Epididymal Sperm Protein Research Articles

SP22: a novel fertility protein from a highly conserved gene family.

Three nucleotide sequences encoding SP22, a protein originally identified in detergent extracts of cauda epididymal sperm, were isolated from a rat testis cDNA library. While two of these cDNA sequences differed only in their 5' untranslated regions, a third cDNA was predicted to contain an additional 13 amino acids of coding sequence. Amino acid sequences obtained following Edman degradation of purified SP22 protein and cDNA sequence data both indicated that SP22 was a member of a highly conserved and widely expressed gene family found in organisms as diverse as human and Escherichia coli. Interestingly, while a 1-kb mRNA transcript was widely expressed in somatic tissues, a unique pattern of testicular expression was observed, including the appearance of a novel 1.5-kb transcript and an increase in the abundance of the 1-kb transcript during spermatogenic cell development. Anti-SP22 peptide antiserum was shown to recognize a family of 22-kDa proteins on western blots of detergent-extracted cauda epididymal sperm protein, suggesting that multiple charge variants of SP22 coexist. Moreover, affinity-purified anti-SP22 peptide immunoglobulin localized in a highly specific manner to the anterior-ventral surface of the equatorial segment of the sperm head. This is an extremely intriguing finding as SP22 was originally shown to be highly correlated with, and predictive of, the fertilizing ability of cauda epididymal sperm. Although no conclusive function has been attributed to any members of the SP22 gene family, the localization of SP22 over a discrete region of the sperm head suggests a pivotal role in sperm-egg interactions.

Shedding of a rat epididymal sperm protein associated with infertility induced by ornidazole and alpha-chlorohydrin.

The protein composition of epididymal fluid and sperm extracts of rats treated with the nitroimidazole compound ornidazole was investigated by two-dimensional gel electrophoresis. Epididymal luminal fluid from the corpus and cauda regions of male animals rendered infertile by ornidazole treatment contained a prominent protein (contraception-associated protein 1, CAP1) with a molecular mass of approximately 25 kDa and an isoelectric point (pI) of 5.8; it was not found in fluids, but was present in sperm, from fertile vehicle-fed rats. Infrared matrix-assisted laser desorption/ionization mass spectrometry indicated that the molecular mass of CAP1 was 20420+/-120 daltons. Analysis of 17 amino acids demonstrated 49% homology to a diuretic hormone from an insect (Acheta domesticus). Densitometric quantitation of CAP1 on silver-stained gels indicated its presence in greater amounts in cauda than in corpus fluid from treated animals, whereas fluid from the rete testis lacked CAP1. In vitro incubations of tissue from the caput, corpus, and cauda epididymidal regions with [35S]methionine gave no hint that CAP1 was a secretion product of the epididymal epithelium. The absence of CAP1 from luminal fluid obtained from the sperm-depleted corpus epididymidis of efferent duct-ligated ornidazole-fed rats suggested a spermatozoal origin. CAP1 was present in spermatozoa from the caput epididymidis but not from the rete testis in control animals. Less CAP1 was present in detergent extracts of cauda sperm from ornidazole-treated rats than in sperm from control animals, suggesting a contraceptive-related displacement of protein from sperm to fluid. The association of ornidazole- and alpha-chlorohydrin-induced infertility with the presence of CAP1 in epididymal fluid, probably originating from spermatozoa, suggests a critical role for this protein in fertilization.

Assembly of AKAP82, a Protein Kinase A Anchor Protein, into the Fibrous Sheath of Mouse Sperm

