Structural and electrical characterization of Bi2Se3 nanostructures grown by metal-organic chemical vapor deposition.

We characterize nanostructures of Bi(2)Se(3) that are grown via metal-organic chemical vapor deposition using the precursors diethyl selenium and trimethyl bismuth. By adjusting growth parameters, we obtain either single-crystalline ribbons up to 10 µm long or thin micrometer-sized platelets. Four-terminal resistance measurements yield a sample resistivity of 4 mΩ·cm. We observe weak antilocalization and extract a phase coherence length l(ϕ) = 178 nm and spin-orbit length l(so) = 93 nm at T = 0.29 K. Our results are consistent with previous measurements on exfoliated samples and samples grown via physical vapor deposition.

Characterization of the guanidinobenzoatase in the epididymal fluids of the mouse.

Guanidinobenzoatase (GB), a proteolytic enzyme found in the epididymal fluids of mice, was purified to apparent homogeneity by molecular sieving and affinity chromatography. It has a molecular mass of 71 kDa and its enzymatic activity is heat labile and sensitive to EGTA. Its kinetic parameters (K(m) of 6.66 microM and a Vmax of 4.38 nmol/min/mg) were determined using 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) as the substrate. GB activity is concentrated in the cauda epididymal region of the genital tract. Heat-solubilized whole zonae, biologically active ZP3, and several serine proteinase inhibitors, including a proteinase inhibitor endogenous to the male genital tract, effectively block the ability of GB to hydrolyze MUGB. Pretreating cumulus-free, zonae intact oocytes with purified GB reduces, in a concentration-dependent manner, the number of sperm able to bind to the zonae. The function of the soluble enzyme is not known. Its ability to bind both trypsin inhibitors and ZP3 suggests a possible role in gamete recognition.

Induction of physiological acrosome reactions in caput epididymal spermatozoa of mice.

Mouse caput spermatozoa are considered immature and thus unable to fertilize oocytes. In this study, we determined whether washing mouse caput spermatozoa increased their ability to acrosome react in response to a physiological stimulus. The results obtained showed that mouse caput spermatozoa incubated in Earles' modified medium containing calcium chloride and supplemented with BSA and pyruvate for 1 h at 37 degrees C and then washed acrosome reacted in response to both solubilized zonae and immunoaggregation of a zona binding site. In addition, the material removed from caput spermatozoa by washing blocked induced acrosome reactions of cauda spermatozoa. The data indicate that mouse caput epididymal spermatozoa, if incubated and washed, can undergo physiological acrosome reactions.

Binding of a murine proteinase inhibitor to the acrosome region of the human sperm head.

Proteinase inhibitors are present in the various glands, tissues, and secretions of the male reproductive tract. Some of these inhibitors bind to the acrosomal region of the sperm, and their release during in vitro or in utero incubation suggests that they may play a role in capacitation. In the mouse, the binding site for a trypsin-acrosin inhibitor, the acceptor, has been implicated in capacitation, zona binding, and the acrosome reaction. This presentation demonstrates that a component, molecular weight approximately 20,000, on the human sperm head may recognize the murine inhibitor. Furthermore, the acrosome reaction can be induced in capacitated human sperm by immunoaggregation of bound murine inhibitor. The data indicate that the proteinase inhibitor binding site on the human sperm head may, as with a similar site on murine sperm, play a role in the early events of fertilization.

Competition between zonae pellucidae and a proteinase inhibitor for sperm binding.

Murine sperm bind a proteinase inhibitor of seminal vesicle origin at ejaculation. The inhibitor binds in the acrosomal region of the sperm head and is removed during in utero or in vitro incubation. Adding inhibitor to sperm reduces their ability to bind zonae, while adding the purified inhibitor binding site to cumulus-free, zona-intact oocytes reduces the ability of the oocytes to bind sperm. Immuno-aggregation of the inhibitor binding site results in exocytosis of the acrosome. These observations suggest that the inhibitor binding site may participate in zona binding and the acrosome reaction. If the inhibitor binding site binds both the zona and the seminal inhibitor, then these components should compete with each other for that site on the sperm. We show that purified seminal inhibitor, as well as other proteinase inhibitors, block zona-induced acrosome reactions. Likewise, zona glycopeptides block inhibitor/anti-inhibitor-induced acrosome reactions in a concentration-dependent fashion. The inhibitor/anti-inhibitor-induced acrosome reaction is sensitive to pertussis toxin and proteinase inhibitor and thus is similar to zona-induced reactions. These findings support the suggestion that the trypsin inhibitor binding site on the head of the sperm functions to insure sperm-zona binding and induction of the acrosome reaction.

