Sterol sulfates in the epididymis; synthesis and possible function in the reproductive process

Sterol sulfates are present in relatively high concentrations in the male reproductive tract. Cholesteryl sulfate is the major sterol sulfate in the human epididymis while desmosteryl sulfate is the major sterol sulfate in the hamster epididymis. While the testis is the major source of sterol sulfate in the human, the epididymis of the hamster is the source of demosteryl sulfate. This conjugate accumulates along the length of the epididymis and is taken up by the plasma membrane in the acrosomal region of the spermatozoa. Sulfotransferase activity increases along the epididymis and this is due to the actual synthesis of the enzyme. Sterol sulfates are potent and specific inhibitors of the proteolytic enzyme, acrosin. This property could provide protection against the premature release of proteolytic activity within the male reproductive tract. It is proposed that the removal of this inhibition occurs within the female tract via sulfatase activity in order to enable the acrosome reaction and ovum penetration to occur.     

Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p < or = 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.     

Human sperm plasma membranes possess alpha-D-mannosidase activity but no galactosyltransferase activity

Previous studies from this laboratory and others have identified several enzymes on the surface of mammalian spermatozoa. Some of these enzymes, namely a galactosyltransferase and a novel alpha-D-mannosidase, are believed to play a ligand-like role in recognizing and binding to the complementary moiety(ies) present on zona pellucida glycoconjugates. However, little or no information is available about the occurrence of these enzymes in human spermatozoa. In the present report, we show that a very small amount of the total galactosyltransferase activity present in human semen is associated with spermatozoa. Moreover, our failure to find a significant amount of the enzyme on sperm plasma membranes suggests that the enzyme is not associated with the sperm surface. Therefore, it is unlikely that galactosyltransferase in humans has the same ligand-like role in zona binding that is demonstrated in mouse sperm. In contrast, nearly 5% of alpha-D-mannosidase activity was repeatedly found in the salt-washed plasma membrane fraction. The recovery and enrichment of the alpha-D-mannosidase was nearly one-half that observed for adenylate cyclase and nearly one-third that for phosphodiesterase I, the two sperm plasma membrane marker enzymes. The differential enrichment and recovery of the sperm surface alpha-D-mannosidase is consistant with our previous studies in rat spermatozoa, and suggests that alpha-D-mannosidase may be localized on morphologically distinct region(s) of the sperm plasma membranes. The properties of human sperm surface alpha-D-mannosidase are quite similar to those reported by us for rat sperm plasma membrane mannosidase, but quite different from human sperm acid alpha-D-mannosidase. In addition, whereas anti-rat epididymal alpha-D-mannosidase antibody (IgG-fraction) cross-reacted with the human sperm acid alpha-D-mannosidase, no cross-reactivity was observed with the sperm surface mannosidase. A small amount of fucosyltransferase (less than 1% of the enzyme originally present on spermatozoa) was found in the salt-washed plasma membrane, but the enrichment of the enzyme was only one-tenth of that observed for adenylate cyclase. The potential ligand-like role of human sperm surface alpha-D-mannosidase and other sperm surface enzymes during fertilization is discussed.     

ATP-binding cassette transporter G2 activity in the bovine spermatozoa is modulated along the epididymal duct and at ejaculation

