ADAMs are present in fungi: identification of two novel ADAM genes in Aspergillus fumigatus

The ADAMs are a family of integral membrane proteases involved in shedding and fusion events in animal tissues. Here, we report the identification of two ADAMs, ADM-A and ADM-B, in the pathogenic fungus Aspergillus fumigatus. The domain structure of metazoan ADAMs was seen in ADM-A and -B, although with some differences. ADAMs were identified in other filamentous fungi and phylogenetic analysis indicated that the fungal ADAMs were monophyletic and most closely related to metazoan ADAM 10 and 17. Recombinant ADM-B protease specifically cleaved casein and albumin while recombinant propeptide+protease was inactive. A sheddase function is therefore proposed for fungal ADAMs.     

Cryopreservation of canine epididymal sperm using ACP-106c and TRIS

The objective was to cryopreserve sperm recovered from the canine epididymal cauda immediately after an orchiectomy. The sperm was stored for 12 h at 4 °C using ACP-106c and TRIS as extenders. Sixty adult male dogs were used. The testis–epididymis complex (TEC) was removed, immersed in 0.9% saline and transported to the laboratory. The 60 TEC were divided into groups according to the 4 °C cooling time (0 h or 12 h) and according to the extender used for sperm recovery (ACP-106c or TRIS), forming 4 experimental groups: G0h-ACP, G12h-ACP, G0h-TRIS and G12h-TRIS. The sperm were recovered from the epididymal cauda using the retrograde flow technique. Next, 1.0 mL of ACP-106c or 1.0 mL of TRIS (preheated to 37 °C for 5 min) was added to the sperm of each epididymis. One week later, the sperm was thawed at 37 °C for 1 min, and its morphology, functionality and total and progressive sperm motilities were analyzed. Other parameters were obtained by Computer Assisted Semen Analysis (CASA). The data were submitted to multivariate analysis of variance (MANOVA) (P < 0.05). The total motility values were 52.17 ± 1.78 and 49.8 ± 1.93 for groups G0h-ACP and G12h-ACP and 50.7 ± 2.06 and 43.90 ± 2.51 for groups G0h-TRIS and G12h-TRIS, respectively. A decrease in total sperm motility was observed after 12 h of cooling for both extenders (P < 0.05). ACP-106c can be used as an extender for freezing canine epididymal sperm, and the freezing procedure must be performed immediately after sperm recovery.     

Histochemical study of epididymal secretions in the lizard,Lacerta vvipara

Summary The epididymis of lizards elaborates voluminous secretory granules made of a central core and a peripheral vacuole which in the species Lacerta vivipara contain respectively an insoluble protein (protein H) and a soluble protein (protein L). After their discharge these secretions mix with spermatozoa. In order to detect the presence of carbohydrates in these secretions, lectins isolated from Canavalia ensiformis (con A) and from eleven other plants (lentil, soja, pea, gorse and several mushrooms), conjugated to fluorescein isothiocyanate, have been utilized in light-microscopic histochemical investigations of frozen sections from Lacerta vivipara epididymis. Whereas lectins having affinity for Lfucose, lactose, D-galactose and N-acetyl-D-galactosamine bound to central cores, lectins having affinity to D-glucose, N-acetylglucosamine and chitobiose bound to the peripheral vacuole. D-mannose or D-glucose seem to be present both in central cores and in peripheral vacuoles.     

Alpha-mannosidase activity in stallion epididymal fluid and spermatozoa

The expression of α-D-mannosidase activity was fluorometrically and electrophoretically assessed in spermatozoa, epididymal fluid and homogenates of stallion epididymal tissue. Enzyme activity had regional differences; it was higher (P < 0.05) in samples from the cauda epididymal region than in samples from the proximal caput region (largely composed of efferent ducts). Based on enzyme activity, as a function of pH of the assay substrate, electrophoretic analysis in native and native/SDS-PAGE conditions, and the effect of inhibitors or activators, we inferred the presence of at least two catalytically active forms of α-D-mannosidase. The neutral form of the enzyme (α-mannosidase II) was activated by Co²⁺, whereas the acid form (optimum pH 3.5 to 4.0) was sensitive to swainsonine (an inhibitor of α-mannosidase I), stabilized or stimulated by Zn²⁺, and not activated by Co²⁺ (activator of the neutral form). The activity of the acid form of the enzyme was highest in the epididymal fluid, where it seemed to be mainly in a secretory form. This form of the enzyme may have a role in plasma membrane remodeling associated with sperm maturation. In contrast, the activity of α-mannosidase II was higher in mature spermatozoa. It has been postulated that α-mannosidase II may act as a receptor in the recognition and binding of the complementary carbohydrate moieties present on the zona pellucida. With non-denaturing electrophoresis, α-D-mannosidase had an electrophoretic mobility of 0.35 and 0.24. When resolved by 1D and 2D SDS-PAGE (under denaturing conditions) the enzyme had a major protein band of molecular weight 154 kDa in spermatozoa and epididymal samples. Based on its properties under native conditions, we inferred that this enzyme might interact with other proteins and form transitory aggregates.     

