Browsing by Author "Barros, C"

Now showing 1 - 13 of 13
  • No Thumbnail Available
    Item
    A basic 18-amino acid peptide contains the polysulfate-binding domain responsible for activation of the boar proacrosin/acrosin system
    (2000) Moreno, RD; Barros, C
    Proacrosin is the zymogen of acrosin, a serine protease localized in the acrosomal matrix of mammalian sperm. Proacrosin/acrosin binds to solubilized zona pellucida glycoproteins (ZPGs) and various polysulfates in a non-enzymatic mechanism. In addition, both polysulfates and ZPGs induce proacrosin activation once they bind to the polysulfate-binding domain (PSBD) of the enzyme. We show here that the peptide (43)IFMYHNNRRYHTCCGILL(60) inhibited the proacrosin activation induced by either fucoidan or ZPGs. In addition, the peptide was recognized by the monoclonal antibody C5F10, which is directed against the PSBD region. Our data suggest that the PSBD is composed of many "subsites" that may or may not interact with each other.
  • No Thumbnail Available
    Item
    Differential effects of polysulphates between mouse and hamster during in vitro fertilization
    (2001) Moreno, RD; Orihuela, PA; Barros, C
    The interaction between zona pellucida polysulphates and sperm receptors appears to be a widespread mechanism used by mammals during gamete interaction. In this work, the effect of hepar in on binding, penetration and fertilization of mouse and hamster oocytes was assessed. We found that heparin inhibited oocyte penetration and fertilization in both species. Heparin as well as fucoidan (a fucose-sulphate polymer) inhibited the proteolytic: activity of acrosomal enzymes in both species. Our results suggest that zona pellucida penetration in both species may be modulated by polysulphates acting on either the proteolytic rate of degradation of the zona and/or its interaction with acrosome-reacted sperm (secondary binding).
  • No Thumbnail Available
    Item
    Effect of recombinant boar β-acrosin on sperm binding to intact zona pellucida during in vitro fertilization
    (1999) Crosby, JA; Barros, C
    In a previous paper we demonstrated that boar beta-acrosin recombinant proteins were able to bind non-enzymatically to solubilized pig zona pellucida (ZP) glycoproteins, Here we report the participation of boar beta-acrosin in the secondary binding of sperm to intact pig ZP, This was achieved by using two boar recombinant proteins: beta-acrosin and a mutant of the catalytic site, beta-acrosin Ser/Ala(222). Assays of binding between the iodinated recombinant beta-acrosin and whole ZP showed that this binding could be saturated, was specific, and was stable over time. Using autoradiography, we determined that recombinant beta-acrosin bound on the entire surface of the ZP but initially was distributed heterogeneously. This suggests that the ligands for beta-acrosin may not be homogeneously distributed on the ZP. To study the contribution of acrosin in sperm secondary binding to the ZP, we preincubated in vitro-matured oocytes with these recombinant proteins and then performed in vitro fertilization assays, Under the experimental conditions used, binding of beta-acrosin recombinant proteins did not block sperm penetration. These results suggest that there may be other proteins that participate in the secondary binding, and that these proteins may recognize ligands that are different from those blocked by beta-acrosin recombinant proteins.
  • No Thumbnail Available
    Item
    Egg coats of the rock shrimp Rhynchocinetes typus
    (2002) Palomino, J; Moreno, RD; Bustamante, E; Messen, L; Dupré, E; Barros, C
    The aim of the present work was to characterize structurally and ultrastructurally the egg coats of the rock shrimp, Rhynchocinetes typus, and to describe their functional roles during fertilization. Oocytes fixed directly from the ovary, have a total diameter of 549 mum and are covered by a 10-mum-thick transparent envelope. Electron microscope sections (dehydrated) of the egg envelope revealed an electron-dense external coat of 0.4 mum covered by filamentous processesi and a granular inner coat of 4-mum thickness. Oocytes placed for 5 min in seawater had a significantly larger diameter (573 mum), because of the increase in the thickness of the egg coats (32 mum) and the formation of a 16-mum perivitelline space. The diameter of the egg proper was reduced by the same extent as the size of the perivitelline space. All these changes were associated to the loss of the egg fertilizability. SDS-PAGE of isolated and solubilized egg coats with 20% beta-mercaptoethanol or 25 mM dithiothreitol (DTT) showed bands between 58 and 105 kDa and between 44 and 103 kDa, respectively. During normal fertilization, the sperm undergoes a drastic change in shape after first contact with the egg. We observed a similar change when solubilized egg coats were placed with vas deferens sperms. When the solubilized egg coat proteins were ultrafiltrated with a membrane of 10,000 MWCO (pore size) and then assayed for their effect on fertilization, an inhibitory effect of 30%, 41%, and 59% was found when oocytes were incubated with spermatozoa pre-treated with 30, 60, and 120 mug/ml of proteins solubilized with beta-mercaptoethanol. A similar inhibitory effect was found when egg coat proteins solubilized with 25 mM DTT were used. Our results suggest that, in the shrimp, the egg coats play an active role during the morphological changes of the sperm during their passage through them. (C) 2002 Wiley-Liss, Inc.