During their epididymal maturation, stabilizing factors such as cholesterol sulfate are associated with the sperm plasma membrane. Cholesterol is sulfated in epididymal spermatozoa by the enzyme estrogen sulfotransferase. Because of its role in the efflux of sulfate conjugates formed intracellularly by sulfotransferases, the ATP-binding cassette membrane transporter G2 (ABCG2) might have a role in the translocation of this compound across the plasma membrane. In the present study we showed that ABCG2 is present in the plasma membrane overlaying the acrosomal region of spermatozoa recovered from testis, epididymis, and after ejaculation. Although ABCG2 is also present in epididymosomes, the transporter is not transferred to spermatozoa via this mechanism. Furthermore, although epididymal sperm ABCG2 was shown to be functional, as determined by its ability to extrude Hoechst 33342 in the presence of the specific inhibitor Fumitremorgin C, ABCG2 present in ejaculated sperm was found to be nonfunctional. Additional experiments demonstrated that phosphorylation of ABCG2 tyrosyl residues, but not its localization in lipid rafts, is the mechanism responsible for its functionality. Dephosphorylation of ABCG2 in ejaculated spermatozoa is proposed to cause a partial protein relocalization to other intracellular compartments. Prostasomes are proposed to have a role in this process because incubation with this fraction of seminal plasma induces a decrease in the amount of ABCG2 in the associated sperm membrane fraction. These results demonstrate that ABCG2 plays a role in epididymal sperm maturation, but not after ejaculation. The loss of ABCG2 function after ejaculation is proposed to be regulated by prostasomes.     

Zymosterol is located in the plasma membrane of cultured human fibroblasts

Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. 1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. 2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. 3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool.     

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and sterol synthesis by cholesterol sulfate in cultured fibroblasts

Although widely distributed throughout mammalian tissues, the biological function of cholesterol sulfate remains largely unknown. In these studies we have demonstrated that cholesterol sulfate suppresses de novo sterol synthesis in cultured human fibroblasts. It was further shown in these cultured cells that cholesterol sulfate is a potent inhibitor of the enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34), the rate-limiting enzyme in cholesterol biosynthesis and the site at which exogenous cholesterol suppresses endogenous cholesterol synthesis. Because cholesterol sulfate inhibited sterologenesis in steroid-sulfatase deficient fibroblasts derived from patients with recessive X-linked ichthyosis, it was inferred that cholesterol sulfate per se and not cholesterol liberated by intracellular desulfation was the inhibitor in these studies. Cholesterol sulfate may be an endogenous regulator of mammalian cholesterol biosynthesis.     

Desmosteryl sulfate and desmosterol in hamster epididymal spermatozoa

Desmosteryl sulfate composes 95% of the sterol sulfate fraction of hamster epididymal spermatozoa at a level of 10 mg/g dry weight. Desmosterol represents as much as 40% of the free sterol fraction of these cells. Only cholesterol was detected in the free sterol fraction from plasma and liver of the same animals. The present report details the evidence for proof of structure of the two compounds isolated.     

Sterols in spermatogenesis and sperm maturation

Mammalian spermatogenesis is a complex developmental program in which a diploid progenitor germ cell transforms into highly specialized spermatozoa. One intriguing aspect of sperm production is the dynamic change in membrane lipid composition that occurs throughout spermatogenesis. Cholesterol content, as well as its intermediates, differs vastly between the male reproductive system and nongonadal tissues. Accumulation of cholesterol precursors such as testis meiosis-activating sterol and desmosterol is observed in testes and spermatozoa from several mammalian species. Moreover, cholesterogenic genes, especially meiosis-activating sterol-producing enzyme cytochrome P450 lanosterol 14α-demethylase, display stage-specific expression patterns during spermatogenesis. Discrepancies in gene expression patterns suggest a complex temporal and cell-type specific regulation of sterol compounds during spermatogenesis, which also involves dynamic interactions between germ and Sertoli cells. The functional importance of sterol compounds in sperm production is further supported by the modulation of sterol composition in spermatozoal membranes during epididymal transit and in the female reproductive tract, which is a prerequisite for successful fertilization. However, the exact role of sterols in male reproduction is unknown. This review discusses sterol dynamics in sperm maturation and describes recent methodological advances that will help to illuminate the complexity of sperm formation and function.     

Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation

The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [³H]cholesterol and of [³H]cholesteryl sulfate from labeled spermatozoa. Efflux of [³H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [³H]cholesterol and [³H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (VLDL), high-density lipoproteins (HDL), as well as the albumin fraction (d > 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [³H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60–70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [³H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [³H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [³H]sterol binding capacity that is 2—3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.     