Lack of tyrosylprotein sulfotransferase-2 activity results in altered sperm-egg interactions and loss of ADAM3 and ADAM6 in epididymal sperm

Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility.     

Essential roles of Meltrin beta (ADAM19) in heart development

Morphogenesis of the heart requires development of the endocardial cushion tissue that gives rise to the membranous septa and valves. Here we show that Meltrin beta/ADAM19, a novel metalloprotease-disintegrin, participates in the development of the endocardial cushion. Mice lacking Meltrin beta exhibit ventricular septal defect (VSD) and immature valves, and most of the animals die soon after birth. During development of the endocardial cushion, epithelial-mesenchymal transformation (EMT) of endocardial epithelial cells generates most of the cushion mesenchymes that constitute the main components of the septa and valves. Meltrin beta is expressed in both the epithelia and the mesenchymes of the endocardial cushion. In the absence of Meltrin beta, the cushion is small or thin in the septum-forming region and show poor remodeling of cardiac jelly components; both of these characteristics suggest impaired growth and differentiation of the endocardial cushion. When embryonic fibroblasts are cultured sparsely, Meltrin beta-lacking cells exhibit aberrant ectodomain shedding of type I Neuregulin, one of the ErbB ligands expressed in endocardial cells. These results suggest the necessity of proteolytic regulation of ErbB ligands by Meltrin beta for proper heart development.     

Effects of cholesterol-loaded cyclodextrins on the quality of frozen-thawed equine epididymal sperm

Equine epididymal sperm are known to be severely sensitive to cryopreservation, in terms of sperm quality and pregnancy rate. The objective of this study was to examine the effects of cholesterol loaded cyclodextrins (CLCs) on the quality of stallion epididymal sperm during cryopreservation.In experiment I, sperm were treated with different concentrations of CLCs: (1) 0 mg (control), (2) 1.5 mg, (3) 3 mg, and (4) 6 mg per 120 × 10⁶ sperm. The sperm viability and amount of cholesterol were determined at 15, 30 and 45 min after CLC treatment using viability markers (Ethidium homodimer-1 and Calcein AM) and gas chromatography, respectively. In experiment II, CLC treated sperm (1.5 mg CLC per 120 × 10⁶ sperm) were fixed and stained with filipin to examine the cholesterol distribution. In experiment III, sperm were treated with CLCs at concentrations of 1.5, 3.0, 6.0 mg per 120 × 10⁶ sperm for 15 min, then equilibrated with freezing extender at 4 °C for 1 h prior to cryopreservation. Epididymal sperm without CLC loading (0 mg) were used as the control group. The sperm quality was examined at post-equilibration and 10 min, 2 h and 4 h after freezing and thawing.The cholesterol was successfully loaded into the plasma membrane of stallion epididymal sperm. The amount of cholesterol was increased in a manner of dose and time dependence, and the filipin–sterol complexes were increasingly labeled over the sperm head. CLCs at 1.5 mg/120 × 10⁶ sperm significantly improved sperm quality during sperm equilibration and cryopreservation compared to other doses of CLCs and non-CLC control. An increasing concentration and incubation time of CLCs was detrimental to sperm quality.It is concluded that cholesterol loading to the sperm plasma membrane via CLCs decreases chilling sensitivity and also improves epididymal sperm cryopreservability.     

