Changes in the protein composition of rat spermatozoa during maturation in the epididymis

Spermatozoa from the testis and cauda epididymidis were solubilized by detergent treatment and electrophoresis on SDS polyacrylamide gels revealed that the relative amounts of 13 detergent-extractable proteins decreased during passage of spermatozoa through the epididymis, 6 increased, whilst the remainder showed little or no change. Lactoperoxidase-catalysed iodination of plasma membrane proteins showed that the components carrying most of the label in testicular spermatozoa had Mr values of 110 000, 94 000, 84 000, 55 000 and 42 000 whereas on cauda epididymal spermatozoa the Mr values were 47 000, 24 000, 17 000, 14 500 and 13 500. Substantial differences were also noted in the protein composition of rete testis fluid and cauda epididymal plasma. The results support the concept that there is a considerable reorganization of the molecular architecture of the plasma membrane of spermatozoa during maturation in the epididymis.     

Development of a maturation antigen on the plasma membrane of rat spermatozoa in the epididymis and its fate during fertilization

The ontogeny of a surface membrane antigen on rat spermatozoa has been investigated using the monoclonal antibody, 2D6. Using indirect immunofluorescence microscopy the 2D6 antigen was first detected on spermatozoa from the proximal corpus epididymidis; no reaction was present on testicular cells. The 2D6 antibody also bound to spermatozoa flushed from the uterus of mated rats and to a sperm-derived antigen on the surface of newly fertilized eggs. When frozen sections of epididymal tissues were stained with 2D6 monoclonal antibody immunofluorescence was confined to the epithelium lining the duct in the proximal and distal corpus epididymidis. Fluorescence in the tissue was androgen-dependent. Immunoblots of proteins in luminal secretions collected by micropuncture from different sites along the epididymal duct showed that in the proximal corpus epididymidis the 2D6 monoclonal antibody recognized a 32 kD antigen, but in secretions from the distal corpus and cauda epididymidis the monoclonal antibody also recognized antigens with molecular weights of 28, 23 and 20 kD. Immunoblots of proteins from spermatozoa collected from the corpus epididymidis revealed a reaction over a 32 kD antigen, while on spermatozoa from the cauda epididymidis the 2D6 monoclonal antibody recognized only a 23 kD antigen. Two hypotheses are proposed to account for the varied reactivity of the monoclonal antibody and their relative merits are discussed.     

Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.     

Labelling of membrane glycoproteins on rat spermatozoa collected from different regions of the epididymis.

1. Charge-shift electrophoresis showed that cholera toxin and its subunits have no hydrophobic surfaces. 2. Amino-acid composition and sequence data suggested that the proteins have no masked hydrophobic regions. 3. The A subunit of cholera toxin may interact with polar molecules in the membrane to exert its effect inside the cell. 4. The only hydrophobic part of tetanus toxin was the H-chain.     

Lipid diffusion in the plasma membrane of ram and boar spermatozoa during maturation in the epididymis measured by fluorescence recovery after photobleaching

Maturation of spermatozoa in the epididymis involves remodelling of many protein and lipid components of the plasma membrane. In this investigation we have examined whether (a) diffusion of lipid molecules in the surface membrane changes during epididymal maturation; (b) diffusion is spatially restricted; and (c) differences in lipid diffusion can be related to known changes in membrane composition. For this purpose we have used the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusion of the lipid reporter probe ODAF (5‐(octa‐decanoyl)aminofluorescein) in spermatozoa from two species: ram, where substantial changes in membrane lipids occur during passage through the epididymis, and boar, where there are relatively few changes. Results on ram spermatozoa show that between the testis and cauda epididymidis, diffusion coefficients values (D) for ODAF increase significantly in all the surface domains. Percentage recovery values (%R) remain constant irrespective of maturational status. In boar spermatozoa, however, D and %R values do not change significantly between epididymal regions. Cholesterol, which has widespread effects on the behaviour of lipid molecules in cell membranes, was visualized by binding of filipin. In both species filipin was concentrated over the acrosomal domain and cytoplasmic droplet of testicular spermatozoa, but in the epididymis it had a heterogenous distribution over the whole head and tail. These results are discussed in relation to the establishment and maintenance of lipid domains in spermatozoa and their influence on development of fertilizing capacity. Mol. Reprod. Dev. 52:207–215, 1999. © 1999 Wiley‐Liss, Inc.     

