Role of phospholipase A2 pathway in regulating activation of Bufo arenarum oocytes.

Transient increases in the concentration of cytosolic Ca(2+) are essential for triggering egg activation events. Increased Ca(2+) results from its rapid release from intracellular stores, mainly mediated by one or both intracellular calcium channels: the inositol trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). Several regulatory pathways that tailor the response of these channels to the specific cell type have been proposed. Among its many modulatory actions, calcium can serve as an activator of a cytosolic phospholipase A(2) (cPLA2), which releases arachidonic acid from phospholipids of the endoplasmic reticulum as well as from the nuclear envelope. Previous studies have suggested that arachidonic acid and/or its metabolites were able to modulate the activity of several ion channels. Based on these findings, we have studied the participation of the phospholipase A(2) (PLA(2)) pathway in the process of Bufo arenarum oocyte activation and the interrelation between any of its metabolites and the ion channels involved in the calcium release from the intracellular reservoirs at fertilization. We found that addition of both melittin, a potent PLA(2) activator, and arachidonic acid, the main PLA(2) reaction metabolite, was able to induce activation events in a bell-shaped manner. Differential regulation of IP3Rs and RyRs by arachidonic acid and its products could explain melittin and arachidonic acid behaviour in Bufo arenarum egg activation. The concerted action of arachidonic acid and/or its metabolites could provide controlled mobilization of calcium from intracellular reservoirs and useful tools for understanding calcium homeostasis in eggs that express both types of receptors.

Involvement of G protein and purines in Rhinella arenarum oocyte maturation.

We investigated the participation of G(αi) protein and of intracellular cAMP levels on spontaneous and progesterone-mediated maturation in Rhinella arenarum fully grown follicles and denuded oocytes. Although progesterone is the established maturation inducer in amphibians, Rhinella arenarum oocytes obtained during the reproductive period (competent oocytes) resume meiosis with no need for an exogenous hormonal stimulus if deprived of their enveloping follicular cells, a phenomenon called spontaneous maturation. In amphibian oocytes, numerous signalling mechanisms have been involved in the rapid, non-genomic, membrane effects of progesterone, but most of these are not fully understood. The data presented here demonstrate that activation of the G(αi) protein by Mas-7 induced maturation in non-competent oocytes and also an increase in GVBD (germinal vesicle breakdown) in competent oocytes. Similar results were obtained with intact follicles independent of the season. The activation of adenylyl cyclase (AC) by forskolin seems to inhibit both spontaneous and progesterone-induced GVBD. In addition, the high intracellular levels of cAMP caused by activation of AC by forskolin treatment or addition of db-cAMP inhibited maturation that had been induced by Mas-7 and in a dose-dependent manner. Treatment with H-89, a protein kinase A (PKA) inhibitor, was able to trigger GVBD in a dose-dependent manner in non-competent oocytes and increased the percentages of GVBD in oocytes competent to mature spontaneously. The results obtained with whole follicles and denuded oocytes were similar, which suggested that effects on AC and PKA were not mediated by follicle cells. The fact that Mas-7 was able to induce maturation in non-competent oocytes in a similar manner to progesterone and to increase spontaneous maturation suggests that G(αi) activation could be an important step in meiosis resumption. Thus, the decrease in cAMP as a result of the regulation of the G proteins on AC and the inactivation of PKA by H-89 could contribute to the activation of MPF (maturation promoting factor) and induce maturation of the oocytes of Rhinella arenarum.

Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation.

Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP3Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP3Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP3Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP3Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP3Rs, probably through a CICR mechanism. Both receptors play a role in Ca²+ release mechanisms although their relative contribution to the activation process is unclear.

Activation of amphibian oocytes by sperm extracts.

In the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.

Behaviour of the vitelline envelope in Bufo arenarum oocytes matured in vitro in blockade to polyspermy.

During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.