Rat sperm AS-A: subcellular localization in testis and epididymis and surface distribution in epididymal sperm

In this study, we investigated the subcellular compartmentalization of arylsulfatase-A (AS-A) in the testis and epididymis as well as the surface distribution in rat epididymal sperm. Testicular AS-A was compartmentalized specifically to the area underneath the outer acrosomal membrane of the acrosomal granule and to the dorsal aspect of the sperm acrosome. Epididymal AS-A was synthesized in the endoplasmic reticular (ER) network of principal cells and secreted into epididymal lumen as evident by its reactivity in the apical cytoplasm and vesicles therein underneath stereocilia. In clear cells, AS-A reactivity was found throughout the cytoplasmic machineries involved in endocytosis. Surface distribution of AS-A was initially detectable at the concave ridge as early as in sperm of the initial segment (IS). AS-A was additionally localized to the post-acrosomal region in caput (CP), corpus (CO) and cauda (CD) epididymal sperm. The expression levels of surface AS-A gradually increased during sperm transit from IS to CD epididymidis. These results favored the adsorption of AS-A from epididymal fluid onto the sperm surface, rather than shunting from the acrosome as a consequence of capacitation-associated membrane priming.This work was supported by Research Initiate Grant funded by Faculty of Science, Mahidol University to W.W.     

The development of signal transduction pathways during epididymal maturation is calcium dependent

Capacitation has been correlated with the activation of a cAMP-PKA-dependent signaling pathway leading to protein tyrosine phosphorylation. The ability to exhibit this response to cAMP matures during epididymal maturation in concert with the ability of the spermatozoa to capacitate. In this study, we have addressed the mechanisms by which spermatozoa gain the potential to activate this signaling pathway during epididymal maturation. In a modified Tyrode's medium containing 1.7 mM calcium, caput spermatozoa had significantly higher [Ca2+]i than caudal cells and could not tyrosine phosphorylate in response to cAMP. However, in calcium-depleted medium both caput and caudal cells could exhibit a cAMP-dependent phosphorylation response. The inhibitory effect of calcium on tyrosine phosphorylation was also observed in caudal spermatozoa using thapsigargin, a Ca(2+)-ATPase inhibitor that increased [Ca2+]i and precipitated a corresponding decrease in phosphotyrosine expression. We also demonstrate that despite the activation of tyrosine phosphorylation in caput spermatozoa, these cells remain nonfunctional in terms of motility, sperm-egg recognition and acrosomal exocytosis. These results demonstrate that the signaling pathway leading to tyrosine phosphorylation in mouse spermatozoa is negatively regulated by [Ca2+]i, and that maturation mechanisms that control [Ca2+]i within the spermatozoon are critically important during epididymal transit.     

ADAMs, a disintegrin and metalloproteinases, mediate shedding of oxytocinase

Placental leucine aminopeptidase (P-LAP), a type-II transmembrane protease responsible for oxytocin degradation during pregnancy, is converted to a soluble form through proteolytic cleavage. The goal of this study was to determine the nature of the P-LAP secretase activity. The hydroxamic acid-based metalloprotease inhibitors GM6001 and ONO-4817 as well as the TNF-alpha protease inhibitor-2 (TAPI-2) reduced P-LAP release, while tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2, which are matrix metalloproteinase inhibitors, had no effect on P-LAP release in Chinese hamster ovary (CHO) cells stably overexpressing P-LAP, thus indicating possible involvement of ADAM (a disintegrin and metalloproteinase) members in P-LAP shedding. Furthermore, overexpression of ADAM9 and ADAM12 increased P-LAP release in P-LAP-CHO transfectants. Immunohistochemical analysis in human placenta demonstrated strong expression of ADAM12 in syncytiotrophoblasts, while little expression of ADAM9 was detected throughout the placenta. Our results suggest ADAM members, at least including ADAM12, are involved in P-LAP shedding in human placenta.     

Zinc deficient diet exacerbates the testicular and epididymal damage in type 2 diabetic rat: Studies on oxidative stress-related mechanisms

Zinc (Zn) is one of the most important trace elements in the body and is required for insulin secretion and release. Zn is also required for the growth and development of the reproductive system. Alteration in the Zn levels can cause moderate to severe damage to various organs, including the reproductive system. Most of type 2 diabetic patients have altered Zn levels/signaling. So, here we investigated the role of Zn-deficient diet (ZDD) in type 2 diabetes. Type 2 diabetes in the rat was induced by the combination of high-fat diet (HFD) and a single low dose of streptozotocin (STZ, 35 mg/kg, i.p.). Control animals were fed normal pellet diet throughout the study, while ZDD was given for four consecutive weeks to the diabetic rats, which were earlier kept on HFD for 16 weeks. The present findings showed that ZDD further decreased the serum Zn, plasma insulin and serum testosterone levels, whereas it increased cholesterol, triglycerides, BUN, %HbA1c in diabetic rats. Oxidative stress in testes was increased by ZDD as evidenced by decreased glutathione, catalase and SOD1 levels. ZDD-induced several abnormalities in sperm head morphology, altered sperm decondensation, sperm chromatin and protamine content, along with significant histopathological alterations in testes and epididymis. Further, ZDD altered protein levels of MT, MTF-1, Keap1, Nrf2, Nf-κB, GPX4 and GPX5 levels in the testes and epididymis of diabetic rat. The present results demonstrated that dietary Zn deficiency could exacerbate type 2 diabetes-induced germ cell damage.     