The assembly of the mammalian sperm flagellum is a complex developmental event requiring the sequential activation of genes encoding the component parts and the coordinated assembly of these proteins during the differentiation of the haploid spermatid. In this study, the mechanism underlying the assembly of the fibrous sheath surrounding the axoneme was examined. The subject of the study was the major fibrous sheath protein of the mouse sperm flagellum, AKAP82, a member of theA KinaseAnchorProtein (AKAP) family of polypeptides that bind the regulatory (RII) subunit of protein kinase A (PK-A). Immunoelectron microscopy demonstrated that AKAP82 is present throughout the transverse ribs and longitudinal columns of the fibrous sheath. Since AKAP82 is initially synthesized as a precursor (pro-AKAP82) during spermiogenesis, an antiserum was raised against a peptide from the processed region of pro-AKAP82 (Mr97,000). In immunoblotting experiments, the antibody detected pro-AKAP82 in condensing spermatids but not in epididymal sperm. In addition, two other immunoreactive proteins ofMr109,000 (p109) andMr26,000 (p26, representing the “pro” domain of the precursor) were present in epididymal sperm. Alkaline phosphatase treatment of epididymal sperm proteins demonstrated that p109 was a phosphorylated form of pro-AKAP82 that remained in sperm. By immunofluorescence, pro-AKAP82 was localized to the entire length of the principal piece in testicular sperm, while in epididymal sperm p109 and p26 were present only in the proximal portion of the principal piece. Pro-AKAP82 was solubilized when germ cells were extracted with Triton X-100. However, in sperm, both AKAP82 and p109 were almost totally resistant to these extraction conditions and remained in the particulate fraction even after extraction with Triton and dithiothreitol. Similar to pro-AKAP82, the RII subunit of PK-A was present in the Triton X-100-soluble fraction of developing germ cells. In sperm, much of the RII also became particulate, consistent with the hypothesis that AKAP82 anchors RII in the flagellum. These data indicate that pro-AKAP82 is synthesized in the cell body, transported down the axoneme to its site of assembly in the fibrous sheath, and then proteolytically clipped to form mature AKAP82.

Cases of Human Infertility are Associated with the Absence of P34H, an Epididymal Sperm Antigen1

The interaction of spermatozoa with the zona pellucida is a critical step of fertilization. Specific sperm surface proteins involved in this process can be added or modified during epididymal transit. We have previously described a 34-kDa human epididymal sperm protein (P34H) that we proposed to be involved in sperm-zona pellucida interaction. In this study, Western blot analysis were performed to determine the level of P34H protein present on the spermatozoa of 16 men with idiopathic infertility. These levels were compared with the amount of P34H protein found in men of proven fertility. In addition, a sperm-zona pellucida binding assay was performed with spermatozoa from fertile and infertile men. Spermatozoa obtained from different semen samples from a given individual had similar P34H levels. However, the amount of P34H varied from one man to another. Nine of 16 infertile men had a P34H level that was less than 30% of the normal value based on a population of fertile men, while the remaining 7 males were in the normal range. Sperm from infertile subjects with a normal P43H determination bound to zonae pellucidae as efficiently as those from controls. However, spermatozoa from subjects with a low amount of P34H exhibited a dramatic diminution in their ability to interact with zonae pellucidae. Our results show that the quantity of the epididymal protein P34H varied from one male to another and that low levels of this epididymal sperm protein are associated with certain cases of idiopathic infertility. Results are discussed with regard to the function of human epididymis in sperm maturation.

Surface localization of P34H an epididymal protein, during maturation, capacitation, and acrosome reaction of human spermatozoa.

During epididymal transit, spermatozoa acquire new surface antigens that are involved in the acquisition of their fertilizing ability. We have previously described a 34-kDa (P34H) human epididymal sperm protein that shows antigenic and functional homologies with the hamster P26h. P34H is localized on the acrosomal cap of human spermatozoa and has been proposed to be involved in the interaction with the zona pellucida. The aim of this study was to document the expression of P34H on the sperm surface during transit along the male and female genital tracts. Immunohistochemical techniques were performed on human testes and epididymides by means of an antiserum specific for P34H. No labelling was detected on those spermatozoa found within the seminiferous tubules or in the vasa efferentia. P34H first appeared in the caput epididymidis and was restricted to the acrosomal cap. Signal intensity then increased considerably from the proximal corpus to the cauda region of the epididymis. After ejaculation, the same pattern of P34H distribution was observed, but the intensity was much lower than that characterizing the cauda epididymal spermatozoa. Strong labeling was restored after incubation in B2 medium and was maximal after 5 h of capacitation. After acrosomal exocytosis induced by a Ca2+ ionophore, the percentage of P34H-labeled spermatozoa decreased proportionally to the number of acrosome-reacted spermatozoa as determined by Pisum sativum-fluorescein isothiocyanate (FITC) labeling. P34H appeared to be strongly anchored to the sperm plasma membrane during epididymal transit as indicated by the requirement for detergent to extract this surface antigen from ejaculated spermatozoa. This confirms the importance of P34H binding to the sperm plasma membrane during epididymal maturation. We have previously proposed that P34H is involved in sperm-zone pellucida interaction. The appearance and accumulation of P34H on the sperm plasma membrane during epididymal maturation, followed by its inaccessibility associated with ejaculation, its unmasking during capacitation, and finally its elimination after the acrosome reaction, are in agreement with te proposed function of this sperm antigen.

Human sperm-zona pellucida interaction is inhibited by an antiserum against a hamster sperm protein.

