Abstracts

Fertilization

Program/Abstract # 264A deep water sea urchin: Evolution X FertilizationLivia Loiola Dos Santos 1, M.O. Castro 1, C.R. Garcia 1,N. Hirohashi 3, A.C.E.S. Vilela-Silva 1, P.A.S. Mourão 1

1 Laboratório de Tecido Conjuntivo, HUCFF, UFRJ, Brazil2 Instituto de Bioquímica Médica, UFRJ, Brazil3 Ochanomizu University, Japan

The jelly coat (JC) that surrounds sea urchin eggs is mostlycomposed of a sulfated polysaccharide responsible for induc-tion of sperm acrosome reaction, which is an obligatory eventfor successful fertilization. The structure of the sulfated poly-saccharide varies among species, determining the species-specificity in gamete recognition process. The JC also possessespeptides and a sialoglycoprotein, a molecule that enhances thepotency of acrosome reaction. In this work, we have studied theJC composition of Glyptocidaris crenularis, a deep water seaurchin collected in the coast of Japan. This species contains asulfated galactan which is structurally different from the uniqueone described so far, in Echinometra lucunter. We have alreadyshown that each species requires its own sulfated polysacchar-ide structural feature for sperm recognition. From the momentwe have characterized a new sulfated galactan, we could be ableto verify if E. lucunter sperm would have been responsive to anheterologous sulfated galactan. We observed that E. lucuntersperm was not able to recognize G. crenularis galactan, evenboth galactans presenting one similar structural unit. Alsorelevant is the fact that among the many species already studiedin our laboratory, this is the first description of a sea urchin thatdoes not present the sialoglycoprotein. This data jeopardizes therole of this molecule on fertilization process. Besides that, asthis genus is primitive, it may suggest that could be part of theevolutional process the presence of sialoglycoprotein.

doi:10.1016/j.ydbio.2007.03.564

Program/Abstract # 265Structural differences in sulfated polysaccharides:Significance for the fertilization successMichelle O. Castro, L.L. Santos, C.R. Garcia,A.C.E. Vilela-Silva, P.A.S. MouraoLab. Tec. Conjuntivo, IBQM, UFRJ, Brazil

For many years our laboratory has been studying the sulfatedpolysaccharides (SP) present in the egg jelly layer of sea urchinseggs. We have already shown that these SP are the responsiblefor induction of sperm acrosome reaction, a fundamental eventfor sperm-egg binding and fusion. Interestingly, different seaurchin species possess SP with structural distinctions, whichcould be the monosaccharide residue, the glycosidic linkageand/or sulfation pattern. In fact, these singularities are thedeterminant for the species-specificity in gamete recognition. Inthe present work we analyzed what structural feature is res-ponsible for this species-specific identification. We made use ofsperm from two species of sea urchins which were put in contactwith several SP from the egg jelly of many others, whichstructure we have already characterized, and observed by twodifferent techniques: the measure of sperm intracellular calciumconcentration, which rises right after the beginning of acrosomereaction, and the recognition of positive acrosome reactionitself, through the observation of an actin filament that isexposed in sperm during the reaction. We verified that eachspecies has its own structural requirements in order to obtain asuccessful fertilization. It seems that for Echinometra lucunter,the sugar residue of SP is not important for sperm recognition.For Lytechinus variegatus, the sulfation pattern of SP seems tobe decisive for sperm activation.

doi:10.1016/j.ydbio.2007.03.563

Program/Abstract # 266Molecular characterization of a Bufo arenarum oviductalproteaseDaniel Barrera, Ricardo J. Llanos, Pablo A. Valdecantos,Dora C. MiceliInst. Sup. de Invests. Biológs. INSIBIO (CONICET-UNT),Tucumán, Argentina

In amphibians, the envelope surrounding the egg exhibitsdifferences according to the functional state of the femalegamete. The eggs obtained from the coelomic cavity are notfertilized, but they are after their passage through the ParsRecta (PR) portion of oviduct. This acquisition of eggfertilizability is due to an oviduct-induced alteration of eggenvelope. The biochemical and ultrastructural vitelline

Developmental Biology 306 (2007) 387–389www.elsevier.com/locate/ydbio

doi:10.1016/j.ydbio.2007.03.285

envelope modifications are caused by the action of a trypsin-like protease named oviductin. The aim of this work was toclone the oviductin complete cDNA and to analyze theirfunctional domains. Total RNAwas isolated from oviductal PRof hormonally stimulated animal. Sets of primers weredesigned based on homology sequences. We first amplified,cloned and sequenced an internal 530 pb partial cDNA. The 5′cDNA end was amplified using a new group of specificdesigned primers. To complete the mRNA sequence, a 3′ rapidamplification of cDNA ends (3′ RACE) was performed. Theoverlapping sequence showed a 3203-bp-long oviductin cDNAwith one open reading frame coding for a 980 aminoacidsprotein. The molecular structure comprise two proteasedomains (α and β) and three CUB domains. The α domainhas three important aminoacids for catalytic activity (His, Asn,Ser), while in the β domain a His residue was replaced by Asn.Thus, this domain is not likely to be proteolytically active.These results would indicate that the Bufo arenarum oviductinα domain produce the partial hydrolysis of the envelopeglycoproteins. At this moment, the exact function of the CUBdomains and β domain is still unknown.