Spink13, an Epididymis-specific Gene of the Kazal-type Serine Protease Inhibitor (SPINK) Family,Is Essential for the Acrosomal Integrity and Male Fertility

Sperm maturation involves numerous surface modifications by a variety of secreted proteins from epididymal epithelia. The sperm surface architecture depends on correct localization of its components and highlights the importance of the sequence of the proteolytic processing of the sperm surface in the epididymal duct. The presence of several protease inhibitors from different families is consistent with the hypothesis that correctly timed epididymal protein processing is essential for proper sperm maturation. Here we show that the rat (Rattus norvegicus) epididymis-specific gene Spink13, an androgen-responsive serine protease inhibitor, could bind to the sperm acrosome region. Furthermore, knockdown of Spink13 in vivo dramatically enhanced the acrosomal exocytosis during the process of capacitation and thus led to a significant reduction in male fertility, indicating that Spink13 was essential for sperm maturation. We conclude that blockade of SPINK13 may provide a new putative target for post-testicular male contraceptives.     

Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp wastes

A protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa MN7. The strain was found to produce proteases when it was grown in media containing only shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. The use of 60 g/l SWP resulted in a high protease production. Elastase, the major protease produced by P. aeruginosa MN7, was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration, and ultrafiltration using a 10-kDa cut-off membrane, with a 5.2-fold increase in specific activity and 38.4% recovery. The molecular weight of the purified elastase was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for protease activity were 60 degrees C and 8.0, respectively. The activity of the enzyme was totally lost in the presence of ethylene glycol tetraacetic acid, suggesting that the purified enzyme is a metalloprotease. The purified enzyme was highly stable in the presence of organic solvents, retaining 100% of its initial activity after 60 days of incubation at 30 degrees C in the presence of dimethyl sulfoxide and methanol. The lasB gene, encoding the MN7 elastase, was isolated and its DNA sequence was determined.     

Biochemical characterization and epididymal localization of the maturation-dependent ram sperm surface antigen ESA152

We examine here the biochemical properties and epididymal localization of a maturation dependent ram sperm surface antigen. A monoclonal antibody, ESA152, identifies an antigen that is present on the surface of ejaculated sperm, but is absent from testicular sperm. Crosslinking of the ESA152 antigen with bivalent antibodies induces the acrosome reaction, redistributing the antigen into the anterior region of the sperm head where it associates with the fusion product of the plasma membrane and the outer acrosomal membrane. The ESA152 antigen appears as a polypeptide of 18 kDa on immunoblots of SDS-polyacrylamide gels. The ESA152 epitope includes the sialic acid termini of N-linked oligosaccharides, as shown by its sensitivity to neuraminidase and endoglycosidase F. The ESA152 antigen is a highly hydrophobic integral membrane protein that resists aqueous extraction, partitions into the detergent phase of Triton-X-114, and solubilizes in chloroform-methanol mixtures. The anchoring of ESA152 is unaffected by phosphtidylinositol specific phospholipase C. The antigen is absent from extracts of caput and corpus epididymidis but appears abruptly in the first segment of the cauda. Immunofluorescence reveals that the ESA152 epitope first appears in clusters of cells in the luminal epithelium of the proximal cauda, prior to or concurrent with its appearance on sperm. © 1993 Wiley-Liss, Inc.     

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.     

