Differential localization of ADAM1a and ADAM1b in the endoplasmic reticulum of testicular germ cells and on the surface of epididymal sperm

Although fertilin is a heterodimeric complex between ADAM1 (A Disintegrin And Metalloprotease 1, fertilin alpha) and ADAM2 (fertilin beta) located on the sperm surface, two different ADAM1 isoforms, ADAM1a and ADAM1b, are present in the mouse testis. In this study, we have examined the localization of ADAM1a and ADAM1b in testicular germ cells and epididymal sperm. ADAM1a was restrictedly present within the endoplasmic reticulum of germ cells, whereas epididymal sperm contained only ADAM1b on the cell surface. The precursors of ADAM1a and ADAM1b formed a heterodimeric complex with that of ADAM2 in the endoplasmic reticulum of germ cells. The heterodimeric complex between the mature forms of ADAM1b and ADAM2 was also found on the sperm surface. These data imply the potential roles of ADAM1a and ADAM1b in spermatogenesis and fertilization, respectively.     

Mouse sperm lacking ADAM1b/ADAM2 fertilin can fuse with the egg plasma membrane and effect fertilization

Fertilin, a heterodimeric protein complex composed of alpha (ADAM1) and beta (ADAM2) subunits on the sperm surface, is believed to mediate adhesion and fusion between the sperm and egg plasma membranes. Here we have shown that mutant male mice lacking ADAM1b are fertile and that the loss of ADAM1b results in no significant defect in sperm functions such as migration from the uterus into oviduct, binding to egg zona pellucida, and fusion with zona pellucida-free eggs. ADAM1b-deficient epididymal sperm showed a severe reduction of ADAM2 on the cell surface, despite the normal presence of ADAM2 in testicular germ cells. The appearance of ADAM1b and ADAM2 on the sperm surface depended on formation and abundance of ADAM1b/ADAM2 fertilin in testicular germ cells. These results suggest that mouse ADAM1b/ADAM2 fertilin may play a crucial role not in the sperm/egg fusion but in the appearance of these two ADAMs on the sperm surface.     

Identification of an ADAM2-ADAM3 complex on the surface of mouse testicular germ cells and cauda epididymal sperm

Male mice lacking ADAM2 (fertilin beta) or ADAM3 (cyritestin) are infertile; cauda epididymal sperm (mature sperm) from these mutant mice cannot bind to the egg zona pellucida. ADAM3 is barely present in Adam2-null sperm, despite normal levels of this protein in Adam2-null testicular germ cells (TGCs; sperm precursor cells). Here, we have explored the molecular basis for the loss of ADAM3 in Adam2-null TGCs to clarify the biosynthetic and functional linkage of ADAM2 and ADAM3. A small portion of total ADAM3 was found present on the surface of wild-type and Adam2(-/-) TGCs at similar levels. In the Adam2-null TGCs, however, surface-localized ADAM3 exhibited an increased amount of an endoglycosidase H-resistant form that may be related to instability of ADAM3. Moreover, we found a complex between ADAM2 and ADAM3 on the surface of TGCs and sperm. The intracellular chaperone calnexin was a component of the testicular ADAM2-ADAM3 complex. Our findings suggest that the association with ADAM2 is a key element for stability of ADAM3 in epididymal sperm. The presence of the ADAM2-ADAM3 complex in sperm also suggests a potential role of ADAM2 with ADAM3 in sperm binding to the egg zona pellucida.     

Calsperin is a testis-specific chaperone required for sperm fertility

Calnexin (CANX) and calreticulin (CALR) are homologous lectin chaperones located in the endoplasmic reticulum and cooperate to mediate nascent glycoprotein folding. In the testis, calmegin (CLGN) and calsperin (CALR3) are expressed as germ cell-specific counterparts of CANX and CALR, respectively. Here, we show that Calr3(-/-) males produced apparently normal sperm but were infertile because of defective sperm migration from the uterus into the oviduct and defective binding to the zona pellucida. Whereas CLGN was required for ADAM1A/ADAM2 dimerization and subsequent maturation of ADAM3, a sperm membrane protein required for fertilization, we show that CALR3 is a lectin-deficient chaperone directly required for ADAM3 maturation. Our results establish the client specificity of CALR3 and demonstrate that the germ cell-specific CALR-like endoplasmic reticulum chaperones have contrasting functions in the development of male fertility. The identification and understanding of the maturation mechanisms of key sperm proteins will pave the way toward novel approaches for both contraception and treatment of unexplained male infertility.     