The binding of sterol sulfates to hamster spermatozoa.

Spermatozoa obtained from the cauda epididymidis possess twice the ability to take up sterol sulfates in vitro when compared to sermatozoa obtained from the caput. This would suggest that a modification of the membrane composition of the spermatozoa occurs during passage through the epididymis. Free sterols are taken up in a similar pattern. Radioautographic studies reveal that, for sterol sulfates, this uptake occurs selectively in the regions of the head and mid-piece of the spermatozoa whereas the free sterols are distributed evenly throughout the length of the spermatozoa. The binding of sterol sulfates to spermatozoa appears to involve sites that are unsaturable. The possibility exists that sterol sulfates, previously implicated in membrane stabilization, may play a similar role in spermatozoa.     

Distribution of filipin-sterol complexes in the plasma membrane of stallion spermatozoa during the epididymal maturation process

The presence and distribution of cholesterol in mature and immature epididymal spermatozoa was analyzed using filipin as a cytochemical tool in freeze-fracture replicas and thin section preparations. The polyenic-antibiotic filipin formed complexes with 3, beta -OH sterols, producing characteristic protrusions, or pits, that were heterogeneously distributed in the plasma membrane of stallion spermatozoa, revealing a specific organization in a functionally specialized area of the gamete. The acrosomal region of the sperm head presented a significantly higher density of filipin sterol complexes than the postacrosomal region, which was usually free of these complexes. The plasma membrane of the flagellum also showed filipin sterol complexes randomly distributed in freeze-fracture replicas. The strong filipin labeling observed in the membrane of spermatozoa obtained from the caput region of the epididymis decreased significantly during epididymal passage. The significance of these changes is not completely understood, but they might contribute to establishing the molecular organization necessary for sperm transit and storage in the epididymis as well as to development of motile spermatozoa that are able to fertilize the oocyte and induce normal embryonic development.     

The side-chain cleavage of cholesterol sulfate--III. The effect of adrenodoxin, membrane phospholipids and Tween 80 on the kinetics of oxidation of the sterol sulfate by a reconstituted cholesterol desmolase system

This paper reports the Km values of a reconstituted cholesterol side-chain cleavage system for cholesterol sulfate, cholesterol, and adrenodoxin, determined under several experimental conditions. The Km values for adrenodoxin change depending on whether cholesterol or its sulfate is used as the substrate. Moreover, the Km values for both of the substrates and for adrenodoxin are greatly modulated by both membrane phospholipids, isolated from adrenal mitochondria, and Tween 80, 0.002%. In the absence of detergents or phospholipids, the enzyme system shows a high affinity for cholesterol sulfate, but is inhibited when high concentrations of the sterol sulfate are added to the incubation mixture. Raising the concentration of adrenodoxin in the assay mixture prevents the substrate inhibition. When cholesterol sulfate is incorporated into micelles containing the phospholipids, the enzyme system does not display substrate inhibition, and the kinetics of cleavage of the sterol sulfate are relatively independent of the concentration of adrenodoxin in the assay mixture. In the absence of phospholipids, the apparent kinetics of cleavage of cholesterol and its sulfate are quite different from each other, but when incorporated into micelles containing phospholipids, the kinetics of cleavage of the two substrates are similar to each other.     

Bicarbonate Induces Membrane Reorganization and CBR1 and TRPV1 Endocannabinoid Receptor Migration in Lipid Microdomains in Capacitating Boar Spermatozoa

