Marcelo O. Cabada

ACROSOME BREAKDOWN IN LEPTODACTYLUS CHAQUENSIS (AMPHIBIA ANURA) SPERMATOZOA

Acrosome breakdown in Leptodactylus chaquensis is described: during this process acrosome enlarges, becomes round‐shaped and finally disrupts. Low tonicity media (0.025 M sucrose and 1/10 Holtfreters solutions) favor acrosome breakdown and sperm fertility loosing. High tonicity media (0.250 M sucrose and Holtfreters solutions) maintain acrosomes in an unreacted stage and sperm fertilizing capacity is preserved. Sperm motility does not seem to be a sufficient condition for the sperm to fertilize and also does not seem to be related with acrosome breakdown. The presence of lectins in the incubation media does not modify the time‐course of acrosome breakdown.

Purification and biological characterization of N‐acetyl β‐D glucosaminidase from Bufo arenarum spermatozoa

Fertilization in Bufo arenarum requires the sperm to penetrate the egg envelopes. The incubation of isolated vitelline envelopes with sperm induces the acrosome reaction, releasing proteases and glycosidases to the media. In the present work N‐acetyl‐β‐D‐glucosaminidase, β‐D‐galactosidase, β‐D‐glucosidase, α‐D‐mannosidase, α‐L‐fucosidase, and α‐D‐glucosidase activities are measured in spermatozoa. N‐acetyl‐β‐D‐glucosaminidase is the major sperm glycosidase activity assayed. However, N‐acetyl‐β‐D‐galactosamine show competitive inhibitory effect. The glycosidase pH optimum is 3.5 being inhibited at pHs higher than 7.5. In our study, N‐acetyl‐β‐D‐glucosaminidase is the only glycosidase that in vitro binds to vitelline envelopes in conditions that resemble natural fertilization media. The isolation of the active enzyme will allow studies of its role in fertilization. The enzyme has been purified in a two‐step procedure. After native gel electrophoresis, the activity‐stained band was cut out and the eluted enzyme was finally subjected to ConA–sepharose chromatography. In SDS‐PAGE, the denatured enzyme migrates as a single band with a molecular mass of 45 kDa. Furthermore, analysis by size‐exclusion on HPLC showed a peak of activity at around 45 kDa. Preliminary localization studies showed higher relative activity in the acrosomal content. In addition, 10% of the N‐acetyl‐β‐D‐glucosaminidase activity was associated with the reacted sperm. By in vitro fertilization assay, it was observed that the inhibition of the enzyme results in the inhibition of fertilization. This last study shows that N‐acetyl‐β‐D‐glucosaminidase plays an important role in toad fertilization. Mol. Reprod. Dev. 57:194–203, 2000.

Transmembrane adenylyl cyclase regulates amphibian sperm motility through protein kinase A activation

Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation.

Sperm binding glycoprotein (SBG) produces calcium and bicarbonate dependent alteration of acrosome morphology and protein tyrosine phosphorylation on boar sperm

The oviduct is a dynamic organ which modulates gamete physiology. Two subpopulations of sperm have been described in the oviduct of sows, a majority with normal appearance in the deep furrows and a minority, centrally located, and showing damaged membranes. Sperm–oviduct interaction provides the formation of a sperm storage and allows the selection of sperm with certain qualities. Pig (Sus scrofa) oviductal sperm binding glycoprotein (SBG) binds to sperm and exposes Galβ1‐3GalNAc. This disaccharide may be recognized by boar spermadhesin AQN1, which seems to be involved in sperm interaction with the oviduct. SBG is present at the apical surface of the epithelial cells that surround the lumen of the oviduct rather than at the bottom of the crypts. These characteristics imply it could be involved in sperm interaction with this organ. In this study, we evaluate the effect of SBG over boar sperm. We show that the presence of SBG produces alterations of the acrosome morphology of sperm only when they are incubated in capacitating conditions. SBG binds to the periacrosomal region of sperm undergoing capacitation. Its presence induces an increase on the tyrosine‐phosphorylation of a polypeptide of apparent molecular mass 97 kDa, as occurs with a 95 kDa protein in other mammalian sperm upon acrosomic reaction. Altogether, these results suggest that SBG might be involved in sperm selection by alteration of the acrosome of sperm that have already begun the capacitation process when they arrive to the oviduct. J. Cell. Biochem. 103: 1413–1423, 2008.

Primary structure and developmental expression of Bufo arenarum cellular nucleic acid‐binding protein: Changes in subcellular localization during early embryogenesis

A Bufo arenarum cellular nucleic acid‐binding protein (bCNBP) full‐length cDNA was cloned. bCNBP is a 19.4 kDa protein containing seven CCHC zinc finger motifs, an RGG box and a Ser‐rich region. Amino acid comparisons showed high values of homology in vertebrates and smaller values in insects or inferior eukaryotes. Northern blot analysis during oogenesis and early development revealed two transcripts with different expressions of pattern behavior. One of them is present in all stages analyzed, whereas the other is only detected from the beginning of zygotic transcription. Immunocytochemistry assays carried out on sections of ovary and early embryos showed that there was no specific staining of previtellogenic oocytes. In early vitellogenic oocytes, in oocytes at stages V/VI and in embryos at early blastula stage, reaction was observed inside the cytoplasm. At mid‐blastula stage, CNBP was mainly detected in the epiblast. At the late gastrula stage, two layers of cells were stained in the archenteron roof, in which the internal one presented as strong staining. Nuclei in this layer were stained even stronger than the cytoplasm. Changes in mRNA expression patterns, accompanied by changes in subcellular localization, suggest that CNBP might interact with both nuclear and cytoplasmic nucleic acids.