During epididymal transit, mammalian spermatozoa acquire new surface antigens that may participate in gamete interaction. We have previously described a 26-kDa (P26h) epididymal hamster sperm protein that we propose to be involved in fertilization. In this study, we have searched for an antigenically related protein in the human, and have found that an anti-P26h antiserum recognizes a 34-kDa (P34H) protein on Western blot of human sperm proteins. Immunostaining showed that this protein is localized on the acrosomal cap of human epididymal spermatozoa but not on testicular gametes. The effect of the anti-P26h antiserum on the fertilizing ability of human spermatozoa was evaluated by use of a human zona pellucida binding assay. Compared to the preimmune serum, the antiserum caused a highly significant decrease in the number of sperm bound per zona pellucida. This inhibition was not due to the induction of a premature acrosomal reaction nor to an effect on the motility of the spermatozoa. The antiserum recognizing the P34H human sperm protein had no effect on gamete fusion as determined by the zona-free hamster test. Our results suggest that the human spermatozoon acquires an epididymal protein that shares a common epitope(s) with the P26h hamster sperm protein. The possible involvement of this human sperm antigen in the binding to the zona pellucida is discussed.

Cyclic AMP-dependent phosphorylation of epididymal mouse sperm proteins during capacitation in vitro: identification of an M(r) 95,000 phosphotyrosine-containing protein.

Cyclic AMP-dependent changes in phosphorylation of epididymal mouse sperm suspensions were examined in media designed to manipulate capacitation and the expression of parameters associated with full fertilizing ability, i.e. hyperactivated motility and the acrosome reaction. After initial assessment of cAMP-dependent protein kinase activity in frozen-thawed and lyophilized sperm suspensions using exogenous substrate, phosphorylation of endogenous sperm phosphoproteins was examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography or immunoblotting. Numerous phosphoproteins were detected in both incapacitated and capacitated suspensions, the majority of which were probably concerned with motility; full expression of fertilizing ability appeared to involve an increase in the amount of endogenous phosphorylation as deduced from the decreased amount of 32P incorporation in these suspensions. The addition of the cAMP-dependent protein kinase inhibitors, H8 and PKI (6-22) amide, demonstrated that most of the phosphoproteins detected were phosphorylated in a cAMP-dependent manner. Of particular interest was a phosphoprotein with an M(r) of about 95,000 which was consistently observed in capacitated suspensions. Evidence suggests that this may be phosphorylated on tyrosine residues, since the inclusion of orthovanadate, a phosphoryltyrosine phosphatase inhibitor, altered phosphorylation of this protein. Furthermore, immunodetection using the antiphosphotyrosine antibody, PY-20, identified five proteins with approximate M(r) 116,000, 105,000, 95,000, 86,000, and 76,000, and possibly a sixth at 54,000. The 95,000 protein was consistently diminished in ionophore-treated spermatozoa, indicating that the protein was located in the acrosomal cap region. These results suggest that the protein may be the same phosphotyrosine-containing protein as that described by Leyton and Saling (1989) which has been proposed to play a role in acrosomal exocytosis.

A study of rat epididymal sperm adenosine 3',5'-monophosphate-dependent protein kinases: maturation differences and cellular location.

The photoaffinity analog [32P]8-N3 cAMP (8-azido adenosine 3',5'-monophosphate was used to analyze the membrane sidedness of rat sperm cAMP binding proteins during epididymal maturation. Evidence is presented here which supports the hypothesis that 35-45% of the regulatory subunits of the Type I and Type II cAMP-dependent protein kinases are readily available to externally added cyclic nucleotide. It was observed by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and autoradiography that only two rat sperm proteins (Mr = 49K and 55K) were photolabeled which comigrated on gels with partially purified Type I and Type II regulatory subunits, respectively. Both of these photolabeled epididymal sperm proteins were saturated at physiological titers of [32P]8-N3cAMP and photoincorporation of [32P]8-N3 cAMP was specific since other SDS-resolvable sperm proteins did not photoincorporate the analog. Caput and cauda sperm protein photoincorporation could be effectively blocked by low levels of cAMP, but not by cGMP, ATP or GTP. Sperm epididymal maturation coincided with changes in the cAMP-dependent protein kinase subunits since cauda sperm contained more available Type II than did caput sperm. A subcellular analysis of cAMP-dependent protein kinase regulatory subunit in head and tail fractions was done for caput and cauda sperm and demonstrated that the tail fractions showed more photo-labeling of both Type I and II regulatory subunits than did the head fractions.