Mammalian spermatozoa acquire full fertilizing ability only after a morphofunctional maturation called "capacitation." During this process the high level of bicarbonate present within the upper female genital tract or in culture medium induces a marked reorganization of sperm membranes characterized by a biphasic behavior: In a few minutes, it promotes membrane phospholipid scrambling preliminary to the apical translocation of sterol that, 2-4?h later, enables spermatozoa to recognize zona pellucida after albumin-mediated cholesterol extraction. In the present research it was demonstrated that spermatozoa incubated with bicarbonate in protein-free media underwent a marked reorganization of lipid microdomains present in a detergent-resistant membrane fraction (DRM) isolated by ultracentrifugation on sucrose density gradient. In fact, bicarbonate exposed sperm (ES) cells, compared with ejaculated spermatozoa (nonexposed sperm [nES] cells), displayed an increase in protein DRM content and, in particular, in Cav-1 and CD55, markers of caveolae and lipid rafts, as well in acrosin-2, a marker of the outer acrosomal membrane (OAM). Moreover, the amount of certain proteins involved in capacitation, such as the endocannabinoid system receptors cannabinoid receptor type 1 (CBR1) and transient receptor potential cation channel 1 (TRPV1), increased in DRM obtained from ES. These data allow us to hypothesize that sperm membrane reorganization takes place even in the absence of extracellular proteins; that not only the plasma membrane but also the OAM participate in this process; and that important molecules playing a key role in inside-out signaling, such as the endocannbinoid receptors TRPV1 and CBR1, are involved in this event, with potentially important consequences on sperm function.     

Isolation and characterization of a protein with homology to angiotensin converting enzyme from the periacrosomal plasma membrane of equine spermatozoa

The periacrosomal plasma membrane of spermatozoa is involved in sperm binding to oviductal epithelial cells and to the zona pellucida. A protein of 68–70 kD molecular mass was purified biochemically from the isolated periacrosomal plasma membrane of equine spermatozoa as a possible receptor for adhesion of spermatozoa to oviductal epithelial cells. A polyclonal antibody raised in rabbits against the purified equine sperm membrane protein recognized the 70 kD and an antigenically related 32 kD protein in preparations of isolated periacrosomal sperm plasma membrane and in detergent extracted ejaculated and epididymal spermatozoa. A larger protein (∼110 kD) was detected in equine testis. Two antigenically related proteins (64 and 45 kD) were recognized on the plasma membrane of cynomolgus macaque spermatozoa. In vitro sperm-binding assays were performed in the presence of antigen-binding fragments or IgG purified from the polyclonal antiserum to investigate a possible function of the isolated protein in binding of equine spermatozoa to homologous oviductal epithelial cells or zona pellucida. Incubation with antigen-binding fragments or IgG purified from the antiserum did not inhibit binding of equine spermatozoa either to oviductal epithelial cells or to the zona pellucida. On ultrastructural examination, the antibody bound exclusively to the cytoplasmic side of the periacrosomal plasma membrane of equine and macaque spermatozoa. Microsequence analysis of 13 residues of sequence showed strong homology with a number of angiotensin converting enzymes: An 84% identity was identified with testis specific and somatic forms of human and mouse angiotensin-converting enzyme. Immunocytochemistry and immunoblot analysis established that the protein is specific for the periacrosomal membrane of ejaculated, epididymal, and testicular stallion spermatozoa. Mol. Reprod. Dev. 48:251–260, 1997. © 1997 Wiley-Liss, Inc.     

The side-chain cleavage of cholesterol sulfate--II. The effect of phospholipids on the oxidation of the sterol sulfate by inner mitochondrial membranes and by a reconstituted cholesterol desmolase system

