Investigation of epididymal sperm maturation in the golden hamster

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 +/- 12.2, 40.6 +/- 20.8, 144 [corrected] +/- 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 +/- 6.4 to 33.8 +/- 4.8 to 70 +/- 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 +/- 7.8% in the proximal caput epididymis to 57.2 +/- 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Major proteins of bovine seminal plasma and high-density lipoprotein induce cholesterol efflux from epididymal sperm

One of the hypotheses to explain the mechanism of capacitation involves the loss of sperm membrane cholesterol. Here, we studied whether or not the major proteins of bovine seminal plasma designated as BSP-A1, -A2, -A3, and -30-kDa (collectively called BSP proteins), which are implicated in sperm capacitation, induce cholesterol efflux. When epididymal sperm were labeled with [3H]cholesterol and incubated with bovine seminal plasma (0.05-2%) or BSP proteins (20-120 microg/ml) for 8 h, the sperm lost [3H]cholesterol (3.6-fold and 3-fold, respectively). The same results in the presence of BSP-A1/-A2 were obtained (3.5-fold) by direct determination of cholesterol on unlabeled epididymal sperm. Analysis of efflux particles by ultracentrifugation on a sucrose gradient revealed a single symmetrical peak of radioactivity at 1.14 g/ml. Immunoblotting of the fractions obtained from size-exclusion chromatography of the efflux particles showed that a portion of the BSP proteins were associated with [3H]cholesterol. Heparin (12 microg/ml) alone did not stimulate cholesterol efflux. In contrast, high-density lipoprotein (HDL, 100 microg/ml) alone stimulated cholesterol efflux up to 3.1-fold after 8 h. When labeled epididymal sperm were preincubated for 20 min with BSP-A1/-A2 (120 microg/ml), washed, and incubated with HDL (100 microg/ml) for 8 h, the total cholesterol efflux of the sperm suspension was 51.8 +/- 5.0% compared to 39.3 +/- 1.2% when HDL alone was used. These results indicate that BSP proteins and HDL play an important role in the sperm sterol efflux that occurs during capacitation. Furthermore, the heparin-induced sperm capacitation did not involve the efflux of sperm membrane cholesterol.     

Development of sperm motility patterns in the murine epididymis

The maturation of sperm motility in the epididymis of the mouse was assessed using a computer-assisted sperm analysis system. Spermatozoa were immotile in the most proximal regions of the epididymis but developed motility rapidly in the proximal caput epididymis; the percentage motility remained high thereafter. In the caput, flagellar beat was erratic with little progression, but in the proximal corpus region circular movement patterns were reflected in reduced linearity (LIN) and straightness (STR) of the sperm tracks, although velocities were little changed and wobble (WOB) increased. In the mid-corpus region, however, all velocities, LIN, STR and WOB, increased markedly. In more distal regions there was little change in these parameters. Distribution curves of the kinematic parameters of spermatozoa obtained from each region indicated that the most heterogeneous population was that from the mid-corpus epididymis; the most homogeneous was that from the mid-cauda region. Individual sperm tracks revealed slowly progressing spermatozoa in the distal caput, transforming to motion in small circles, interrupted by more linear progression. More distally, linear progression was interrupted by looping movements and a generally progressive path was observed thereafter, with less deviation from the average path as the spermatozoa matured. Spermatozoa displaying motion compatible with passing the uterotubal junction were first found in the proximal corpus epididymis, in agreement with earlier in-vivo fertilization studies on where fertilizing capacity is achieved with epididymal spermatozoa.     

Autometallographic demonstration of zinc ions in rat sperm cells

An in-vitro technique for autometallographic (AMG) demonstration of chelatable zinc in electroejaculated sperm cells and spermatozoa from the epididymis is presented and the localization of zinc ions in rat spermatozoa is described. Sperm cells from caput epididymis showed zinc staining in all parts of the tail and a sparse, dispersed staining in the acrosome. Spermatozoa from cauda epididymis showed heavy staining in the acrosome but no staining in the tail, or post-acrosomal part of the sperm head. This distinct acrosomal AMG staining was also found in ejaculated spermatozoa, but additionally a segmentation of the tail was seen based on differences in staining intensity. The membrane penetrating chelator diethyldithiocarbamate (DEDTC) was found to block the AMG staining whereas calcium-EDTA, known not to pass through cell membranes, did not influence the staining, proving that the detected zinc ions are intracellularly located. Two different approaches for demonstrating the presence of a chelatable zinc pool at electron microscope levels are presented, and the ultrastructural presence of AMG grains located in the acrosome and in the mitochondria of the midpiece is demonstrated. It is postulated that an exchange of zinc ions takes place between the epididymal epithelium and the sperm cells as they pass along the epididymal duct.     

