Equine sperm-oocyte interaction: the role of sperm surface hyaluronidase.

The plasma membrane over the sperm head of several mammalian species has been shown to express a glycerolphosphatidylinositol-linked hyaluronidase known as PH-20. This protein has been associated with the sperm's interaction with the oocyte cumulus matrix and zona pellucida. The characteristics of PH-20 in equine sperm have not been clearly defined. In this study, ejaculated gel-free semen from five stallions and epididymal sperm from isolated epididymis from 10 stallions was used to characterize the PH-20 activity in equine sperm. Affinity purified anti-equine PH-20 polyclonal antibody was used to immunodetect sperm surface-associated PH-20 and immunolabel whole sperm. The intracellular calcium indicator, Fluo-3, was used to assess sperm intracellular calcium. Stallion sperm express a surface-associated hyaluronidase localized to the posterior sperm head region in ejaculated sperm. Following in vitro capacitation and acrosomal exocytosis, the inner acrosomal membrane (IAM) displays intense hyaluronidase fluorescence suggesting that the IAM and hyaluronidase plays a significant role in zona penetration by sperm. Sperm incubated in hyaluronan (HA)-containing capacitation medium display an elevated intracellular calcium concentration (P<0.01) that is associated with translocation of PH-20 antigenic sites on the sperm surface in addition to increases in protein tyrosine phosphorylation. Caput- and cauda-derived sperm display developmentally unique PH-20 immunofluorescence expression patterns. These data suggest that the differential expression of PH-20 in ejaculated and epididymal sperm could be involved in cumulus penetration, sperm-egg recognition, and oolemmal fusion in this species.     

Identification of the bull sperm p80 protein as a PH-20 ortholog and its modification during the epididymal transit

We have identified an 80 kDa protein in ejaculated bull spermatozoa (p80) which is found in acrosomal and post-acrosomal areas of the head. It has a hyaluronidase activity and shares homologies with PH-20, a sperm surface glycoprotein involved in sperm-egg interaction. The aim of the present study was to characterize bull sperm p80 protein at the nucleic and amino acid levels to determine whether it is the bovine PH-20 ortholog. The complete nucleotide sequence determined by RT-PCR, 3' and 5' RACE show that bull p80, displays identity with the PH-20 nucleotide and amino acid sequences. Messenger RNA and protein expressions determined by Northern blot and immunohistochemistry revealed that the protein is testicular (expressed in spermatocytes and spermatids). The localization of p80 on spermatozoa, determined by indirect immunofluorescence using a monoclonal antibody, shows the protein in acrosomal and post acrosomal areas of the head with an increase in the signal intensity as sperm progress through the epididymis. Post-translational modifications of the protein were investigated during the epididymal maturation by Western blot on protein extracts from sperm collected in the caput, corpus and cauda portions of bull epididymis. Glycolysation status of sperm p80 protein on proteins from ejaculated and epididymidal sperm was investigated. Result show that the glycosylation status is modified as spermatozoa migrate through the epididymis. Hyaluronidase activity evaluated in protein extracts from spermatozoa of the three different epididymal sections revealed that the activity is higher at pH 7 than 4 and is not affected by epididymal maturation. These data strongly suggest that p80 is the bovine PH-20.     

Evidence that proteolysis of the surface is an initial step in the mechanism of formation of sperm cell surface domains

On terminally differentiated sperm cells, surface proteins are segregated into distinct surface domains that include the anterior and posterior head domains. We have analyzed the formation of the anterior and posterior head domains of guinea pig sperm in terms of both the timing of protein localization and the mechanism(s) responsible. On testicular sperm, the surface proteins PH-20, PH-30 and AH-50 were found to be present on the whole cell (PH-20) or whole head surface (PH- 30, AH-50). On sperm that have completed differentiation (cauda epididymal sperm), PH-20 and PH-30 proteins were restricted to the posterior head domain and AH-50 was restricted to the anterior head domain. Thus these proteins become restricted in their distribution late in sperm differentiation, after sperm leave the testis. We discovered that the differentiation process that localizes these proteins can be mimicked in vitro by treating testicular sperm with trypsin. After testicular sperm were treated with 20 micrograms/ml trypsin for 5 min at room temperature, PH-20, PH-30, and AH-50 were found localized to the same domains to which they are restricted during in vivo differentiation. The in vitro trypsin-induced localization of PH-20 to the posterior head mimicked the in vivo differentiation process quantitatively as well as qualitatively. The quantitative analysis showed the process of PH-20 localization involves the migration of surface PH-20 from other regions to the posterior head domain. Immunoprecipitation experiments confirmed that there is protease action in vivo on the sperm surface during the late stages of sperm differentiation. Both the PH-20 and PH-30 proteins were shown to be proteolytically cleaved late in sperm differentiation. These findings strongly implicate proteolysis of surface molecules as an initial step in the mechanism of formation of sperm head surface domains.     

Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm

PH-20 protein on the plasma membrane (PH-20PM) is restricted to the posterior head of acrosome-intact guinea pig sperm. During the exocytotic acrosome reaction the inner acrosomal membrane (IAM) becomes continuous with the posterior head plasma membrane, and PH-20PM migrates to the IAM. There it joins a second population of PH-20 protein localized to this region of the acrosomal membrane (PH-20AM) (Cowan, A.E., P. Primakoff, and D.G. Myles, 1986, J. Cell Biol. 103:1289-1297). To investigate how the localized distributions of PH-20 protein are maintained, the lateral mobility of PH-20 protein on these different membrane domains was determined using fluorescence redistribution after photobleaching. PH-20PM on the posterior head of acrosome-intact sperm was found to be mobile, with a diffusion coefficient and percent recovery typical of integral membrane proteins (D = 1.8 X 10(-10) cm2/s; %R = 73). This value of D was some 50-fold lower than that found for the lipid probe 1,1-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (C14diI) in the same region (D = 8.9 X 10(-9) cm2/s). After migration to the IAM of acrosome-reacted sperm, this same population of molecules (PH-20PM) exhibited a 30-fold increase in diffusion rate (D = 4.9 X 10(-9) cm2/s; %R = 78). This rate was similar to diffusion of the lipid probe C14diI in the IAM (D = 5.4 X 10(-9) cm2/s). The finding of free diffusion of PH-20PM in the IAM of acrosome-reacted sperm supports the proposal that PH-20 is maintained within the IAM by a barrier to diffusion at the domain boundary. The slower diffusion of PH-20PM on the posterior head of acrosome-intact sperm is also consistent with localization by barriers to diffusion, but does not rule out alternative mechanisms.     

Sperm exocytosis increases the amount of PH-20 antigen on the surface of guinea pig sperm

Evidence has been presented that the PH-20 protein functions in sperm adhesion to the egg zona pellucida (Primakoff, P., H. Hyatt, and D. G. Myles, 1985, J. Cell Biol., 101:2239-2244). The PH-20 protein migrates from its original surface domain to a new surface domain after the acrosome reaction (Myles, D. G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). The acrosome reaction is an exocytotic event that results in insertion of a region of the secretory granule membrane, the inner acrosomal membrane (IAM), into the plasma membrane. After the acrosome reaction, PH-20 protein migrates to the IAM from its initial domain on the posterior head surface. We have now found a new dynamic feature of the regulation of PH-20 protein on the sperm surface; exocytosis increases the surface expression of PH-20 protein. After the acrosome reaction there is an approximately threefold increase in the number of PH-20 antigenic sites on the sperm surface. These new antigenic sites are revealed on the surface by insertion of the IAM into the plasma membrane. Our evidence indicates that before the acrosome reaction an intracellular population of PH-20 antigen is localized to the IAM. When migration of the surface population of the PH-20 protein is prevented, PH-20 protein can still be detected on the IAM of acrosome-reacted sperm. Also, PH-20 protein can be detected on the IAM of permeabilized acrosome-intact sperm by indirect immunofluorescence. Thus, the sperm cell regulates the amount of PH-20 protein on its surface by sequestering about two-thirds of the protein on an intracellular membrane and subsequently exposing this population on the cell surface by an exocytotic event. This may be a general mechanism for regulating cell surface composition where a rapid increase in the amount of a cell surface protein is required.     

Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism

The redistribution of membrane proteins on the surface of cells is a prevalent feature of differentiation in a variety of cells. In most cases the mechanism responsible for such redistribution is poorly understood. Two potential mechanisms for the redistribution of surface proteins are: (1) passive diffusion coupled with trapping, and (2) active translocation. We have studied the process of membrane protein redistribution for the PH-20 protein of guinea pig sperm, a surface protein required for sperm binding to the egg zona pellucida (P. Primakoff, H. Hyatt, and D. G. Myles (1985). J. Cell Biol. 101, 2239-2244). PH-20 protein is localized to the posterior head plasma menbrane of the mature sperm cell. Following the exocytotic acrosome reaction, PH-20 protein moves into the newly incorporated inner acrosomal membrane (IAM), placing it in a position favorable for a role in binding sperm to the egg zona pellucida (D. G. Myles, and P. Primakoff (1984), J. Cell Biol. 99, 1634-1641). To analyze the mechanistic basis for this protein migration, we have used fluorescence microscopy and digital image processing to characterize PH-20 protein migration in individual cells. PH-20 protein was observed to move against a concentration gradient in the posterior head plasma membrane. This result argues strongly against a model of passive diffusion followed by trapping in the IAM, and instead suggests that an active process serves to concentrate PH-20 protein toward the boundary separating the posterior head and IAM regions. A transient gradient of PH-20 concentration observed in the IAM suggests that once PH-20 protein reaches the IAM, it is freely diffusing. Additionally, we observed that migration of PH-20 protein was calcium dependent.     

Characterization of PH-20 in canine spermatozoa and testis

The purpose of this study was to characterize the sperm membrane protein PH-20 in the dog. Canine spermatozoa were extracted with Triton X- 100 and the presence of PH-20 was determined by immunoblot with an antibody against recombinant macaque PH-20. The hyaluronidase activity of canine PH-20 was determined with substrate gel electrophoresis based upon digestion of hyaluronic acid (HA) incorporated into the separating gels. Hyaluronidase activity was also quantified using a microplate assay. Sperm extracts were incubated at pH 4 or 7 in wells containing agarose and HA. For immunolabeling of PH-20 on canine sperm membranes, canine sperm were fixed and incubated with R-10 primary antibody, and an anti-rabbit IgG-FITC secondary antibody. Samples were visualized by fluorescence microscopy. Non-reducing SDS-PAGE and Western blot of detergent-extracted canine sperm revealed a major band at 50 kDa, and three other bands at 42, 124, and >209 kDa. Substrate PAGE revealed translucent bands of hyaluronidase activity of similar size to bovine testicular hyaluronidase. These bands were markedly more pronounced at pH 4 than at pH 7. The microplate assay also demonstrated that hyaluronidase activity was over four times greater at the acidic pH. Immunolabeling of canine spermatozoa demonstrated that PH-20 is localized to the anterior head region and appeared in the Golgi area of round spermatids as detected by the immunohistochemical staining of the testis. This study provides evidence that PH-20 is present on the membrane of canine spermatozoa and in round spermatids. Canine PH-20 exhibits hyaluronidase activity that is markedly more pronounced at acidic pH.     

Is prnt a Pseudogene? Identification of Ram Prt in Testis and Ejaculated Spermatozoa

A hallmark of prion diseases or transmissible spongiform encephalopaties is the conversion of the cellular prion protein (PrP(C)), expressed by the prion gene (prnp), into an abnormally folded isoform (PrP(Sc)) with amyloid-like features that causes scrapie in sheep among other diseases. prnp together with prnd (which encodes a prion-like protein designated as Doppel), and prnt (that encodes the prion protein testis specific - Prt) with sprn (shadow of prion protein gene, that encodes Shadoo or Sho) genes, constitute the "prion gene complex". Whereas a role for prnd in the proper functioning of male reproductive system has been confirmed, the function of prnt, a recently discovered prion family gene, comprises a conundrum leading to the assumption that ruminant prnt is a pseudogene with no protein expression. The main objective of the present study was to identify Prt localization in the ram reproductive system and simultaneously to elucidate if ovine prnt gene is transcribed into protein-coding RNA. Moreover, as Prt is a prnp-related protein, the amyloid propensity was also tested for ovine and caprine Prt. Recombinant Prt was used to immunize BALB/c mice, and the anti-Prt polyclonal antibody (APPA) immune response was evaluated by ELISA and Western Blot. When tested by indirect immunofluorescence, APPA showed high avidity to the ram sperm head apical ridge subdomain, before and after induced capacitation, but did not show the same behavior against goat spermatozoa, suggesting high antibody specificity against ovine-Prt. Prt was also found in the testis when assayed by immunohistochemistry during ram spermatogenesis, where spermatogonia, spermatocytes, spermatids and spermatozoa, stained positive. These observations strongly suggest ovine prnt to be a translated protein-coding gene, pointing to a role for Prt protein in the ram reproductive physiology. Besides, caprine Prt appears to exhibit a higher amyloid propensity than ovine Prt, mostly associated with its phenylalanine residue.     