  • No Thumbnail Available
    Item
    Fertilizing characteristics of bovine sperm with flattened or indented acrosomes
    (2001) Thundathil, J; Palomino, J; Barth, A; Mapletoft, R; Barros, C
    Frozen semen from a control bull (C: 89% morphologically normal sperm) and two bulls with acrosomal defects (K1: 92% flattened acrosomes; K2: 82% indented acrosomes) were used to investigate the fertilizing ability of bull sperm with flattened or indented acrosomes. In experiment 1, frozen-thawed sperm were evaluated for acrosomal integrity with fluorescent microscopy. In experiment 2, proteolytic activity of the acrosomal contents of sperm was evaluated through a gelatin digestion assay. In experiment 3, an IVF test system was used to determine the ability of sperm with flattened or indented acrosomes to bind to bovine oocytes and penetrate the zona pellucida. In experiment 4, IVM zona-free bovine oocytes (ZFO) were fertilized and examined to evaluate sperm chromatin decondensation. In experiment 1, bulls K1 and K2 had a lower proportion of sperm with intact acrosomes (0 and 13.6 +/- 4.5%, respectively) than bull C (30.2 +/- 5.6%) after 2 h of incubation. In experiment 2, the proportion of sperm with proteolytic activity, as indicated by gelatin digestion around sperm heads, did not differ among bulls (C: 55%, n = 410; K1: 43%, n = 426; K2: 48%, n = 324). In experiment 3, a lower proportion of sperm with flattened (K1) or indented acrosomes (K2) bound to oocytes than sperm from the control bull, C. The percentage of zona penetrated (55%, n = 20; 13%, n = 23; 4%, n = 25) and the mean (+/-S.E.M.) number of sperm penetrating these zona pellucida (19.7 +/- 2.5; 6.9 +/- 1.0; and 2.6 +/- 0.5) was higher (P < 0.05) for bull C than for bulls K1 or K2, respectively. In experiment 4, the percentage of ZFO penetrated (95%, n = 20; 52%, n = 30; 30%, n = 33) and the mean (+/-S.E.M.) number of sperm with chromatin decondensation (7.8 +/- 1.6; 0.8 +/- 0.2; and 0.3 +/- 0.1) were also higher (P < 0.05) for the control bull, C than for bulls K1 or K2, respectively. Results suggest that although sperm with the flattened or indented acrosomes had a tendency to undergo spontaneous acrosome reaction on incubation after thawing, the proteolytic activity of the acrosomal contents appeared to be normal. Sperm with the flattened or indented acrosomes also appeared to have a reduced ability to fuse with oolemma as demonstrated by confocal microscopy. This would impair the ability to penetrate ooplasm and undergo sperm chromatin decondensation. (C) 2001 Elsevier Science B.V. All rights reserved.