Glycoproteins from Bufo arenarum vitelline envelope with fertility-impairing effect on homologous spermatozoa

When spermatozoa from Bufo arenarum are incubated with molecules extracted from the vitelline envelopes of homologous oocytes, they lose their fertilizing capacity. Those molecules are glycoproteins, and the elimination of mannoside residues from them results in activity loss, while digestion of the proteic moiety did not alter their biological effect. Sepharose-concanavalin A columns were used to purify the glycoproteins, since the active fraction binds to the column. The fertility-impairing effect observed does not seem to be mediated by an acrosome reaction-inducing effect.

Sperm binding glycoprotein is differentially present surrounding the lumen of isthmus and ampulla of the pig's oviduct

In several mammals a sperm reservoir is formed at the isthmus of the Fallopian tube, providing viable, potentially fertile sperm for an extensive period. In pig (Sus scrofa) the spermadhesin AQN-1 seems to be involved in the establishment of the sperm reservoir. The pig oviductal protein, sperm binding glycoprotein (SBG), binds to sperm and exposes carbohydrate groups that can be recognized by AQN-1. In this study we obtain anti-SBG polyclonal antibodies and use them to localize SBG in the oviduct. Immunohistochemical analysis shows that SBG is present at the apical surface of isthmic and ampullar epithelial cells. The presence of SBG is limited to the upper two-thirds of the crypts of the isthmus and to cells located near the oviductal lumen in the ampulla. The ratio of the amount of SBG detected by western blot is 1:3 (ampulla:isthmus). Sperm entering the Fallopian tube probably contact the epithelial cells at the lumen before they reach the cells at the bottom of the folds. In vitro sperm can bind to isthmus and, at less extent, to ampulla. Thus, the localization and the relative amount of SBG in the isthmus and ampulla of pig’s oviduct are compatible with its possible function in sperm binding to oviductal epithelial cells.

Vitelline Envelope of Bufo arenarum: Biochemical and Biological Characterization

Abstract Vitelline envelopes (VEs) of Bufo arenarum were isolated in order to study their composition and their role in fertilization. VEs are composed of four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa. To characterize its biological properties, we quantitatively determined sperm-VE binding and the induction of the acrosome reaction. Heterologous binding of B. arenarum sperm to Xenopus laevis VE components was observed with about one-third the efficiency of homologous binding. Equivalent binding of X. laevis sperm to the B. arenarum VE was observed. When B. arenarum sperm were incubated with fluorescein isothiocyanate-labeled VE, the labeled glycoproteins bound to the anterior end of the sperm head, showing a lateral distribution. Induction of the acrosome reaction was evaluated by incubating sperm in hypotonic saline media with VE glycoproteins. VEs induced the acrosome reaction in a time- and concentration-dependent manner. The acrosome reaction was maximal after 10 min. The half-maximal effect was obtained at a glycoprotein concentration of 1 μg/ml. Specificity was determined using fertilization envelope glycoproteins, which failed to induce the acrosome reaction. The B. arenarum VE is biochemically similar to other egg envelopes. It also seems that its biological properties are similar to other species in regard to sperm binding and induction of the acrosome reaction. However, as far as we are aware, this is the first observation of the VE inducing the sperm acrosome reaction in amphibians. The relatively small differences observed in heterologous sperm-VE binding in X. laevis and B. arenarum are inconsistent with the current paradigm that species specificity in fertilization is regulated at the sperm-VE binding step.

Diffusible highly glycosylated protein from Bufo arenarum egg-jelly coat: Biological activity

L‐HGP is a highly glycosylated protein from Bufo arenarum egg‐jelly coat that diffuses into the surrounding medium when the strings of oocytes are incubated in saline solutions. L‐HGP was purified from egg water and the estimated percentage of L‐HGP/total protein in egg water was estimated in 30%. In the present study we examine, by indirect immunofluorescence, the effect of L‐HGP on acrosome status of homologous spermatozoa. A high percentage (77%) of sperm lost the acrosome when incubated in 10% Ringer solution buffered with 10 mM Tris‐HCl, pH 7.6, during 60 min, a condition that resembles egg‐jelly osmolarity. The addition of purified L‐HGP to the incubation medium prevents acrosome breakdown. The acrosome integrity is maintained for at least 1 hr. This effect is specific for L‐HGP at concentration ranging from 0.01 to 0.1 mg/ml since neither BSA nor fetuin seems to have similar activity at similar concentrations. The same effect was observed when spermatozoa were incubated in egg water. Preliminary results suggest that L‐HGP binds to B. arenarum spermatozoan membranes. Mol. Reprod. Dev. 56:392–400, 2000.

Effect of heparin on in vitro capacitation of boar sperm

Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 microM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 microg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.