Characterization of an 80-kilodalton bull sperm protein identified as PH-20

This paper presents the partial characterization and the identification of an 80-kDa protein detected in bull spermatozoa using a monoclonal antibody directed against a 16-amino acid long peptide from the N-terminal domain of the protooncogene p60(src) from the Rous Sarcoma Virus When subjected to two-dimensional electrophoresis, this 80-kDa protein migrated as several isoforms, with an isoelectric point ranging from 7.4 to 8.2. Amino acid sequence analysis of a peptide obtained following trypsin digestion of the bull sperm protein showed homology to the PH-20/hyaluronidase precursor sperm protein. As for PH-20, this bull sperm 80-kDa protein is located at the plasma membrane surface in the postacrosomal region of the head. An increased immunolabeling in the anterior head region of fixed/permeabilized spermatozoa was observed when these cells were incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react lost their labeling almost completely. As for the PH-20 protein, the 80-kDa bull sperm protein possesses a hyaluronidase activity that is higher at pH 7.0 than at pH 4.0 in an in-gel assay. Unlike what has been observed in the guinea pig, mouse, and human PH-20, this 80-kDa protein was not released from the surface of bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C or with trypsin. However, this protein was not sedimented by a 100,000 x g centrifugation after nitrogen cavitation, which suggests that the 80-kDa protein is loosely attached to the sperm membrane by a yet-unknown mechanism. These results suggest that the 80-kDa bull sperm protein shares many homologies with the sperm PH-20 protein reported in the literature and, most likely, is the bull sperm homologue of the PH-20.     

Posttranslational processing of PH-20 during epididymal sperm maturation in the horse.

It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n = 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal anti-equine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.     

Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion

Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The mAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 micrograms/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75%. The average number of sperm fused per egg was also reduced by 75%. In contrast a control mAb, PH-1, preincubated with sperm at 400 micrograms/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two 125I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes. Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears. After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to approximately 49 kD and approximately 33 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44- and 60-kD bands. In the absence of detergent, the purified protein precipitates, suggesting that either 60 or 44 kD could be an integral membrane polypeptide.     

The guinea pig sperm plasma membrane protein, PH-20, reaches the surface via two transport pathways and becomes localized to a domain after an initial uniform distribution

The PH-20 protein is first detected in the Golgi complex at the start of differentiation of round spermatids into a polarized cell (spermiogenesis), and next appears in the membrane of the developing secretory granule (the acrosome). Thereafter, a second population of PH-20 is inserted directly into the plasma membrane. Initially, both the acrosomal membrane (PH-20AM) and the plasma membrane (PH-20PM) populations are uniformly distributed in each membrane. Subsequently, PH-20AM is restricted to the inner acrosomal membrane, and during epididymal passage PH-20PM becomes localized to the posterior head surface domain. Therefore, the PH-20 protein does not become localized to either domain by intracellular sorting and insertion into a localized domain, but by restriction following uniform insertion. When the sperm undergoes Ca2+-regulated exocytosis (the acrosome reaction), the inner acrosomal membrane becomes confluent with the plasma membrane. Consequently, the population of PH-20AM is now inserted into the plasma membrane. The PH-20 protein isolated from developing testicular cells contains a major form, approximately 66 kDa, and a minor form, approximately equal to 56 kDa, but it remains to be determined if each form enters only one or both pathways. The developmental control of surface expression of PH-20 during spermiogenesis in the guinea pig may reflect the regulation of a protein involved in sperm-egg adhesion. (Primakoff, P., Hyatt, H., and Myles, D. g. (1985), J. Cell. Biol. 101, 2239-2244).     

Rearrangement of the PH-20 protein on the surface of macaque spermatozoa following exposure to anti-PH-20 antibodies or binding to zona pellucida