Role of monoclonal antibody J-23 in inducing acrosome reactions in capacitated mouse spermatozoa.

A monoclonal antibody (J-23) to the 15 kDa component on the sperm head, the acceptor, which functions in zona binding, was shown to induce the acrosome reaction in capacitated cells, but not in fresh cells. The antibody recognized its epitope in the acrosomal cap region of fresh spermatozoa and in the equatorial region on washed and capacitated spermatozoa. However, equatorial expression did not depend on the acrosome reaction, since washing fresh spermatozoa increased the percentage with equatorial fluorescence, but did not increase the percentage with reacted acrosomes. The data indicate that the acrosome reaction can be induced in capacitated spermatozoa in the absence of zona glycoproteins.

Acrosome reaction induced by immunoaggregation of a proteinase inhibitor bound to the murine sperm head.

ZP3, a glycoprotein of the murine zona pellucida, functions both to bind acrosome intact sperm and to induce the acrosome reaction. Solubilized whole zonae as well as purified ZP3 are able to induce acrosome reactions in capacitated sperm. Pronase digests of whole zonae yield glycopeptides that bind to sperm but are unable to induce acrosome reactions. However, immunoaggregation of these glycopeptides results in the exocytosis of the acrosome in the majority of treated sperm. The data suggest that ZP3 triggers the acrosome reaction by the aggregation of ZP3 binding sites on the sperm head. If aggregation of ZP3 binding sites is important in the induction of the acrosome reaction, then it may be possible to induce the acrosome reaction in the absence of zona by immunoaggregation of the sites. This presentation deals with the immunoaggregation of a proteinase inhibitor of seminal vesicle origin (SVI) that binds to a site on the sperm head known to participate in zona binding. We show that capacitated murine sperm, pretreated with the SVI, will acrosome react, as determined by Coomassie brilliant blue staining, when incubated with rabbit antiinhibitor antiserum (anti-SVI). The percentage of SVI-treated sperm displaying an acrosome reaction is dependent on the concentration of the immune serum. Sperm stain positive for intact acrosomes when anti-SVI Fab fragments or normal rabbit serum is substituted for the immune serum. However, when capacitated sperm, treated with both SVI and anti-SVI Fab fragments, are incubated with goat antirabbit IgG, the majority of sperm acrosome react. The data suggest that the aggregation of SVI bound to the sperm surface, in the absence of zona glycoproteins, is sufficient to induce the acrosome reaction.

Effects of in vitro incubation on a zona binding site found on murine spermatozoa.

Murine cauda epididymal sperm possess a site, the acceptor, on the plasma membrane over the apical cap region of the acrosome which recognizes both a proteinase inhibitor of seminal vesicle origin and homologous zonae. The acceptor site may participate in both capacitation and zona binding. This presentation explores the effect of in vitro incubation in a medium known to induce capacitation on the binding capabilities of this site. Approximately 80% of fresh cauda epididymal sperm will bind the seminal inhibitor in vitro. Incubating sperm, pretreated with inhibitor for 2 hr in a medium (M199-M) known to support capacitation, reduces by 60% the number of sperm showing evidence of the inhibitor. No such decrease is seen when sperm are incubated in a medium (M199) that does not support capacitation. During the 2-hr incubation in either medium, 60-70% of the sperm retain two diverse components on the plasma membrane over the acrosome: a receptor for the Fc portion of IgG and an epitope recognized by a monoclonal antibody to the acceptor site. These observations suggest that the plasma membrane in the acrosome region of the cell remains structurally intact during incubation. Furthermore, sperm retain the ability to bind the seminal inhibitor during incubation. After a 2-hr incubation in M199-M, sperm pretreated with heat-solubilized zonae no longer bind the inhibitor. These sperm, however, retain the plasma membrane over the acrosomal cap region. When the sperm are incubated in M199, no decrease in inhibitor binding due to zona treatment is noted.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody to a zona pellucida-proteinase inhibitor binding component on murine spermatozoa.