  • No Thumbnail Available
    Item
    Functional interactions between sulphated polysaccharides and proacrosin: implications in sperm binding and digestion of zona pellucida
    (1999) Moreno, RD; Hoshi, M; Barros, C
    Acrosin is a serine protease located within mammalian acrosome as inactive proacrosin. Sulphated polymers bind to proacrosin and acrosin, to a domain different from the active site. Upon binding, these polymers induce proacrosin activation and some of them, such as fucoidan, inhibit sperm binding to the zona pellucida. In this work we have studied the interaction of solubilised zona pellucida glycoproteins (ZPGs), heparin and ARTS (Acrosome Reaction Inducing Substance of Starfish) with boar and human acrosin. We have found that ARIS, solubilised ZPGs and fucoidan, but not heparin, inhibit the binding of the monoclonal antibody against human acrosin C5F10 to boar or human proacrosin. These results suggest that fucoidan, solubilised ZPGs and ARIS bind to a related domain on the proacrosin surface. Moreover, ARIS was able to induce human proacrosin activation. On the other hand, neither ARIS nor heparin from porcine intestinal mucosa or bovine lung induced hamster sperm acrosome reaction or sperm motility. Recent data showed that acrosin is involved in dispersal of the acrosomal matrix after acrosome reaction. Thus, the control of the ZPG glycan chains over proacrosin activation may regulate both sperm penetration rate and limited proteolysis of zona pellucida proteins.
  • No Thumbnail Available
    Item
    Gametogenesis, histological gonadal cycle and in vitro fertilization in the whitemouth croaker (Micropogonias furnieri, Desmarest, 1823)
    (2004) Berois, N; Bolatto, C; Brauer, MM; Barros, C
    The whitemouth croaker (Micropogonias furnieri) is a demersal teleostean fish inhabiting the Atlantic Ocean from northern Venezuela to southern Argentina. In terms of biomass, the whitemouth croaker is the dominant sciaenid in the Rio de la Plata and constitutes an important renewable resource for the Uruguayan and Argentinean fisheries. In the present study, analyzed were the gametogenesis and histological gonadal cycle in female and male whitemouth croakers collected at the Uruguayan coast of the Rio de la Plata, between March 1997 and February 1998. In addition, the experimental conditions required to obtain mature viable gametes and achieve in vitro fertilization in this species were investigated. These studies indicate that the whitemouth croaker inhabiting the Rio de la Plata is a multiple spawner, which reproduces on the Uruguayan coast between October and February. In vitro studies showed that following activation, sperm remain motile for up to 40 min, and under optimal dilution conditions that they retain a high fertilization capability for 15 min. Taken together these results could support future aquaculture research and exploitation of this species.
  • No Thumbnail Available
    Item
    Inhibition of mouse in vitro fertilization by an antibody against a unique 18-amino acid domain in the polysulfate-binding domain of proacrosin/acrosin
    (2002) Moreno, RD; Bustamante, E; Schatten, G; Barros, C
    Objective: To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration.
  • No Thumbnail Available
    Item
    Larval development and metamorphosis of cultured Tetrapygus niger (Echinodermata Echinoidea)
    (2000) Fuentes, I; Barros, C
    This study provides a qualitative and quantitative description of larval development of Tetrapygus niger to metamorphosis. After fertilization, the length of the planktonic interval (developmental time) for T. niger at room temperature (14-18 degrees C) ranged from 94 to 120 days. Competent larvae were induced to metamorphose by transferring them into culture vessels containing bacterial films and diatoms. About 40% of the larvae reached the juvenile stage, with complete metamorphosis from the feeding larval stage to the feeding adult stage taking 4-6 days. Although the morphology of T. niger larvae is atypical compared with most regular extant echinoid species, it is similar to another arbacioid species, Arbacia punctulata. This suggests that larval morphology needs to be included in studies aimed at establishing phylogenetic relationships. The large larval size (>3.0 mm close to metamorphosis) and uniform pattern of ciliation observed in T. niger larvae have also been observed in non-feeding larvae. However, as T. niger larvae feed, we believe these characteristics reflect a functional solution to the swimming and feeding requirements of a long planktonic life. The maximum size (>4.5 mm) achieved by T. niger larvae in this in vitro study represents the largest recorded for echinoid larvae.
  • No Thumbnail Available
    Item
    Purification and biochemical characterization of a trypsin-like enzyme present in the sperm of the rock shrimp, Rhynchocinetes typus
    (2001) Bustamante, E; Palomino, J; Amoroso, A; Moreno, R; Barros, C
    The aim of the present work was to isolate, purify and characterize a trypsin-like enzyme from the sperm of the rock shrimp, Rhynchocinetes typus. Sperm proteins were extracted with 1 mM HCl in 10% glycerol at pH 3.0. Purification of the trypsin-like substance was effected by affinity chromatography using SBTI-agarose, yielding a specific activity on BAEE as substrate of 787 U/mg, with a recovery rate of 34%. Enzymatic activity was maximal at 27 degreesC, pH of 8.0, 50 mM Ca2+ and 30 mM Mg2+. One hundred percent inhibition of enzymatic activity was obtained at 0.05 mM Zn2+. Kinetic analysis showed that the K-M on BAEE as substrate at pH 8.0 was 2.5 x 10(-5) M and the V-MAX reached was 198 U. It was also found that the enzyme had a substrate inhibition at concentrations higher than 0.06 mM of BAEE. These findings suggest that thin enzyme has similar characteristics to other trypsin-like enzymes including acrosin.