Capacitated cynomolgus macaque sperm have a surface hyaluronidase (PH-20) that is evenly distributed over the entire head and can be visualized at the ultrastructural level using a secondary antibody labeled with colloidal gold . Exposure of sperm to mono-specific, bivalent polyclonal antibodies to PH-20 causes a rapid clustering of PH-20 . The predominant morphological consequence of PH-20 redistribution is its aggregation along the lateral edge of the sperm head. Monovalent Fab fragments of the anti-PH-20 antibody bound to the sperm head but did not induce a change in PH-20 distribution. PH-20 aggregation was observed in almost all sperm following treatment with the polyclonal antibody, but only about 20% of the sperm had morphological acrosome reactions, regardless of the time of exposure or the concentration of antibody. There was morphological evidence of swelling of the acrosomal matrix in over 50% of the sperm following exposure to anti-PH-20 antibodies. Anti-PH-20 Fab fragments did not induce the acrosome reaction or acrosomal matrix swelling. Sperm bound to macaque zona pellucida also showed aggregation of the PH-20 protein as soon as 30 sec after sperm-zona interaction. This aggregation was not observed when macaque sperm were bound to hamster zona pellucida. When macaque sperm were surface-labeled with biotin and then incubated with anti-PH-20 antibodies or macaque zona pellucida, there was no evidence of a global surface protein rearrangement, although PH-20 protein was aggregated on the surface of the same sperm cells. An increase in levels of internal sperm Ca⁺⁺ was measured in association with the antibody-induced PH-20 aggregation. Fab fragments did not increase Ca⁺⁺ levels, but when they were crosslinked with anti-Fab antibody there was a significant Ca⁺⁺ increase and induction of acrosome reactions. Anti-PH-20 Fab fragments did not block macaque sperm binding to macaque zona pellucida or the zona-induced acrosome reaction. We conclude that PH-20 on the sperm surface is involved in sperm-zona pellucida interaction and the zona-induced acrosome reaction. Mol. Reprod. Dev. 50:207–220, 1998. © 1998 Wiley-Liss, Inc.     

The dual functions of GPI-anchored PH-20: hyaluronidase and intracellular signaling

The ovulated mammalian oocyte is surrounded by the "cumulus ECM", composed of cells embedded in an extracellular matrix that is rich in hyaluronic acid (HA). The cumulus ECM is a viscoelastic gel that sperm must traverse prior to fertilization. Mammalian sperm have a GPI-anchored hyaluronidase which is known as PH-20 and also as SPAM 1. PH-20 is located on the sperm surface, and in the lysosome-derived acrosome, where it is bound to the inner acrosomal membrane. PH-20 appears to be a multifunctional protein; it is a hyaluronidase, a receptor for HA-induced cell signaling, and a receptor for the zona pellucida surrounding the oocyte. The zona pellucida recognition function of PH-20 was discovered first. This function is ascribed to the inner acrosomal membrane PH-20, which appears to differ biochemically from the PH-20 on the sperm surface. Later, when bee venom hyaluronidase was cloned, a marked cDNA sequence homology with PH-20 was recognized, and it is now apparent that PH-20 is the hyaluronidase of mammalian sperm. PH-20 is unique among the hyaluronidases in that it has enzyme activity at both acid and neutral pH, and these activities appear to involve two different domains in the protein. The neutral enzyme activity of plasma membrane PH-20 is responsible for local degradation of the cumulus ECM during sperm penetration. Plasma membrane PH-20 mediates HA-induced sperm signaling via a HA binding domain that is separate from the hyaluronidase domains. This signaling is associated with an increase in intracellular calcium and as a consequence, the responsiveness of sperm to induction of the acrosome reaction by the zona pellucida is increased. There is extensive evidence that GPI-anchored proteins are involved in signal transduction initiated by a diverse group of cell surface receptors. GPI-anchored proteins involved in signaling are often associated with signaling proteins bound to the cytoplasmic leaflet of the plasma membrane, typically Src family, non-receptor protein tyrosine kinases. PH-20 appears to initiate intracellular signaling by aggregating in the plasma membrane, and a 92-kDa protein may be the cell signaling molecule linked to PH-20.     

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.     

A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida

After the acrosome reaction, the PH-20 surface antigen of guinea pig sperm migrates from its original location on the posterior head surface to a new location on the inner acrosomal membrane (Myles, D.G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). We have isolated three monoclonal antibodies (MAbs) of the IgG1 subclass, PH-20, PH-21, and PH-22, that bind to the PH-20 antigen. The PH-20 MAb strongly inhibited (approximately 90%) sperm binding to the guinea pig egg zona pellucida at saturating antibody concentrations (greater than 20 micrograms/ml). Half-maximal inhibition of sperm binding to the zona was obtained with approximately 2 micrograms/ml PH-20 MAb. The PH-21 MAb at saturating concentration (50 micrograms/ml) partially inhibited (approximately 45%) sperm-zona binding, and the PH-22 MAb (50 micrograms/ml) did not inhibit (0%) sperm-zona binding. Essentially the same amounts of the three MAbs were bound to sperm under the conditions where inhibition (PH-20, PH-21) or no inhibition (PH-22) of sperm-zona binding was observed, which indicates that the different levels of inhibition did not arise from different levels of MAb binding. Competition binding assays with 125I-labeled MAbs showed that PH-21 binding to sperm was not affected by the binding of PH-20 or PH-22. However, that PH-20 and PH-22 blocked each other's binding to sperm suggests that their recognized determinants may be relatively close to one another. The results indicate that the migrating PH-20 antigen has a required function in sperm binding to the zona pellucida and that the PH-20 MAb affects is active site.     