Changes in distribution of phosphomannosyl receptors during maturation of rat spermatozoa

The aim of the present work was to study the distribution of the cation-independent (CI) and cation-dependent (CD) mannose-6-phosphate receptors (MPRs) in spermatozoa obtained from either rete testis or three regions of rat epididymis. We observed that both receptors underwent changes in distribution as spermatozoa passed from rete testis to cauda epididymis. CI-MPR was concentrated in the dorsal region of the head in rete testis sperm and that this labeling extended to the equatorial segment of epididymal spermatozoa. CD-MPR, however, changed from a dorsal distribution in rete testis, caput, and corpus to a double labeling on the dorsal and ventral regions in cauda spermatozoa. The percentages of spermatozoa that showed staining for either CI-MPR or CD-MPR increased from rete testis to epididymis. The observed changes were probably the result of a redistribution during transit rather than an unmasking of receptors. The fluorescence corresponding to CD-MPR and CI-MPR on the dorsal region disappeared when caudal spermatozoa underwent the acrosomal reaction. Receptors were localized on the plasmalemma of spermatozoa, as observed by immunoelectron microscopy. Changes in distribution may be related to a maturation process, which suggests new roles for the phosphomannosyl receptors.     

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.     

Changes in the acrosome of guinea pig spermatozoa during passage through the epididymis

Summary Electron microscopic observations have been made on the structure of guinea-pig spermatozoa in successive segments of the epididymal duct. Marked changes are observed in the shape and internal structure of the acrosome as the sperm move through the epididymis. The morphological basis for this continuing differentiation of the sperm is traced back to certain unusual features of acrosome formation in the early spermatids of this species. The possible relation of the progressive development of the acrosome to the known increase in fertilizing capacity of the spermatozoa during their passage through the epididymis is discussed.Supported in part by grant RG-6729 and by GM-10182-8 from the Division of General Medical Sciences, National Institutes of Health, United States Public Health Service.It is a pleasure to acknowledge the technical assistance of Arthur Mitchell who made some of the initial observations that stimulated this investigation.     

Distribution of carnitine and acylcarnitine in the hamster epididymis and in epididymal spermatozoa during maturation

The highest levels of carnitine and acylcarnitine were found in the cauda epididymidis, and spermatozoa from the cauda contained greater amounts of total carnitine (free carnitine plus acylcarnitine) than those removed from the corpus or caput epididymidis. Spermatozoa from the distal cauda contained significantly greater amounts of both free and total carnitine than those removed from the proximal cauda epididymidis. The acylcarnitine:carnitine ratio was 1.7 and 0.37 in caput and cauda spermatozoa, respectively and 1.7 and 1.3 in caput and cauda fluid, respectively. It is suggested that the accumulation of carnitine is involved in sperm maturation and that acylcarnitine serves as an energy substrate for epididymal spermatozoa.     

Biogenesis of plasma membrane glycoproteins. Purification and properties of two rat liver plasma membrane glycoproteins

As a preliminary to a study of the biogenesis of individual plasma membrane glycoproteins, the marker enzyme nucleotide pyrophosphatase (NPPase) and a major rat liver plasma membrane sialoprotein, subsequently found to be identical with the enzyme dipeptidyl peptidase IV (DPP IV), were purified 10,000- and 2,000-fold, respectively, from rat liver. Both were amphipathic proteins which formed defined micellar complexes with detergents and aggregated in their absence. Gel filtration, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the Triton X-100 complex of NPPase to contain a single 150,000-dalton peptide, while that of DPP IV was composed of two 120,000-dalton subunits; each complex also contained about 150,000-dalton Triton X-100. Trypsin cleaved the detergent complexes with release of major hydrophilic fragments which no longer bound detergent micelles; the accompanying change in peptide size was small for NPPase and undetectable for DPP IV, which also retained the dimer structure of its native form. DPP IV was the only major glycoprotein in rat liver plasma membrane which bound strongly to wheat germ agglutinin. Monospecific rabbit antibodies against NPPase and DPP IV precipitated the antigens without affecting their enzymatic activities.     

