RESUMEN

Seasonal variations in the morphology of the parenchymal mass and function of the albumen gland/capsule gland complex have been studied in Pomacea canaliculata, together with the cellular types involved in the synthesis and secretion of perivitellin fluid components. The two major parenchymal cell types, albumen secretory cells (AS) and labyrinthic cells (LC), undergo seasonal variations throughout the annual reproductive cycle, which is divided into three periods. Both cellular types show maximal development and structural complexity during the reproductive period (spring and summer). AS cells have a well-developed Golgi complex and rough endoplasmic reticulum and their secretory granules show electron-dense particles of about 20 nm (probably galactogen). These cells are uniquely involved in ovorubin and PV2 perivitellin synthesis and their secretory granules are the single storage site for these two major perivitellins, as revealed by immunoelectron microscopy. AS also possess calcium deposits that infiltrate the cytoplasmic matrix. The luminal surfaces of LC exhibit long cilia intermingled with sparce short microvilli. Basally, the plasma membrane shows deep irregular folds that extend through the cytoplasm up to the subapical region. Calcium deposits infiltrate the cytoplasm and accumulate in the extracellular space of the basal labyrinth. Nerve terminals seem to be involved in the regulation of parenchymal cell secretion. At the post-reproductive period, AS markedly change their aspect following the release of most of the secretory granules into the acinar lumen. LC decrease in volume, the number of their cilia decreases, their cytoplasmic folds are much thinner and their extracellular spaces lack calcium particles. At the pre-reproductive period (winter), AS and LC recover and prepare for the subsequent period.

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A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization

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RESUMEN

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization

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RESUMEN

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization.

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Serum steroid binding properties of mature Bufo arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo arenarum sex binding protein (Ba SBP), which binds 5 alpha-dihydrotestosterone (DHT), testosterone (T), and estradiol-17 beta (E2) with high affinity (10(-7) M-1 - 10(8) M-1) and fair capacity (10(-6) M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady-state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E2. Estriol and estrone competed poorly, while diethylstilbestrol and C21 steroids did not compete. The binding capacity of this protein is under estrogenic control.

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1. Proteins secreted by toad oviductal pars recta are involved in fertilization and their biological activity is regulated by estrogens. 2. Effect of 17 beta-estradiol on protein synthesis was examined on castrated animals by different in vivo and in vitro experimental approaches. 3. Different routes of L-[3H]leucine administration were assessed. An intralymphatic route was the most efficient for incorporating radioactivity per mg of trichloroacetic acid insoluble proteins. 4. Ligature of pars recta induces protein synthesis at a similar level to exogenous estradiol. 5. Electrophoretic pattern of radioactive proteins did not show synthesis of a specific protein related to the zone with biological activity. 6. Pars recta releases newly synthesized proteins in vivo into its fluid secretion as much as in vitro into culture medium.

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