A novel isoform of human cyclic 3',5'-adenosine monophosphate-dependent protein kinase, c alpha-s, localizes to sperm midpiece

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human C alpha catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine C alpha gene. Sequence comparison revealed similarity to an ovine C alpha variant characterized by protein purification and micropeptide sequencing, C alpha-s, identifying the cloned human cDNA as the C alpha-s isoform. The C alpha-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The C alpha-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a C alpha-s-specific antibody indicated that C alpha-s was localized in the midpiece region of the spermatozoon. The majority of C alpha-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the C alpha-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RI alpha, RII alpha, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target C alpha-s. This novel C alpha-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.     

Purification of the guinea pig sperm PH-20 antigen and detection of a site-specific endoproteolytic activity in sperm preparations that cleaves PH-20 into two disulfide-linked fragments

Previous work has indicated that the guinea pig sperm membrane protein, PH-20, functions in sperm-egg adhesion and that its surface expression is regulated by the acrosome reaction. The PH-20 protein was purified by monoclonal antibody affinity chromatography. Sixty-seven to one hundred percent of the PH-20 antigenic activity present in an octylglucoside (OG) extract of sperm was recovered in the purified protein. From 10(10) sperm, approximately 0.4 mg of PH-20 protein was obtained, which was about 0.24% of the total protein in the OG extract. The purified protein retained the ability to bind the three anti-PH-20 monoclonal antibodies we have isolated. Silver staining of purified PH-20 on overloaded sodium dodecyl sulfate (SDS) gels allowed the estimate that silver-stainable contaminants were present at a level of one part in 2000. The purified PH-20 protein exists in three forms separable on SDS-polyacrylamide gel electrophoresis: a major form with a molecular mass of 64 kDa, a minor form of 56 kDa, and an endoproteolytically cleaved form composed of two disulfide-linked fragments of 41-48 kDa and 27 kDa. Cleveland digests of the 64 kDa and 56 kDa polypeptides indicated that they were structurally related. A proportion of the 64 kDa polypeptide in each purified preparation had undergone endoproteolysis at a specific site, so that it was cleaved into the two disulfide-linked fragments, 41-48 kDa and 27 kDa. It is speculated that the site-specific endoproteolysis of PH-20 may occur during the acrosome reaction and have biological significance.     

ATP-induced reactivation of ram testicular, cauda epididymal, and ejaculated spermatozoa extracted with Triton X-100

It was possible to demembrante and reactivate not only freshly collected testicular, cauda epididymal, and ejaculated ram sperm but also sperm that had been stored for several days at 0 degrees C and for several months at -196 degrees C in rete testis fluid or egg yolk citrate media. Sperm were usually washed free of seminal plasma before demembranation, but this was not essential for reactivation. Bovine serum albumin (1.0%) in the wash medium increased the survival of sperm, but more than 0.25% in the extraction medium decreased reactivation. A macro-molecular component of cauda epididymal fluid also inhibited the reactivation of testicular sperm. Triton X-100 concentrations between 0.01% and 1.00% in the extraction medium were satisfactory for demembranating the sperm. Rapid cooling (i.e., cold shock) mimicked the effect of detergent in making the sperm responsive to added ATP and demonstrated that damage to ram sperm in cold shock does not involve the axoneme. Ejaculated and cauda sperm were reactivated immediately on addition of ATP and activity persisted for up to 10 min. Testicular sperm, on the other hand, required about 4 min to become fully reactivated. The optimal ATP concentration for activation of sperm was 0.1-1.0 mM. Magnesium ions (0.1-1.0 mM) were important for reactivation, and testicular sperm required a higher magnesium concentration than did cauda or ejaculated sperm. Manganese ions were almost as effective as magnesium for reactivating cauda epididymal and ejaculated sperm. Cobalt and cadmium ions were much less active for cauda and ejaculated sperm and none of these ions were effective for testicular sperm. Fluoride (25-50 mM) inhibited reactivation. The presence of 50 microM cAMP in the extraction medium or preincubation of testicular sperm with theophylline or caffeine increased low levels of activation, but this was not evident with ejaculated or cauda sperm. We conclude that the motor apparatus is already functionally assembled in spermatozoa on leaving the testis, but some fine adjustment must take place during maturation in the epididymis.