  • No Thumbnail Available
    Item
    Subcellular localization of catalase in sea urchin (Tetrapigus niger) gametes
    (1997) Figueroa, C; Kawada, ME; Munizaga, A; González, S; Barros, C; Koenig, C; Santos, MJ
    Peroxisomes are essential subcellular organelles that appear to be derived from pre existing organelles. To test the presence of peroxisomes in sea urchin (Tetrapigus niger) sperm and eggs, we performed biochemical and morphological experiments to evaluate the subcellular distribution of catalase as the typical peroxisomal marker. In sea urchin sperm, we found that catalase is localized in the cell cytosol. In contrast, sea urchin eggs contain sedimentable catalase, presumably contained in peroxisome-like structures detected by immunomicroscopy and by cytochemistry. Our results show, for the first time, evidence for the presence of peroxisome-like structures in sea urchin eggs and provide evidence for the peroxisome biogenesis hypothesis by division of preexisting organelles. (C) 1997 Elsevier Science Inc.
  • No Thumbnail Available
    Item
    The polysulphate binding domain of human proacrosin/acrosin is involved in both the enzyme activation and spermatozoa zona pellucida interaction
    (1998) Moreno, RD; Sepulveda, MS; de Ioannes, A; Barros, C
    Mammalian acrosin is a protease present as a zymogen in the acrosome of a non-reacted mammalian sperm, and in vitro is able to carry out limited hydrolysis of homologous and heterologous zonae pellucidae. On the other hand, sulphated polymers and zona pellucida glycoproteins bind to acrosin on a domain different from the active site, named the polysulphate binding domain (PSBD). Thus it is believed that acrosome-reacted spermatozoa bind to glycan chains bf the zona pellucida through PSBD participating as secondary binding receptor. The aim of the present work was to study the role of PSBD during both human gamete interaction and acrosin activation. In this work we present evidence that the anti-human acrosin monoclonal antibody C5F10 is directed to an epitope located on or near the PSBD on human proacrosin/acrosin. Moreover, we show that this antibody is able to inhibit both proacrosin activation induced by fucoidan and the sperm binding to the zona pellucida. Our results suggest that the same PSBD is involved in both sperm secondary binding, during zona pellucida penetration, and proacrosin activation.
  • No Thumbnail Available
    Item
    Trypsin-like enzymes during fertilization in the shrimp Rhynchocinetes typus
    (1997) Rios, M; Barros, C
    The spermatozoon of Rhynchocinetes typus is atypical because it is nonmotile and lacks head and tail. The body has a rigid spike. Neither an acrosome-like structure nor changes during gamete interaction which could be interpreted as an acrosome reaction have been observed in this species. Nevertheless, the spermatozoon exerts a lytic effect on the extracellular envelope of the egg, and in this way it penetrates through egg-coats, forming a channel. In this research we found that crude spermatozoa. extracts analyzed by gelatin SDS-PAGE developed one band of protease activity that was completely inhibited by SBTI (soybean trypsin inhibitor) and pAB (p-aminobenzamidine). In sperm extracts an enzymatic activity was determined, using BAEE (N-benzoil-L- arginine ethyl ester), but not ATEE, as substrate, This activity was inhibited by SBTI and pAB. We observed that in vitro fertilization was inhibited by spermatozoon incubation with the trypsin inhibitors SBTI, PMSF (phenyl-methanesulphonyl fluoride), and pAB. Additionally, we observed that when whole isolated egg-coats were incubated with sperm extract and then analyzed by SDS-PAGE, one band of the egg-coats disappeared, These results have been interpreted as sperm trypsinlike enzyme participation in R. typus sperm passage through the egg-coats. (C) 1997 Wiley-Liss, Inc.