A monoclonal antibody, J-23, was produced to an epitope of a binding (acceptor) component on the plasma membrane in the acrosomal cap region of the mouse sperm head. The component binds a proteinase inhibitor of seminal vesicle origin at ejaculation and participates in the in vitro binding of capacitated sperm to the zona pellucida. The antibody, an IgM molecule, recognizes affinity purified acceptor, crude acceptor and whole sperm as determined by ELISA methodology. The antibody reacts with a 15,000 molecular weight component, the size previously determined for the acceptor, found in the supernatants of frozen-thawed cauda epididymal sperm. In addition, it binds to a 21,000 molecular weight component generated by mixing an excess of purified inhibitor (6400 daltons) with a crude acceptor preparation. J-23 binds to an epitope in the same region of the sperm head as does the inhibitor. This epitope becomes fully expressed during epididymal maturation and is found only in the lumen of epididymal tissues. Pretreating sperm with J-23 inhibits their ability to bind the inhibitor as well as the zona pellucida. Pretreating sperm with the inhibitor has little effect on the binding of J-23. These data indicate that J-23 recognizes an epitope on the acceptor but the epitope is not directly involved with inhibitor binding.

Features of a seminal proteinase inhibitor- zona pellucida-binding component on murine spermatozoa.

Murine cauda epididymal sperm contain sites on the plasma membrane over the apical portion of the acrosome that recognize proteinase inhibitors and the homologous zona pellucida. Ten times more of the component can be extracted from cauda and ductus sperm than from equal numbers of caput and corpus sperm. Likewise, few sperm from the upper epididymal regions are able to bind seminal inhibitor, while the majority of sperm from the cauda and ductus do bind. Cauda epididymal and ductus sperm lose little of their ability to bind inhibitor after a 4-hour in vitro incubation in either a capacitating or a noncapacitating medium. The percentage of naturally inseminated sperm with the seminal inhibitor bound to their surface decreases to about 10 after 4 hours in utero. Approximately 80% of these sperm show positive fluorescence when given the opportunity to rebind the the inhibitor, and these sperm do have an intact plasma membrane over the apical portion of the acrosome. Furthermore, after 4 hours in utero, the inhibitor bound in the same region of the sperm head as it did on freshly ejaculated sperm. The seminal inhibitor inhibits the binding of sperm to the zona if added during the first 15 minutes of incubation but has no effect on attachment. The data indicate that sperm gain the ability to bind the seminal inhibitor during the epididymal sojourn. Furthermore, this binding capacity is not lost during in vitro or in utero incubation. The site is not involved in sperm-zona attachment but does participate in the binding of sperm to the zona.

Competition between seminal and exogenous proteinase inhibitors for sites on murine epididymal sperm.

Sixteen proteinase inhibitors were tested for their ability to compete with the natural seminal inhibitor for binding to the surface of murine epididymal sperm. The most effective competitors, 4-methylumbelliferyl-p-quanidinobenzoate (MUGB) and p-nitrophenyl-p-guanidinobenzoate (NPGB), are also effective inhibitors of both murine acrosin and in vitro fertilization of mouse gametes. The data support the suggestion that the inhibition of fertilization by these inhibitors may be effected by their action on the sperm surface rather than binding to enzymes located within the acrosome. Since the surface acceptor molecule recognizes a number of inhibitor types, as well as substrates for such enzymes as trypsin and acrosin, the acceptor's binding site may be similar to the active site on the enzyme.

Evidence for a proteinase inhibitor binding component associated with murine spermatozoa.

The supernatants of frozen-thawed murine epididymal sperm suspensions contain a heat-labile component capable of binding a low molecular weight, acid-stable proteinase inhibitor of seminal vesicle origin. The substance has a molecular weight of approximately 15,000 and can be isolated by affinity chromatography using the inhibitor as the ligand. Although the substance has no inherent enzymatic properties, it will decrease the activity of the seminal inhibitor in the standard N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) assay. Enzyme-linked immunosorbent assays (ELISA) indicate that the substance, when bound to microtiter plates, is capable of binding the seminal vesicle inhibitor. Turkey egg white trypsin inhibitor will decrease the amount of the seminal inhibitor that will bind to the substance, while noninhibitor proteins, e.g., bovine serum albumin or insulin, have no effect. Turkey egg white and lima bean trypsin inhibitor will also decrease the amount of seminal vesicle inhibitor capable of binding to washed sperm. These data indicate the presence of an inhibitor acceptor site associated with murine epididymal spermatozoa.