Processing and subcellular localization of ADAM2 in the Macaca fascicularis testis and sperm

Fertilin, a heterodimeric protein complex composed of ADAM1 and ADAM2 located on the sperm surface, is involved in sperm–egg interaction. In our study, we examined the physiological processing and subcellular localization of M. fascicularis ADAM2 during spermatogenesis in the testis and epididymal tract. M. fascicularis ADAM2 was initially synthesized as a 100 kDa precursor in testicular germ cells. After passing into 50 kDa intermediate form in the epididymal tracts, the precursor form was finally processed into a 47 kDa protein in sperm. We found that M. fascicularis ADAM2 is localized on the sperm surface and contributes to the formation of a candidate fertilin complex. In particular, Far-Western blot analysis revealed that M. fascicularis ADAM2 cystein-rich domain may be related to protein–protein interaction. Therefore, the cystein-rich domain of ADAM2 could provide a mechanism to form a fertilin complex.     

Aberrant distribution of ADAM3 in sperm from both angiotensin-converting enzyme (Ace)- and calmegin (Clgn)-deficient mice

Male mice deficient for the calmegin (Clgn) or the angiotensin-converting enzyme (Ace) gene show impaired sperm migration into the oviduct and loss of sperm-zona pellucida binding ability in vitro. Since CLGN is a molecular chaperone for membrane transport of target proteins and ACE is a membrane protein, we looked for ACE on the sperm membranes from Clgn-/- mice. ACE was present and showed normal activity, indicating that CLGN is not involved in transporting ACE to the sperm membranes. The ablation of the Adam2 and Adam3 genes generated animals whose sperm did not bind the zona pellucida, which led us to examine the presence of ADAM2 and ADAM3 in Clgn-/- and Ace-/- sperm. ADAM3 was absent from Clgn-/- sperm. In the Ace-/- mice, while ADAM2 was found normally in the sperm, ADAM3 disappeared from the Triton X-114 detergent-enriched phase after phase separation, which suggests that ACE is involved in distributing ADAM3 to a location where it can participate in sperm-zona pellucida binding. This diminished amount of ADAM3 in the Triton X-114 detergent-enriched phase may explain the inability of Clgn-/- and Ace-/- sperm to bind to the zona pellucida.     

Cyclic 3',5'-AMP causes ADAM1/ADAM2 to rapidly diffuse within the plasma membrane of guinea pig sperm

Because sperm cannot synthesize new proteins as they journey to the egg, they use multiple mechanisms to modify the activity of existing proteins, including changes in the diffusion coefficient of some membrane proteins. Previously, we showed that during capacitation the guinea pig heterodimeric membrane protein ADAM1/ADAM2 (fertilin) transforms from a stationary state to one of rapid diffusion within the lipid bilayer. The cause for this biophysical change, however, was unknown. In this study we examined whether an increase in cAMP, such as occurs during capacitation, could trigger this change. We incubated guinea pig cauda sperm with the membrane-permeable cAMP analog dibutyryl cAMP (db-cAMP) and the phosphodiesterase inhibitor papaverine and first tested for indications of capacitation. We observed hypermotility and acrosome-reaction competence. We then used fluorescence redistribution after photobleaching (FRAP) to measure the lateral mobility of ADAM1/ADAM2 after the db-cAMP treatment. We observed that db-cAMP caused roughly a 12-fold increase in lateral mobility of ADAM1/ADAM2, yielding diffusion similar to that observed for sperm capacitated in vitro. When we repeated the FRAP on testicular sperm incubated in db-cAMP, we found only a modest increase in lateral mobility of ADAM1/ADAM2, which underwent little redistribution. Interestingly, testicular sperm also cannot be induced to undergo capacitation. Together, the data suggest that the release of ADAM1/ADAM2 from its diffusion constraints results from a cAMP-induced signaling pathway that, like others of capacitation, is established during epididymal sperm maturation.     