Testosterone secretion by the early fetal pig testes in organ culture

The alkaline phosphatase activity was measured in testicular fluid and in epididymal plasma from caput and cauda epididymidis in boars with normal sperm production and in boars in which the number of spermatozoa passing from the testis to the epididymidis was reduced. The testicular fluid and the epididymal plasma from caput epididymidis contained low amounts of alkaline phosphatase in comparison with epididymal plasma from the cauda. This applies to both groups of boars e.g. boars with normal as well as with totally lacking or lowered sperm production. As no fluid resorption takes place between caput and cauda the distal part of the epididymidis must be the main production site for alkaline phosphatase. The production there is not related to the presence of spermatozoa in the duct. In the caput, on the other hand, it seems that the level of alkaline phosphatase in some way is influenced by the sperm supply to the duct.     

In vivo acylation of rat brain myelin proteolipid protein

Examination of brain myelin proteins by sodium dodecyl sulfate-gel electrophoresis followed by fluorography clearly showed that both proteolipid protein (PLP) and DM-20 were acylated 24 h after the intracerebral injection of 30-day-old rats with [3H]palmitic acid. The radioactivity associated with PLP remained after purification, re-electrophoresis, and fluorography. Most of the radioactivity associated with PLP was removed when the gels were treated with hydroxylamine and then fluorographed, indicating that fatty acids were bound to PLP by ester linkage. Cleavage of purified PLP with methanolic sodium hydroxide readily released almost all protein-bound radioactivity. Thin layer chromatography of this material on both silver nitrate and reverse-phase plates provided evidence that most of the radioactivity co-migrated with methyl palmitate (77%) and methyl stearate (19%); however, some radioactivity was associated with methyl oleate (4%). Gas-liquid chromatography of the fatty acids associated with PLP distinctly revealed the presence of methyl palmitate and a detectable peak of methyl stearate.     

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.     

Effects of antifertility drugs on epididymal protein secretion, acquisition of sperm surface proteins and fertility in male rats

The possibility that alpha-chlorohydrin, 6-chloro-6-deoxyglucose (6CDG) and cyproterone acetate (CPA) might affect epididymal protein secretion or acquisition of sperm surface proteins as the cause of their antifertility action in male rats was investigated. Daily administration of 9 mg/kg alpha-chlorohydrin for 7--14 days and 24 mg/kg 6CDG for 14--21 days induced sterility in male rats and imparied the capacity of the cauda epididymal spermatozoa to initiate motility. Treatment with CPA (30 mg/kg/day) for 21--28 days, however, was found to have no effect on fertility and initiation of sperm motility, although the epididymis of the treated animals underwent a loss in weight. The antifertility effects of alpha-chlorohydrin or 6CDG did not seem to be attributed to an interference with epididymal protein secretion. The cauda epididymal fluids of the alpha-chlorohydrin, 6CDG and CPA treated animals have similar protein patterns compared to those of the control animals. However, when the surface proteins of the spermatozoa were labelled with radioactive iodine, the sperm surface proteins alpha-chlorohydrin and 6CDG treated animals were found to differ from those of the control animals. Two peaks (MW 32 000 and 70 000) and one peak (70 000) were significantly reduced in the alpha-chlorohydrin treated and 6CDG treated animals, respectively. Additional bands appeared on the surface of the treated (infertile) animals. In contrast, CPA treatment did not affect the surface protein pattern of the epididymal spermatozoa. It was concluded that the antifertility affects of alpha-chlorohydrin and 6CDG are not due to an interference with epididymal secretion of specific proteins but to an intervention of the subsequent acquisition of these proteins by epididymal spermatozoa. This results in a decrease in the capacity of the epididymal sperm to initiate motility and hence a loss of fertilizing capacity.     