Distribution of a proteinase inhibitor of epididymal origin in the tissues and secretions of the male reproductive tract of mice.

Monoclonal antibodies to a low molecular weight, acid-stable acrosin-trypsin inhibitor isolated from epididymal homogenates were used to localize the inhibitor in tissues and secretions of the male reproductive tract of mice. The inhibitor, identified by indirect immunofluorescence, is present in the testes and in the apical portion of the epithelial cells from the caput region of the epididymis of both intact and efferentiectomized animals. Sperm isolated from the testes and caput epididymal region show inhibitor-positive fluorescence on the anterior acrosomal region. The inhibitor could not be localized on ductus or electroejaculated sperm or ductus sperm previously incubated in a purified inhibitor solution. Furthermore, the inhibitor was not visible in the copulatory plug of recently inseminated animals or on sperm recovered from the uterus or from an artificial capacitating medium. The inhibitor could be detected by an enzyme-linked immunosorbent assay in the supernatants of detergent treated or frozen-thawed caput sperm but not of ductus sperm. The data suggest that the inhibitor, made in both the testes and caput epididymis where it associates with the sperm, is lost from the sperm or irreversibly masked during the epididymal sojourn.

Immunofluorescent localization of a murine seminal vesicle proteinase inhibitor.

The indirect immunofluorescent technique was used to localize a proteinase inhibitor isolated from murine seminal vesicles. The inhibitor was found in the lumen and in the apical epithelium of the seminal vesicle but not in the testes, epididymides, ductus deferens or Cowper's glands. It was also associated with the anterior acrosomal region of ejaculated sperm and sperm recovered from the female tract within 5 min of coitus. The inhibitor is removed from uterine sperm between 2 to 4 h postcoitus, however sperm recovered from the uterus 2 h postcoitus will rebind inhibitor. The inhibitor is not normally associated with epididymal or ductus sperm although these sperm will bind purified inhibitor in vitro.

Immunofluorescent localization of a seminal vesicle proteinase inhibitor in the female reproductive tract of naturally inseminated mice.

The indirect immunofluorescent technique was used to localize a low molecular weight, acid-stable proteinase inhibitor of seminal vesicle origin in the female reproductive tract of mice. In recently inseminated animals (0, 2, and 4 hr postcoitus) the inhibitor was localized in the copulatory plug, on the epithelia of the vaginal fornix and cervix, in the uterine lumen, and on the apical surface of the uterine epithelium. Ten hours postcoitus the inhibitor was found in localized areas on the uterine epithelium, in a sperm-leucocyte mass in the uterine lumen, and in the copulatory plug. The inhibitor was not found in females 24 hr postcoitus. The inhibitor could not be localized in the oviducts of any of the animals tested. The data are interpreted to mean that the inhibitor, transported to the female at ejaculation, coats the surface of the female reproductive tract protecting it from acrosomal enzymes or from invasion by spermatozoa or pathogens.

Fine structure distribution of non-specific acid phosphatase in the head region of mouse spermatozoa from various regions of the male reproductive tract.

The fine structure distribution of non-specific acid phosphatase was determined in the head region of mouse spermatozoa from the testes, the caput, corpus and cauda epididymidis and the ductus deferens. Enzymatic localization was achieved by the Gomori technique. The postacrosomal dense lamina, the nuclear side of the inner acrosomal membrane and the space between the plasmalemma and the outer acrosomal membrane showed reaction product in spermatozoa from the testis and caput epididymidis. Spermatozoa from the cauda epididymidis exhibited reaction product only between the plasmalemma and the outer acrosomal membrane. Spermatozoa from the corpus epididymidis and from the ductus deferens showed no reaction product in the head region. The changes observed in the distribution of acid phosphatase in the sperm head during epididymal transport may reflect maturational events.