Calmegin is required for fertilin alpha/beta heterodimerization and sperm fertility.

Loss of the endoplasmic reticulum resident chaperone calmegin leads to the production of sterile sperm that do not bind to the egg zona pellucida (M. Ikawa et al., 1997, Nature 387, 607-611). In the present study, we demonstrate that calmegin -/- sperm were defective in migrating into the oviducts and in binding to the egg plasma membrane. Despite the impaired adhesive function, calmegin -/- sperm could fertilize eggs when zonae pellucidae were partially dissected, and eggs fertilized in this manner could develop normally to term. Since these sperm characteristics were similar to those found in fertilin beta -/- sperm, we investigated the interaction of calmegin with fertilin beta. Using immunoprecipitation techniques, calmegin was found to bind to sperm membrane proteins, fertilin alpha and beta, during spermatogenesis. The binding was specific to calmegin: another endoplasmic reticulum chaperone calnexin, a calmegin homologue, was not able to bind to fertilin alpha and beta. In the calmegin -/- mice, a loss of heterodimerization of fertilin alpha and beta was observed and fertilin beta was not detectable in mature sperm. The data not only explain why the calmegin and fertilin beta knockout mouse lines share a common infertile phenotype, but also reveal the importance of the maturation of sperm membrane proteins in the endoplasmic reticulum.     

Localization and expression of ADAM2 in the dromedary camel testis,epididymis and sperm during rutting season

ADAM2 (fertilin β) is a sperm surface protein reported in several mammalian species. However, the presence of ADAM2 in the male reproductive system and sperm of the camel is not well known. The present study was to clarify the localization and expression of ADAM2 in the dromedary camel testis, epididymis and spermatozoa during rutting season using immunohistochemistry (IHC) and the quantitative real-time polymerase chain reaction (qPCR). Tissue samples were obtained from the testis (proximal and distal) and epididymis (caput, corpus, and cauda) from eight mature male camels. Epididymal and ejaculated sperms were collected from four other fertile camels. IHC analysis clearly showed the localization of ADAM2 protein in the spermatocytes and the round and elongated spermatids of the testis, in the epithelial cells along the epididymis tract, on the posterior head of the sperm within the cauda epididymis, and on the acrosomal cap of both the epididymal and ejaculated sperm. The expression of camel ADAM2 mRNA was significantly higher (P < 0.05) in the testis when compared with the epididymis. These findings may suggest an important role of ADAM2 in the fertility of male dromedary camels.     

Mice expressing aberrant sperm-specific protein PMIS2 produce normal-looking but fertilization-incompetent spermatozoa

Eight kinds of gene-disrupted mice (Clgn, Calr3, Pdilt, Tpst2, Ace, Adam1a, Adam2, and Adam3) show impaired sperm transition into the oviducts and defective sperm binding to the zona pellucida. All of these knockout strains are reported to lack or show aberrant expression of a disintegrin and metallopeptidase domain 3 (ADAM3) on the sperm membrane. We performed proteomic analyses of the proteins of these infertile spermatozoa to clarify whether the abnormal function is caused exclusively by a deficiency in ADAM3 expression. Two proteins, named PMIS1 and PMIS2, were missing in spermatozoa from Clgn-disrupted mice. To study their roles, we generated two gene-disrupted mouse lines. Pmis1-knockout mice were fertile, but Pmis2-knockout males were sterile because of a failure of sperm transport into the oviducts. Pmis2-deficient spermatozoa also failed to bind to the zona pellucida. However, they showed normal fertilizing ability when eggs surrounded with cumulus cells were used for in vitro fertilization. Further analysis revealed that these spermatozoa lacked the ADAM3 protein, but the amount of PMIS2 was also severely reduced in Adam3-deficient spermatozoa. These results suggest that PMIS2 might function both as the ultimate factor regulating sperm transport into the oviducts and in modulating sperm-zona binding.     