This study compares the side-chain cleavage of aqueous suspensions of cholesterol sulfate with the side-chain cleavage of cholesterol sulfate which is incorporated into phospholipid vesicles. Three different cholesterol desmolase systems are examined: the membrane-bound cholesterol side-chain cleavage system present in inner mitochondrial membranes isolated from bovine adrenal mitochondria; a soluble, lipid-depleted, reconstituted side-chain cleavage system prepared from cytochrome P-450scc, adrenodoxin and adrenodoxin reductase; a membrane associated side-chain cleavage system prepared by adding phospholipid vesicles, prepared from adrenal mitochondrial, to the reconstituted system. Soluble cholesterol sulfate, in low concentration, is a good substrate for the lipid-depleted reconstituted side chain cleavage system. However, at concentrations above 2 microM, in the absence of phospholipids, the sterol sulfate appears to bind at a non-productive site on cytochrome P-450scc which leads to substrate inhibition. Phospholipids, while inhibiting the binding of cholesterol sulfate to the cytochrome, also appear to prevent non-productive binding of the sterol sulfate to the cytochrome. Thus the addition of phospholipids to the lipid-depleted enzyme system leads to an activation of side-chain cleavage of high concentrations of the sterol sulfate. Soluble cholesterol sulfate is a good substrate for both the native and reconstituted membrane-bound systems and no substrate inhibition is observed when the membrane bound enzyme systems are employed in the assay of side-chain activity. However, the cleavage of cholesterol sulfate, which is incorporated into phospholipid vesicles, by both membrane bound enzyme systems appears to be competitively inhibited by the phospholipids of the vesicles. The results of this study suggest that the regulation of the side-chain cleavage of cholesterol sulfate may be entirely different than the regulation of the side-chain cleavage of cholesterol, if cholesterol sulfate exists intracellularly as a soluble non-complexed substrate. If, on the other hand, cholesterol sulfate is present in the cell in lipid droplets as a complex with phospholipids, its metabolism may be under the same constraints as the side-chain cleavage of cholesterol.     

Receptor-mediated regulation of guanylate cyclase activity in spermatozoa

Two peptides, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), which activate sperm respiration and motility and elevate cyclic GMP concentrations in a species-specific manner, were tested for effects on guanylate cyclase activity. The guanylate cyclase of sea urchin spermatozoa is a glycoprotein and it is localized entirely on the plasma membrane. When intact sea urchin sperm cells were incubated with the appropriate peptide for time periods as short as 5 s and subsequently homogenized in detergent, guanylate cyclase activity was found to be as low as 10% of the activity of cells not treated with peptide. The peptides showed complete species specificity and analogues of one peptide (speract) caused decreases in enzyme activity coincident with their receptor binding properties. The peptides did not inhibit enzyme activity when added after detergent solubilization of the enzyme. When detergent-solubilized spermatozoa were incubated at 22 degrees C, guanylate cyclase activity declined in previously nontreated cells to the peptide-treated level. The rate of decline was dependent on temperature and protein concentration. When spermatozoa were first incubated with 32P, the decrease in guanylate cyclase activity was accompanied by a shift in the apparent molecular weight of a major plasma membrane protein (160,000-150,000) and a loss of 32P label from the 160,000 band. Other agents (Monensin A, NH4Cl) which were capable of stimulating sperm respiration and motility also caused decreases of guanylate cyclase activity when added to intact but not detergent-solubilized spermatozoa. The maximal decrease in guanylate cyclase activity occurred 5-10 min after addition of these agents. The enzyme response to Monensin A required extracellular Na+ suggestive that the ionophore caused the effect on guanylate cyclase activity by virtue of its ability to catalyze Na+/H+ exchange. These studies demonstrate that guanylate cyclase activity of sperm cells can be altered by the specific interaction of egg-associated peptides with their plasma membrane receptors.     

Cellular sterol ester synthesis in plants is performed by an enzyme (phospholipid:sterol acyltransferase) different from the yeast and mammalian acyl-CoA:sterol acyltransferases