Biogenesis of plasma membrane glycoproteins. Tracer kinetic study of two rat liver plasma membrane glycoproteins in vivo

Antibodies to purified nucleotide pyrophosphatase (NPPase) and dipeptidyl peptidase IV (DPP IV) were used to study the biogenesis of these rat liver plasma membrane glycoproteins in vivo. Following injection of tritiated leucine, the radioactivity in NPPase and DPP IV decayed at markedly different rates in the plasma membrane, with apparent half-lives of about 1 and 5 days, respectively. In short term experiments, labeling of total plasma membrane proteins was rapid and insensitive to colchicine, while labeling of both NPPase and DPP IV showed a lag of about 15 min, followed by colchcine-sensitive/cycloheximide-insensitive increases to half-maximal and maximal values at about 1 and 2 h, respectively. A peak of labeled DPP IV in rough microsomes at 15 min showed increased mobility on polyacrylamide gels and was largely inaccessible to antibodies in intact microsomes, consistent with its being an underglycosylated precursor, exposed on the cisternal side of the rough endoplasmic reticulum. In contrast, the behavior of unlabeled DPP IV in preparations of rough microsomes and Golgi was consistent with its being contributed by contaminating right-side-out plasma membrane vesicles. This conclusion was also necessary to fit the tracer kinetic data to a simple membrane-flow model, which gave precursor pools (1 microgram/g of liver) and fluxes (1 microgram/h/g of liver) for both DPP IV and NPPase which were about 3 orders of magnitude less than those for the synthesis of rat serum albumin. Thus, unlike hepatoma tissue culture cells (Doyle, D., Baumann, H., England, B., Friedman, E., Hou, E., and Tweto, J. (1978) J. Biol. Chem. 253, 967-973), normal rat liver does not contain large amounts of preformed intracellular plasma membrane precursors.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na⁺ + K⁺)-ATPase, leucine aminopeptidase, 5′-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na⁺ + K⁺)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48–108 h and in 5′-nucleotidase activity at 12–24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Topographical rearrangement of a plasma membrane antigen during capacitation of rat spermatozoa in vitro

We have previously described an antigen (termed 2B1) on rat spermatozoa that is present on the plasma membrane overlying the tail domain. The antigen is mobile within the plane of the plasma membrane and a mAb to it blocks fertilization in vitro. In the present study we describe some dynamic properties of this antigen in relation to its topographical distribution. When spermatozoa were incubated in vitro in a capacitation medium and stained with 2B1 mAb/FITC-rabbit anti-mouse F(ab')2, strong fluorescence appeared over the acrosomal domain. Acute exposure of fresh spermatozoa to dissociating reagents (1 M NaCl or 5 mM 2-mercaptoethanol) or inducers of the acrosome reaction (lysolecithin + Ca2+ or A23187 + Ca2+) failed to mimic these effects. Spermatozoa prelabeled with FITC-2B1 IgG and then capacitated in the presence of excess "cold" 2B1 IgG also showed accumulation of fluorescence on the acrosomal domain, suggesting that the antigen had migrated from the tail. Migration was selective and Ca2(+)- and temperature-dependent but was not inhibited by metabolic poisons (NaF or NaN3). Motility was not obligatory for migration. Immunogold-labeling studies at the ultrastructural level showed that 2B1 antigen was restricted to the surface membrane over both the tail and the acrosomal domains and that during migration it did not change the type of membrane into which it was inserted. From a quantitative analysis of fluorescence on spermatozoa prelabeled with FITC-2B1 IgG and then capacitated, the amount of antigen that appeared on the acrosomal domain was approximately equivalent to that lost from the midpiece domain. The Mr of 2B1 antigen extracted from capacitated spermatozoa was 300-500 Da less than that extracted from noncapacitated cells, suggesting that the molecule had undergone processing concomitant with migration. These results are discussed in relation to mechanisms for targeting antigens to sites where they become physiologically active and are correctly positioned to participate in gamete recognition processes.     