Osmetrichia in the grey brocket deer (Mazama gouazoubira)

Osmetrichia have been defined as hairs specialized in the storage of secretions used in olfactory communication between conspecifics (Müller-Schwarze, et al. 1977). These authors found highly specialized osmetrichia in the tarsal gland tufts of black-tailed but not white-tailed deer. Chemical communication appears to be well developed in grey brocket deer: the bucks scent mark by rubbing their foreheads on bushes, and all deer urinate and defecate almost exclusively on dung heaps. Brocket deer also possess tarsal tufts. The purpose of this study was to examine hairs from several glandular areas in this species. Osmetrichia, similar to those found in black tailed deer, were found in tarsal tufts and in interdigital gland hairs; these hairs possessed open scales with deep pockets suitable for holding secretions, in comparison to the flat scales seen on control hairs. Hairs with different morphological characteristics (slightly open scales) were found over the frontal gland. Specialized hairs were not found in the tarsal tufts of one specimen of a related species, the red brocket deer (Mazama americana). The similarities in the hairs of grey brocket and black-tailed deer are remarkable in light of the ecological and behavioral differences between these two species

Osmetrichia in the grey brocket deer (Mazama gouazoubira)

Osmetrichia have been defined as hairs specialized in the storage of secretions used in olfactory communication between conspecifics (M³ller-Schwarze, et al. 1977). These authors found highly specialized osmetrichia in the tarsal gland tufts of black-tailed but not white-tailed deer. Chemical communication appears to be well developed in grey brocket deer: the bucks scent mark by rubbing their foreheads on bushes, and all deer urinate and defecate almost exclusively on dung heaps. Brocket deer also possess tarsal tufts. The purpose of this study was to examine hairs from several glandular areas in this species. Osmetrichia, similar to those found in black tailed deer, were found in tarsal tufts and in interdigital gland hairs; these hairs possessed open scales with deep pockets suitable for holding secretions, in comparison to the flat scales seen on control hairs. Hairs with different morphological characteristics (slightly open scales) were found over the frontal gland. Specialized hairs were not found in the tarsal tufts of one specimen of a related species, the red brocket deer (Mazama americana). The similarities in the hairs of grey brocket and black-tailed deer are remarkable in light of the ecological and behavioral differences between these two species

"Osmetrichia" in the grey brocket deer (Mazama gouazoubira).

Osmetrichia have been defined as hairs specialized in the storage of secretions used in olfactory communication between conspecifics (Müller-Schwarze, et al. 1977). These authors found highly specialized osmetrichia in the tarsal gland tufts of black-tailed but not white-tailed deer. Chemical communication appears to be well developed in grey brocket deer: the bucks scent mark by rubbing their foreheads on bushes, and all deer urinate and defecate almost exclusively on dung heaps. Brocket deer also possess tarsal tufts. The purpose of this study was to examine hairs from several glandular areas in this species. Osmetrichia, similar to those found in black tailed deer, were found in tarsal tufts and in interdigital gland hairs; these hairs possessed open scales with deep pockets suitable for holding secretions, in comparison to the flat scales seen on control hairs. Hairs with different morphological characteristics (slightly open scales) were found over the frontal gland. Specialized hairs were not found in the tarsal tufts of one specimen of a related species, the red brocket deer (Mazama americana). The similarities in the hairs of grey brocket and black-tailed deer are remarkable in light of the ecological and behavioral differences between these two species.

[quot ]Osmetrichia[quot ] in the grey brocket deer (Mazama gouazoubira).

Osmetrichia have been defined as hairs specialized in the storage of secretions used in olfactory communication between conspecifics (M³ller-Schwarze, et al. 1977). These authors found highly specialized osmetrichia in the tarsal gland tufts of black-tailed but not white-tailed deer. Chemical communication appears to be well developed in grey brocket deer: the bucks scent mark by rubbing their foreheads on bushes, and all deer urinate and defecate almost exclusively on dung heaps. Brocket deer also possess tarsal tufts. The purpose of this study was to examine hairs from several glandular areas in this species. Osmetrichia, similar to those found in black tailed deer, were found in tarsal tufts and in interdigital gland hairs; these hairs possessed open scales with deep pockets suitable for holding secretions, in comparison to the flat scales seen on control hairs. Hairs with different morphological characteristics (slightly open scales) were found over the frontal gland. Specialized hairs were not found in the tarsal tufts of one specimen of a related species, the red brocket deer (Mazama americana). The similarities in the hairs of grey brocket and black-tailed deer are remarkable in light of the ecological and behavioral differences between these two species.