A gene encoding a sterol ester-synthesizing enzyme was identified in Arabidopsis. The cDNA of the Arabidopsis gene At1g04010 (AtPSAT) was overexpressed in Arabidopsis behind the cauliflower mosaic virus 35S promoter. Microsomal membranes from the leaves of overexpresser lines catalyzed the transacylation of acyl groups from phosphatidylethanolamine to sterols. This activity correlated with the expression level of the AtPSAT gene, thus demonstrating that this gene encodes a phospholipid:sterol acyltransferase (PSAT). Properties of the AtPSAT were examined in microsomal fractions from the tissues of an overexpresser. The enzyme did not utilize neutral lipids, had the highest activity with phosphatidylethanolamine, had a 5-fold preference for the sn-2 position, and utilized both saturated and unsaturated fatty acids. Various sterols and sterol intermediates, including triterpenic precursors, were acylated by the PSAT, whereas other triterpenes were not. Sterol selectivity studies showed that the enzyme is activated by end product sterols and that sterol intermediates are preferentially acylated by the activated enzyme. This indicates that PSAT both regulates the pool of free sterols as well as limits the amount of free sterol intermediates in the membranes. Two T-DNA insertion mutants in the AtPSAT gene, with strongly reduced (but still measurable) levels of sterol esters in their tissues, had no detectable PSAT activity in the microsomal fractions, suggesting that Arabidopsis possess other enzyme(s) capable of acylating sterols. The AtPSAT is the only intracellular enzyme found so far that catalyzes an acyl-CoA-independent sterol ester formation. Thus, PSAT has a similar physiological function in plant cells as the unrelated acyl-CoA:sterol acyltransferase has in animal cells.     

Acrosin activation follows its surface exposure and precedes membrane fusion in human sperm acrosome reaction

Evidence has accumulated suggesting multiple roles of acrosin in fertilization, including its participation in early steps of gamete recognition and binding. However, the implication of acrosin in many of these processes is not compatible with its presumptive sequestration within the sperm acrosome until a late phase of the acrosome reaction. In an earlier study (J. Tesarik, J. Drahorad, J. Peknicova, 1988, Fertil. Steril. 50, 133-141), we reported the binding of an anti-acrosin monoclonal antibody (MO-AKR.1) to the plasma membrane overlying the acrosome of human spermatozoa starting the acrosome reaction. In this study, we characterized further this antibody with regard to its reactivity with different forms of acrosin and found that it recognizes specifically an active form of this enzyme and does not react with its proenzyme form. MO-AKR.1 was thus used as a probe for in situ analysis of acrosin activation during the acrosome reaction. When suspensions of living spermatozoa were incubated with MO-AKR.1 and with another monoclonal antibody (T6) directed to an intra-acrosomal cytoskeletal protein, significantly more spermatozoa reacted with the former antibody than with the latter; this indicated that some of the spermatozoa showing acrosin immunoreactivity carried activated acrosin on the cell surface, while their acrosome was still impermeable to intra-acrosomal-directed probes. The size of this particular sperm subpopulation was increased by the action of follicular fluid (a natural acrosome reaction inducer), but not ionophore A23187 (an artificial acrosome reaction inducer); it corresponded to the proportion of spermatozoa showing acrosin immunoreactivity on the plasma membrane but neither intra-acrosomal staining nor perceptible membrane perturbations when examined by immunoelectron microscopy. When spermatozoa were pre-incubated with protease inhibitors before the addition of acrosome reaction-inducing agents, the percentage of cells binding MO-AKR.1 was markedly reduced. These data suggest that limited acrosin activation on the sperm plasma membrane is an early event in the physiological acrosome reaction.     

Contact with basement membrane heparan sulfate enhances the growth of transformed vascular endothelial cells, but suppresses normal cells.

Modulation of vascular endothelial cell growth by basement membrane heparan sulfate was investigated using four lines of normal and transformed cells. The growth of transformed endothelial cells, but not normal cells, on reconstituted basement membrane was severely suppressed when heparan sulfate, one of the components of the membrane, was specifically degraded by an enzyme, heparitinase. Similarly, when cells were grown on surfaces coated with heparan sulfate, as little as 60 pg/cm2 of heparan sulfate caused growth enhancement of transformed cells, but suppression of normal cells. These results together with our previous observations (IMAMURA, T and MITSUI, Y. (1987) Exp. Cell Res., 172: 92-100) argue that transformed cells have reversed a mechanism by which basement membrane heparan sulfate functions as a physiological suppressor for the growth of normal endothelial cells.     

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.