Changes in membrane glycoproteins during fruit-body formation inPhysarum polycephalum

Sporangia formation ofPhysarum polycephalum was induced by starvation and illumination, and the morphogenic process during the differentiation was studied by scanning electron microscopy. Plasma membranes were prepared from these differentiating plasmodia and the membrane proteins were analyzed by polyacrylamide gel electrophoresis. Many glycoproteins appeared during the fruit-body formation. Of these a protein of apparent molecular mass of 66 kD was prominent in sporangia forming stage which showed a high affinity to RCA lectin. Inhibition of the glycosylation and processing of these glycoproteins resulted in the prevention of fruit-body formation suggesting that the synthesis of these membrane components is a prerequisite process for the sporangia formation in the slime mold.     

Changes in the organization of the lipid bilayer of the plasma membrane during spermatogenesis and epididymal maturation

Ram, bull, and mouse sperm cells were stained with several fluorescent membrane probes. In contrast to nonspecific probes, merocyanine 540 (MC540), which displays preferential binding to loosely packed phospholipids in model membranes, was specifically localized to the anterior portion of the head and the midpiece of mature sperm. To establish when during development this distinctive staining pattern was acquired, germ cells from prepubescent and adult mouse testes as well as sperm from the caput, corpus, and cauda epididymides were isolated and examined. Localized staining with MC540 was not observed until sperm reached the corpus epididymidis, where those cells with a completely translocated (i.e., distally located) cytoplasmic droplet fluoresced. Likewise, when sperm were stained with fluoresceinated concanavalin A (fl-ConA), a localized pattern of fluorescence with lectin restricted to the anterior portion of the head was not observed until the corpus epididymidis was reached. However, in contrast to MC540 staining, only a fraction of sperm with completely translocated droplets exhibited this localized staining with fl-ConA, the remainder exhibiting diffuse fluorescence over the entire cell as seen on caput epididymal sperm. These developmental changes in staining patterns are specific to murine cells, since no change in the pattern of staining by either MC540 or fl-ConA was seen on epididymal sperm of the ram. These results are discussed with respect to: 1) species-to-species differences in sperm membrane features; and 2) the hypothesis that domains of loosely packed lipids may be involved in the regionalization of membrane proteins that occurs during sperm development.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat.

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na+ + K+)-ATPase, leucine aminopeptidase, 5'-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na+ + K+)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48--108 h and in 5'-nucleotidase activity at 12--24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane

Seven integral proteins (CE 9, HA 21, HA 116, HA 16, HA 4, HA 201, and HA 301) were isolated from rat hepatocyte plasma membranes by immunoaffinity chromatography on monoclonal antibody-Sepharose. Six of the proteins (all but HA 16) exhibit domain-specific localizations (either bile canalicular or sinusoidal/lateral) about the hepatocyte surface. We identified three of these protein antigens as leucine aminopeptidase (HA 201), dipeptidyl peptidase IV (HA 301), and the asialoglycoprotein receptor (HA 116). We also developed 125I-lectin blotting procedures that, when used in conjunction with chemical and glycosidase treatments, permitted a comparison of the types of oligosaccharides present on the seven proteins. All seven are sialoglycoproteins, based upon the effects of prior neuraminidase and periodate-aniline-cyanoborohydride treatments of blots on labeling by 125I-wheat germ agglutinin. 125I-labeled Ricinus communis agglutinin I and 125I-peanut agglutinin blotting of the desialylated proteins revealed few if any conventional O-linked oligosaccharides, suggesting that the sialyl residues represent termini of N-linked complex-type oligosaccharides. Depending upon the protein, we estimated the presence of 2-26 N-linked oligosaccharides/polypeptide chain from the Mr reductions accompanying chemical or enzymatic deglycosylation. Three of these mature plasma membrane proteins (HA 21, HA 116, and HA 4) have both high mannose-type and complex-type oligosaccharides on every copy of their polypeptide chains. The labeling of these three proteins by 125I-concanavalin A was sensitive to treatment with endoglycosidase H, and each exhibited a quantitative reduction in Mr after the treatment, as assessed independently by 125I-wheat germ agglutinin blotting. At this level of analysis, we were unable to discern differences in the types of oligosaccharides present on these seven glycoproteins that correlate with their patterns of expression within the plasma membrane domains of this polarized epithelial cell.