CASK is in the mammalian sperm head and is processed during epididymal maturation

Upon adhesion to the zona pellucida or egg extracellular matrix, sperm undergo regulated exocytosis of the acrosomal vesicle. CASK is an adaptor protein that has been implicated in coupling neuronal cell adhesion to regulated exocytosis. In neurons, this scaffolding molecule is associated with several types of transmembrane receptor complexes and connects cell adhesion molecules with ion channels, the actin cytoskeleton, and the cell's exocytotic machinery. We hypothesized CASK might also be an important link between zona pellucida binding and the sperm acrosome reaction. RT-PCR experiments indicated CASK is transcribed in mouse testis. The full size (120 kDa) CASK protein was present in testis from mouse and pig. Immunoblots of mature porcine and murine sperm revealed that the 120 kDa molecule was much less abundant than in testis but the antibody also recognized a group of smaller proteins migrating at 55-65 kDa. Immunofluorescence experiments indicated both the full length and smaller CASK immunoreactive products were found only in the acrosomal region of spermatids and mature sperm and not in other testicular cell types. CASK immunofluorescence was lost following the acrosome reaction. During epididymal maturation, the abundance of the full size CASK decreased and the CASK fragments increased. These results suggest that CASK may be proteolytically processed during epididymal maturation. Because sperm acquire the ability to bind the zona pellucida, acrosome react, and fertilize eggs during epididymal maturation, CASK processing may play a role in the acquisition of these functions.     

Localization and significance of molecular chaperones, heat shock protein 1, and tumor rejection antigen gp96 in the male reproductive tract and during capacitation and acrosome reaction

Although the molecular basis of sperm-oocyte interaction is unclear, recent studies have implicated two chaperone proteins, heat shock protein 1 (HSPD1; previously known as heat shock protein 60) and tumor rejection antigen gp96 (TRA1; previously known as endoplasmin), in the formation of a functional zona-receptor complex on the surface of mammalian spermatozoa. The current study was undertaken to investigate the expression of these chaperones during the ontogeny of male germ cells through spermatogenesis, epididymal sperm maturation, capacitation, and acrosomal exocytosis. In testicular sections, both HSPD1 and TRA1 were closely associated with the mitochondria of spermatogonia and primary spermatocytes. However, this labeling pattern disappeared from the male germ line during spermiogenesis to become undetectable in testicular spermatozoa. Subsequently, these chaperones could be detected in epididymal spermatozoa and in previously unreported "dense bodies" in the epididymal lumen. The latter appeared in the precise region of the epididymis (proximal corpus), where spermatozoa acquire the capacity to recognize and bind to the zona pellucida, implicating these structures in the functional remodeling of the sperm surface during epididymal maturation. Both HSPD1 and TRA1 were subsequently found to become coexpressed on the surface of live mouse spermatozoa following capacitation in vitro and were lost once these cells had undergone the acrosome reaction, as would be expected of cell surface molecules involved in sperm-egg interaction. These data reinforce the notion that these chaperones are intimately involved in the mechanisms by which mammalian spermatozoa both acquire and express their ability to recognize the zona pellucida.     

Binding of zona pellucida proteins to a boar sperm polypeptide of Mr 53,000 and identification of zona moieties involved

Experiments have been carried out to identify proteins on boar spermatozoa that bind to components of the zona pellucida. Polypeptides in sodium deoxycholate extracts of boar spermatozoa and in whole seminal plasma have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose sheet by electroblotting and probed with 125I-labelled heat-solubilized zona pellucida from pig oocytes or ovulated eggs. Zona proteins bound avidly and consistently to a polypeptide of Mr 53,000 on blots of capacitated and noncapacitated sperm and weakly to polypeptides of Mr 67,000, 38,000 and 18,000. On blots of seminal plasma the 125I-labelled probes bound to two polypeptides of Mr 65,000 and 19-24,000. Identification of the zona proteins that were binding to the aforementioned proteins on blots showed that all the major zona pellucida glycoproteins were involved, including those acquired from oviduct secretions. Binding of 125I-ovulated zona pellucida to the polypeptide of Mr 53,000 also occurred in extracts of testicular and epididymal boar spermatozoa. The results are discussed in relation to sperm-egg recognition in the pig.     