Ultrastructure of the epididymis of the tammar,Macropus eugenii,and its relationship to sperm maturation

Summary The ductus epididymidis of the tammar is lined by an epithelium composed of principal, mitochondria-rich, apical and basal cells, and intraepithelial leucocytes. The epithelium is structurally differentiated into 6 zones referred to as the initial segment, middle segment (3 subdivisions) and terminal segment (2 subdivisions). The occurrence of the initial, middle and terminal segments corresponds quite closely to the anatomical differentiation of the epididymis into a head, body and tail.The initial segment epithelium in the tammar is lower and has shorter and more slender stereocilia than in other mammals which have been described. Otherwise, the structure of the epithelium has similar characteristics in the tammar to that described in other mammals.Spermatozoa begin to develop the capacity for motility within the initial segment, but only show structural signs of maturation in the middle segment. The sperm head rotates through 90 degrees in the proximal subdivision of the middle segment. The cytoplasmic droplet is detached and spermatozoa develop the capacity for motility in the middle subdivision of the middle segment. The cytoplasmic droplets are phagocytosed by the epididymal epithelium of the middle segment. Sperm storage appears to be the main function of the terminal segment.     

Extracellular ubiquitin system implicated in fertilization of the ascidian, Halocynthia roretzi: isolation and characterization

The ubiquitin-proteasome system is essential for intracellular protein degradation, but there are few studies of this system in the extracellular milieu. Recently, we reported that a 70-kDa sperm receptor, HrVC70, on the vitelline coat is ubiquitinated and then degraded by the sperm proteasome during fertilization of the ascidian, Halocynthia roretzi. Here, we investigated the mechanism of extracellular ubiquitination. The HrVC70-ubiquitinating enzyme activity was found to be released from the activated sperm during the fertilization process. This enzyme was purified from an activated sperm exudate, by chromatography on DEAE-cellulose and ubiquitin-agarose columns, and by glycerol density gradient centrifugation. The molecular mass of the enzyme was estimated to be 700 kDa. The purified enzyme requires CaCl2 and MgATP for activity, and is active in seawater. The purified enzyme preparation, but not the crude enzyme preparation, showed narrow substrate specificity to HrVC70. Moreover, ATP and ubiquitin are released from the activated sperm to the surrounding seawater during fertilization. These results indicate that ascidian sperm release a novel extracellular ubiquitinating enzyme system together with ATP and ubiquitin during penetration of the vitelline coat of the egg, which catalyzes the ubiquitination of the HrVC70, an essential component of ascidian fertilization.     

Purification and characterization of a vitelline coat lysin from Ciona intestinalis spermatozoa.

In Ciona intestinalis a chymotrypsin-like activity is involved in sperm penetration of the egg vitelline coat. A chymotrypsin-like enzyme has been purified from spermatozoa by a protocol including ion exchange chromatography, gel filtration, and native polyacrylamide gel electrophoresis. The purified enzyme resulted homogeneous when analyzed by SDS-PAGE. The molecular weight of the chymotrypsin-like enzyme was estimated to be 35 kDa by gel filtration and 24 KDa by SDS-PAGE in nonreducing conditions. The pH optimum of the enzyme is 8.4 and its activity is enhanced by Ca2+. It shows the highest activity towards the synthetic substrate Suc-Ala-Ala-Pro-Phe-AMC. Furthermore, by electron microscopy, the purified enzyme affects the structure of egg vitelline coat, and thus it fulfills one of the criteria of a lysin.     

Structure and functions of the genital ducts of the male Port Jackson shark,Heterodontus portusjacksoni

Synopsis The genital ducts ofHeterodontus portusjacksoni consist of the sperm carrying ducts (the rete testis, ductuli efferentes, and initial and terminal segments of the ductus epididymidis) and the Leydig glands (anterior opisthonephros). The ducts are lined by a ciliated epithelium which maintains a barrier to the transport of solute between blood and the lumen of the duct. Spermatozoa, Sertoli cell bodies, Sertoli cell cytoplasts and cellular debris are released from spermatocysts into the longitudinal canal of the rete testis. However, only the Sertoli cell cytoplasts persist throughout the sperm ducts. The epithelia lining the initial segment of the ductus epididymidis and secretory tubules of the Leydig glands are specialized for protein secretion and (particularly the Leydig glands) must be the main source of luminal protein in the ductus epididymidis. The epithelium lining the terminal segment of the ductus epididymidis also secretes protein, reabsorbs fluid and sodium, and may carry out heterophagic digestion. Spermatozoa develop the capacity for motility in the extratesticular sperm ducts, but do not undergo structural changes. However, they form spherical bundles in the terminal segment of the ductus epididymidis. It is suggested that the reduction in ratio of sodium:potassium from 48:8 in the ductuli efferentes to 3:4 in the distal end of the terminal segment of the ductus epididymidis may favour sperm survival.