The practical utility of performing peri-ictal SPECT in the evaluation of children with partial epilepsy

The distribution of membrane filipin sterol complexes (FSC) in the plasma membrane of the acrosomal region (PMAR) of rabbit sperm from epididymis and testis, in normal and hypercholesterolaemic rabbits, was examined at ultrastructural level. Membrane FSG were quantitatively analysed on freeze fracture replicas of filipin-treated cells. Cauda epididymal sperm shows a significant increase in filipin sterol complexes concentration in PMAR of hypercholesterolaemic animals compared to normal rabbits. Hypercholesterolaemic animals had 0.53 +/- 0.08 FSC micron-2 in the marginal segment of PMAR and 0.26 +/- 0.03 FSC micron-2 for normal animals. In the principal piece we found 0.70 +/- 0.07 FSC micron-2 for hypercholesterolaemic and 0.43 +/- 0.03 FSC micron-2 for control animals. We also counted 0.58 +/- 0.04 FSC micron-2 in the equatorial segment of PMAR for hypercholesterolaemic and 0.38 +/- 0.03 FSC micron-2 for normal animals respectively. The FSC concentration of testicular sperm, like sperm from corpus and caput of epididymis in hypercholesterolaemic animals, did not differ from the controls. Cholesterol, phospholipids and cholesterol:phospholipid ratio in caudal epididymal sperm from treated males did not differ from controls. Only the sphingomyelin concentration decreases in cauda epididymal sperm from hypercholesterolaemic males compared to controls. The results presented in this paper suggest that the lipidic domains in PMAR of hypercholesterolaemic rabbits changes when the gametes go through the epididymis.     

Surveillance of multiresistant bacteria: justification, role of the laboratory, indicators, and recent French data]

This work demonstrates similarities between epididymal basal cells and macrophages in the mouse. Light microscopic studies of the postnatal development of the murine epididymis showed that basal cells were not present before days 12, 14 and 16 in the cauda, caput and corpus epididymis, respectively. An increase in cell number per unit length of tubule perimeter was demonstrated in all segments between days 20 and 27, when testicular fluid and spermatozoa start entering the epididymis. In the adult, there were more basal cells per unit perimeter in the cauda than caput or corpus epididymis. Conspicuous and consistent expression by basal cells of antigens detected by antibodies against tissue-fixed macrophages (F4/80) and mature macrophages (Mac-1) occurred only after they became established within the epithelium. Basal cells in the cauda epididymis did not display either antigen in the adult, although they persisted in the caput region. Such developmental patterns are compatible with the hypothesis that basal cells play a role in immune defence against sperm autoantigens.     

Assessment of protein synthesis and secretion by rat seminiferous and epididymal tubules in vivo

In vivo microperifusion and micropuncture were used to study tubule protein synthesis and proluminal secretion by the male reproductive tract in vivo. Seminiferous and caput and cauda epididymal tubules were perifused for 3 h. with [35S]-methionine. Perifused interstitial fluid (IF), lumen fluid (LF), and tubule extract (TE) were collected. Proteins were separated by SDS-PAGE, and autoradiograms were developed. Trichloroacetic acid precipitable proteins in each fluid were determined and a protein synthesis index (PSI) was calculated. PSI values demonstrated that the cauda epididymis synthesized less protein in vivo than did either seminiferous or caput tubules. Seminiferous tubules synthesized and secreted into the tubule lumen a relatively constant panel of proteins. Epididymal tubules synthesized and secreted proteins in a region-specific manner. In the caput epididymis the most prominent secreted bands were consistent with the heavy and light chains of epididymal clusterin. In the cauda epididymis, the most prominent synthesized and secreted protein was a 25 kDa protein consistent with the protein D. The above approach to studying protein synthesis and secretion will allow direct study of the physiological and pathophysiological effects on this important epithelial function in vivo.     

