2 expression/activity. According to our expectations, nicotinamide treatment increases FoxL2 transcription. Finally, we show that 11 disease-causing mutations in the ORF of FOXL2 induce aberrant regulation of FOXL2 and/or regulation of the FOXL2 stress-response target gene MnSOD. Taken together, our results establish that FOXL2 is an actor of the stress response and provide new insights into the pathogenic consequences of FOXL2 mutations.

The transcription factor Forkhead box L subfamily member 2 (FOXL2) is involved in craniofacial development and ovarian function. Using 2-DE and immunoblotting, we show that it is highly modified post-translationally. The most outstanding feature of its migration profile is the presence of two distinct modification “trains” and the absence of intermediates. A theoretical analysis of the modification profile of FOXL2 suggests that it undergoes parallel processive/concerted modifications. The absence of intermediates is compatible with the recruitment of poorly modified FOXL2 into a post-translational “modification factory.”

Background The functions of three zona glycoproteins, ZP1, ZP2 and ZP3 during the sperm-zona pellucida (ZP) interaction are now well established in mice. The expression of an additional zona glycoprotein, ZPB/4, in humans, led us to reconsider the classical mouse model of gamete interaction. We investigated the various functions of human ZP (hZP) during the interaction of spermatozoa with fertilised and unfertilised oocytes. Results The hZP of fertilised oocytes retained their ability to bind sperm (albeit less strongly than that from unfertilised oocytes), to induce an intraspermatic calcium influx through voltage-dependent channels similar to that observed with hZP from unfertilised oocytes and to promote the acrosome reaction at a rate similar to that induced by the ZP of unfertilised oocytes (61.6 ± 6.2% vs60.7 ± 9.1% respectively). Conversely, the rate of hZP penetrated by sperm was much lower for fertilised than for unfertilised oocytes (19% vs 57% respectively, p < 0.01). We investigated the status of ZP2 in the oocytes used in the functional tests, and demonstrated that sperm binding and acrosome reaction induction, but not ZP penetration, occurred whether or not ZP2 was cleaved. Conclusion The change in ZP function induced by fertilisation could be different in human and mouse species. Our results suggest a zona blocking to polyspermy based at the sperm penetration level in humans.

IgA and IgG antibodies eluted from the surface of spermatozoa obtained from 11 infertile men were used to analyse the antigens defined by each class of sperm-associated antibodies. An enhanced chemiluminescent Western blot technique was developed to detect the low concentrations of immunoglobulins present in the eluted samples. The same analysis was performed with the sperm membrane-specific antibodies isolated from the sera of 8 of the patients included in the study. Sperm-eluted antibodies reacted with a total of 18 protein bands migrating with molecular masses of between 110 and 18 kDa. Individual antibody-binding patterns differed. Furthermore, IgA and IgG antibodies from any one patient recognised different sets of antigens. In spite of the apparent heterogeneity of the antigens defined by sperm-associated antibodies, the majority of these antibodies reacted with three protein zones of 68/64, 37/36 and 20/18 kDa. The antigens defined by the sperm surface-specific antibodies obtained from the sera of eight infertile patients differed from one patient to another and, in the majority of the patients, differed from those defined by the corresponding sperm-associated antibodies. Nevertheless, two protein zones of 68/64 and 20/18 kDa were recognised by both local and systemic antibodies in 6 and 4 patients, respectively.