doi:10.1016/j.ydbio.2007.03.565

Program/Abstract # 267Capacitation-like changes in external fertilization:correlation of physiological modifications with fertilizingcapacity acquisition in Bufo arenarum spermatozoaDarío Krapf 1, Pablo E. Visconti 2, Silvia E. Arranz 1,Marcelo O. Cabada 1

1 Dept. of Dev. Biol., IBR (UNR-CONICET), Argentina2 Dept. of Vet. and Anim. Sc., UMass, USA

During its short life in the female tract, mammalian spermmust accomplish a series of cellular processes named capacita-tion to acquire fertilizing capacity. In animals with externalfertilization as amphibians, gamete interactions are first estab-lished between sperm and molecules of the egg jelly coatreleased into the medium. Since dejellied oocytes are notnormally fertilized, the aim of this study was to determine if thejelly coat of the toad B. arenarum promotes a “capacitating”activity on homologous sperm. We found that incubation ofsperm in diffusible substances of the jelly coat (Egg Water) for90–180 s is sufficient to render sperm transiently capable offertilizing dejellied oocytes. Similar to mammalian sperm, thefertilizing state was correlated with an increase of proteintyrosine phosphorylation and a decrease of the sperm cho-lesterol content. Inhibition of either the increase in tyrosinephosphorylation or cholesterol efflux affected the acquisition offertilizing capacity. Phosphorylation and fertilization could bepromoted with NaHCO3, and also by addition of the cholesterolbinding compound beta cyclodextrin. Moreover, sperm couldgain the ability to fertilize dejellied oocytes in the presence ofthese compounds. These data indicate that B. arenarum spermshould undergo a series of molecular changes to gain fertilizingcapacity; these changes are reminiscent of mammalian sperm

capacitation. Supported by NIH HD38082 and HD44044 (toPEV); ANPCyT (PICT0108545) and CONICET (PIP6428) (toMOC and SEA).

doi:10.1016/j.ydbio.2007.03.566

Program/Abstract # 268Involvement of calmodulin on guinea pig sperm nucleidecondensationArmando Zepeda-Bastida, Adela MújicaDepartment of Cell Biology, CINVESTAV-IPN

Actin was implicated in diverse nuclear activities includingtranscription, chromatin remodeling and nucleocytoplasmictrafficking. Our group, detected actin myosin, spectrin andcytokeratin as guinea pig sperm nuclear matrix components. Aretarding effect of nuclear decondensation, caused by heparin, isproduced by phalloidin and/or diacetyl-monoxime treatment,suggesting a role for F-actin and myosin in the maintenance ofnuclear stability in sperm. We detected calmodulin (CaM),myosin light chain and tubulin in the pure nuclei proteins ofguinea pig sperm. We found actin–myosin interaction in thenuclear matrix. To define if CaM has a function in nuclearstability, we performed experiments of nuclear decondensationby heparin in absence or in CaM antagonists (W5, W7 or cal-midazolium) presence. All three CaM antagonists assayed re-tarded heparin nuclear decondensation; the effect began to beclear and significant (p<0.05) after 2 min heparin treatment. Thehighest decondensation stable values were observed at 4 min oftreatment; nuclear decondensation values were: 9.4 μm ofdiameter and 51.9 μm2 of area, after this time the sperm numberdecline, which might mean nuclei destruction. Furthermore, aninteresting result is, at 10 min of heparin/CaM antagoniststreatment, approximately 80% (W5: 5.3; W7: 5.7 and calmida-zolium: 5.7×106) of de original nuclei number (6.3×106)remained. While in the sperm nuclei sample treated with heparinor heparin/DMSO, original nuclei number was reduced in 80%(1.3×106 and 1.8×106, respectively).

doi:10.1016/j.ydbio.2007.03.567

Program/Abstract # 269Chromatin remodeling in mouse metaphase II oocytesindependently of meiotic exitNaoko Yoshida, Manjula Brahmajosyula, Shisako Shoji,Manami Amanai, Anthony C. PerryLaboratory of Mammalian Molecular Embryology, RIKENCDB, Kobe, Japan

In mammalian fertilization, paternal chromatin becomesexhaustively remodeled, however, the maternal contribution tothis process is still unclear. To address this, we prevented theinduction of meiotic exit by microinjecting inactivated spermheads into metaphase II (mII) oocytes. This permitted us toexamine oocyte- (as opposed to embryo-) mediated sperm

388 ABSTRACTS / Developmental Biology 306 (2007) 387–389