Morphological changes in the midpiece of wooly opossum spermatozoa during epididymal transit

Several ultrastructural changes were found to occur in the midpiece region of wooly opossum spermatozoa during epididymal maturation. The changes include alterations in mitochondrial morphology, development of structural specializations of the plasma membrane, and acquisition of a prominent extracellular coating. The lamellar membrane network which is wound about the periphery of the mitochondria becomes more densely packed during sperm development and the reticular network of membranes noted in the center of the mitochondria of immature sperm disappears leaving a homogeneous electron dense central zone. During epididymal transit the plasma membrane over the sperm midpiece region shows extensive structural modification. In cross sections of paired spermatozoa the plasma membrane of the midpiece regions shows a very regular, repetitive scalloping. In longitudinal sections the scalloping is observed as continuous parallel ridges which extend slightly obliquely to the flagellar long axis. Each ridge appears to be greater in density than the interridge areas. In the epididymis a prominent extracellular coating of dense material is deposited over the midpiece surface; this material is similar in appearance to dense material seen in restricted areas of the epididymal lumen. At the proximal and distal ends of the midpiece the plasma membrane comes into intimate contact with underlying structural specializations and it is suggested that these zones of fusion may serve to preserve regional differences in membrane composition.     

Physical activity in usual occupation and risk of breast cancer (United States)

OBJECTIVE: To identify whether the cause, site of ductal obstruction, and characteristics of fluid aspirates are associated with the cryosurvival and fertility after thawing of sperm obtained during reconstruction of the excurrent ducts with microsurgical epididymal sperm aspiration, vasal sperm aspiration, or both. DESIGN: Prospective study. SETTING: Andrology center at a tertiary care institution. PATIENT(S): Men undergoing reconstruction of the excurrent duct and sperm aspiration (n = 42) or microsurgical epididymal sperm aspiration (n = 11). INTERVENTION(S): Sperm were tested for an association with the cause and site of obstruction. Fertilization and pregnancy rates after sperm aspiration and intracytoplasmic sperm injection (ICSI) were evaluated for fresh and frozen aspirates. MAIN OUTCOME MEASURE(S): Motile sperm count and percentage motility after thawing. RESULT(S): The motile sperm count before freezing was significantly higher in the caput epididymis than in the corpus. The motile sperm count before freezing was related inversely to the distance from the caput where the sperm were aspirated. Sperm from clear and opaque fluid aspirates had better percent motility than those from cloudy and creamy fluid aspirates. High fertilization and pregnancy rates were achieved using both fresh and frozen epididymal sperm. CONCLUSION(S): None of the factors studied was associated with cryosurvival of aspirated epididymal or vasal spermatozoa. Because motility is low after thawing, these specimens are best used with ICSI.     

Prolonged survival of human spermatozoa when co-incubated with epididymal cell cultures

Human epididymal tissue was recovered from 11 patients undergoing orchidectomy without anti-androgen treatment. Everted epithelial fragments from the caput and corpus epididymis of six patients were successfully cultured in a modified RPMI 1640 medium supplemented with HEPES and androgens for up to 110 days (mean 56 +/- 28) in 5% CO(2) in air at 37 degrees C. Epithelial cells from human oviduct and non-reproductive tract cells (breast epithelial cells, fibroblasts) were also cultured for comparison. The proportion of epididymal epithelial cells in primary cultures assessed by immunofluorescent localization using a cytokeratin monoclonal antibody was shown to be >70% for the first 6-8 weeks of culture. Light and electron microscopy indicated that epithelial cells maintained polarity and some normal morphology during the culture period. Washed epididymal or ejaculated spermatozoa prepared by a 'swim-up' procedure were co-incubated (i) directly with epididymal cells in culture wells, (ii) in 12 mm Millicell inserts within culture wells, thereby preventing contact of spermatozoa with culture cells; and (iii) in culture medium alone. A significant proportion of spermatozoa in direct contact with culture cells or in Millicell inserts were viable after 6 days of co-incubation (30-45%) and exhibited progressive motility, while all spermatozoa in medium alone were non-motile by 3 days. Using computer-assisted sperm analysis it was shown that the progressive motility of viable spermatozoa decreased gradually for the first 5 days in culture and then remained constant (approximately 30 microm/s, average path velocity). After 12 days of co-incubation, 15 +/- 4% of spermatozoa in direct contact with epithelial cells remained motile; in one experiment, a few spermatozoa (<1%) were motile at 17 days. Light and electron microscope observations indicated that prolonged sperm survival was associated with close apposition of spermatozoa (by equatorial segment) to the apical membrane of epithelial cells. Oviductal epithelial cells were also beneficial for sperm survival, but other cell types had no effect.     