The ADAM1a and ADAM1b genes,instead of the ADAM1 (fertilin alpha) gene,are localized on mouse chromosome 5

Fertilin is reported to be a heterodimeric protein composed of A Disintegrin And Metalloprotease 1 (ADAM1, fertilin alpha) and ADAM2 (fertilin beta) located on the sperm surface. In the process of clarifying the molecular basis of mouse ADAM1, we have identified two intron-less mouse genes encoding different isoforms of ADAM1, termed ADAM1a and ADAM1b. The amino acid sequences of ADAM1a and ADAM1b deduced from the DNA sequences were homologous to each other (99% identity) in the pro- and metalloprotease domains, whereas the C-terminal half region of ADAM1a, including the disintegrin and Cys-rich domains, shared only a low degree of identity (37%) with that of ADAM1b. These two genes were both localized on mouse chromosome 5 as a single copy gene, and were expressed specifically in the testis. These data demonstrate the presence of the ADAM1a (Adam1a) and ADAM1b (Adam1b) genes in mouse, instead of the ADAM1 gene, and may imply different roles of ADAM1a and ADAM1b in spermatogenesis, sperm maturation, and/or fertilization.     

A Role for the Disintegrin Domain of Cyritestin, a Sperm Surface Protein Belonging to the ADAM Family, in Mouse Sperm–Egg Plasma Membrane Adhesion and Fusion

Sperm–egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell–cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm–egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (α and β subunits). Fertilin α and β are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin β functions in sperm–egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin α, fertilin β, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin β, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm–egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80–90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin β active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin β also strongly inhibited (80–90%) both sperm–egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin β showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin β in sperm–egg plasma membrane adhesion leading to fusion.     

Cell adhesion and fertilization: steps in oocyte transport,sperm-zona pellucida interactions,and sperm-egg fusion

Fertilization in mammals requires the successful completion of many steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion. In this minireview, we focus on three adhesion steps in this multistep process. The first is oocyte "pick-up," in which the degree of adhesion between the extracellular matrix of the cumulus cells and oviductal epithelial cells controls the successful pick-up of the oocyte-cumulus complex and its subsequent transfer into the oviduct. The second part of this review is concerned with the interaction between the sperm and the zona pellucida of the egg. Evidence is discussed that a plasma membrane form of galactosyltransferase on the surface of mouse sperm binds to ZP3 in the zona pellucida and initiates an acrosome reaction. Additional evidence raises the possibility that initial sperm binding to the zona pellucida is independent of ZP3. Last, we address the relationship between sperm adhesion to the egg plasma membrane and membrane fusion, especially the role of ADAM family proteins on the sperm surface and egg integrins.     

Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta

We produced mice lacking the sperm surface protein cyritestin (ADAM 3) and found mutant males are infertile. Similar to fertilin beta (ADAM 2) null sperm (C. Cho et al., 1998, Science 281, 1857-1859), cyritestin null sperm are drastically deficient in adhesion to the egg zona pellucida (0.3% of wild type) and to the egg plasma membrane (9% of wild type). Thus sperm from male mice with a gene deletion of either ADAM have a loss of adhesive function in at least two steps of fertilization. We found deletion of either ADAM gene resulted in the loss of multiple gene products. This loss of multiple gene products (sperm membrane proteins) appears to result from a novel, developmental mechanism during sperm differentiation. Because the altered sperm protein expression must be responsible for the fertilization defects, our data suggest new models for the molecular basis of the affected steps in fertilization.     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.