Sound-induced activation of auditory cortices in cochlear implant users with post- and prelingual deafness demonstrated by positron emission tomography

Lactoferrin has been for the first time purified from the porcine cauda epididymal fluid as a 70 kDa protein. Both Western and Northern blot analyses show that lactoferrin is synthesized in the regions from the distal caput to the cauda epididymis and secreted into the luminal fluid. Lactoferrin is first secreted as a 75 kDa glycoprotein and its carbohydrate moieties are gradually digested to form 70 kDa protein in the cauda epididymis. Lactoferrin has already bound to the surface of the epididymal sperm because the anti-lactoferrin antiserum induces the mature sperm tail-to-tail agglutination. These results strongly suggest new physiological functions of lactoferrin on the sperm maturation in the epididymis.     

Familial brain wave patterns: study of a 12-sib family

To identify peptide-specific antibodies which define sperm surface antigens, hybridomas were derived from the splenocytes of mice immunized with swollen human spermatozoa which had been subjected to limited proteolytic cleavage under reducing conditions prior to immunization. A total of 13.7% of the hybrid clones secreted antibodies which reacted with deglycosylated human seminal plasma glycoproteins when screened by an ELISA. A monoclonal antibody, designated mAb 4A8 sp., specifying a peptide epitope of human epididymal and a sperm surface glycoprotein was selected which inhibited human sperm-egg binding in a dose-dependent manner, and totally blocked sperm penetration in vitro. This inhibition did not result from an effect of the antibody on the motility of spermatozoa, nor was it due to premature induction of the acrosome reaction. Exclusion of oligosaccharide chains by chemical hydrolysis with trifluoromethane sulphonic acid (TFMS), enzymatic degradation and binding of lectins, did not abrogate the reactivity of mAb 4A8 to the cognate epitope whereas antibody binding was precluded upon digestion with proteolytic enzymes. In Western immunoblots of human seminal plasma glycoproteins, the antigen presented as a set of immunoreactive polypeptides, a major glycoprotein of M(r) 78 kDa and less prominent bands of M(r) 56 and 44 kDa. Immunocytochemical staining of a number of human reproductive and somatic tissues revealed strong immunostaining of the luminal epithelium of the epididymis as well as of spermatozoa in the lumen. Immunolocalization to the plasma membrane of ejaculated human spermatozoa was demonstrated by immunofluorescence, although on undigested spermatozoa the antigen epitope was less accessible. Upon capacitation the antigen persisted on the sperm surface and was present on the head of capacitated acrosome-intact spermatozoa. The pronounced peripheral immunostaining of the sperm head was accentuated after DTT/trypsin treatment, implicating the dynamic accessibility of the epitope on the plasma membrane of capacitated spermatozoa. It is suggested that the protein in question appears on the sperm membrane as a consequence of its modification in the epididymis (insertion and processing), and may be involved in the processes leading to sperm attachment and interaction with the human zona pellucida.     

Rat epididymis-specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin I lectin column. Epididymal fluid antigens have apparent M(rs) of 38-26 kD, whereas the membrane-associated form of the molecule has an M(r) of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secreted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm.     

The acylation of megakaryocyte proteins: glycoprotein IX is primarily myristoylated while glycoprotein Ib is palmitoylated

The acylation of megakaryocyte proteins was studied with special emphasis on the myristoylation and palmitoylation of the glycoprotein (GP) Ib complex. Guinea pig megakaryocytes were purified and separated into subpopulations at different phases of maturation. Cells were incubated with [3H]myristate, [3H]palmitate, or [3H]acetate to study endogenous protein acylation. Cycloheximide was used to distinguish between cotranslational and posttranslational acylation and hydroxylamine to distinguish between thioester and amide linkages. After incubations, delipidated proteins or GPIb complex subunits, immunoprecipitated with PG-1, AN-51 or FMC-25 monoclonal antibody, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assessed by fluorography. Radiolabeled fatty acids bound to GPIX and GPIb were also analyzed by high pressure liquid chromatography (HPLC) and scintillation spectrometry. With [3H]myristic acid and [3H]acetate, GPIX was found to be a major myristoylated protein in megakaryocytes and CHRF-288 cells. Myristic acid was linked to GPIX by an amide bond, and this process occurred cotranslationally. With [3H]acetate, GPIb was primarily palmitoylated, but with [3H]myristate, GPIb was acylated with about equal mounts of myristic acid and palmitic acids. Both fatty acids were linked to GPIb by thioester bonds, and acylation was posttranslational. The myristoylation of GPIX while the palmitoylation of GPIb occurred throughout megakaryocyte maturation. Myristoylation and palmitoylation may have different functions relevant to the assembly of the GPIb complex in megakaryocytes.     

Fatty acylation of the rat and human asialoglycoprotein receptors. A conserved cytoplasmic cysteine residue is acylated in all receptor subunits

Functional rat or human asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomeric integral membrane glycoproteins. Rat ASGP-R contains three subunits, designated rat hepatic lectins (RHL) 1, 2, and 3; human ASGP-R contains two subunits, HHL1 and HHL2. Both receptors are covalently modified by fatty acylation (Zeng, F.-Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21382-21387; Zeng, F.-Y., Oka, J. A., and Weigel, P. H. (1996) Biochem. Biophys. Res. Commun. 218, 325-330). We report here that the single Cys residue in the cytoplasmic domain of each RHL or HHL subunit is fatty acylated. The degree of acylation is >/=90% per subunit. Deacylation of affinity-purified ASGP-Rs with hydroxylamine results in the spontaneous formation of dimers through reversible disulfide bonds, indicating that deacylation concomitantly generates free thiol groups. Reaction of hydroxylamine-treated ASGP-R with [14C]iodoacetamide resulted in the specific incorporation of radioactivity into all RHL and HHL subunits, verifying that fatty acids are attached via thioester linkages. To identify the Cys residue involved in the thioester linkages, 14C-carboxyamidomethylated RHL subunits were separated by SDS-polyacrylamide gel electrophoresis and digested in-gel with trypsin, and the resulting peptides were separated by reverse-phase high performance liquid chromatography. Amino acid sequence of radioactive peptides revealed that Cys35 in RHL1 and Cys54 in RHL2 and RHL3 were radiolabeled and, therefore, are fatty acylation sites. Fatty acylation of HHL subunits was analyzed by site-directed mutagenesis. Metabolic labeling of Cos7 cells transfected with wild type HHL1 cDNA resulted in substantial incorporation of [3H]palmitate into purified HHL1. Incorporation of [3H]palmitate into a C36S mutant of HHL1 was negligible ( approximately 1%) compared with wild type. This result also shows that Cys57 within the transmembrane domain of HHL1 is not normally palmitoylated. We conclude that Cys35 in RHL1, Cys54 in RHL2 and RHL3, and Cys36 in HHL1 are fatty acylated. Cys57 in HHL1 and probably Cys56 in RHL1 are not palmitoylated.     

Biochemical evidence that Semliki Forest virus obtains its envelope from the plasma membrane of the host cell

The data from chemical studies and electron microscopy suggest that Semliki Forest virus obtains its envelope by budding into the medium from the plasma membrane of the host cell. Biochemical evidence for this phenomenon, however, has not been published. Therefore, we undertook a series of pulse-chase studies so that we might quantitatively evaluate the importance of the budding mechanism in the morphogenesis of Semliki Forest virus. Baby hamster kidney cells (clone 13) were grown in culture and infected with Semliki Forest virus. The cells were exposed to [4,5-3H] leucine for 20 min and the subsequent incorporation of the label into virus proteins associated with cytoplasmic membranes and extracellular virus was determined. Initial experiments were conducted with microsomes and a precursor-product relationship was demonstrated between viral proteins in the microsomes and in extracellular virus. Further studies were performed with endoplasmic reticulum and plasma membrane preparations. Maximal incorporation of [3H] leucine was observed in the viral proteins located in the endoplasmic reticulum at the end of a 20-min pulse period; greater than 50% of this activity had disappeared within 2 h. The plasma membrane fraction contained no radioactivity at the end of the pulse period; subsequently, maximal labeling of the viral proteins in the plasma membrane occurred 4 h into the chase period and these labeled proteins had disappeared from this membrane 11 h after the pulse. At this time maximal incorporation of the labeled proteins into extracellular virus was observed. These data are consistent with a precursor-product relationship between the viral proteins in the endoplasmic reticulum which migrate to the plasma membrane and are subsequently incorporated into extracellular virus. All the radioactivity in the extracellular virus appears to have been derived from viral proteins associated with the plasma membrane of the cell. Therefore, mechanisms for the morphogenesis of Semliki Forest virus (in baby hamster kidney cells), other than budding from the plasma membrane, are unlikely to be of quantitative importance.