Involvement of GABAA receptor in Bufo arenarum oocyte maturation

Amphibian oocytes meiotic arrest is released under the stimulus of progesterone; this hormone interacts with the oocyte surface and starts a cascade of events leading to the activation of a cytoplasmic maturation promoting factor (MPF) that induces germinal vesicle breakdown (GVBD), chromosome condensation and extrusion of the first polar body.The aim of this work was to determine whether the activation of a GABAA receptor is able to induce GVBD in fully grown denuded oocytes of Bufo arenarum and to analyse its possible participation in progesterone-induced maturation. We also evaluated the role of purines and phospholipids in the maturation process induced by a GABAA receptor agonist such as muscimol.Our results indicated that the activation of the GABAA receptor by muscimol induces maturation in a dose- and time-dependent manner and that this activation is a genuine maturation that enables oocytes to form pronuclei. Assays with a receptor antagonist, picrotoxine, showed that the maturation induced by muscimol was inhibited. Treatment with picrotoxine, however, shows that the participation of GABAA receptor in progesterone-induced maturation is not significant.In addition, our results indicate that high intracellular levels of purines obtained by the use of db-AMPc and theophylline or the inhibition of the phosphatidylinositol 4,5-bisphosphate (PIP2 hydrolysis by neomycin and PIP2 turn over by LiCl, respectively, inhibited the maturation induced by muscimol. Treatment with H-7 indicated, however, that PKC activation is not necessary for GVBD induced by the GABAA receptor agonist. Results suggest that the transduction pathway used by the GABAA receptor to induce maturation is different from those used by progesterone.     

Membrane currents in the oocyte of the toad Bufo arenarum

The amphibian oocyte cell model is widely used for heterologous expression of ionic channels and receptors. Little is known, however, about the physiology of oocyte cell models other than Xenopus laevis. In this study, the two-electrode voltage clamp technique was used to assess the most common electrical patterns of oocytes of the South American toad Bufo arenarum. Basal membrane resistance, resting potential, and ionic currents were determined in this cell model. The oocyte transmembrane resistance was 0.35 M(Omega), and the resting potential in normal saline was about -33 mV with a range between -20 mV and -50 mV. This is, to our knowledge, the first attempt to begin an understanding of the ion transport mechanisms of Bufo arenarum oocytes. This cell model may provide a viable alternative to the expression of ion channels, in particular those endogenously observed in Xenopus laevis oocytes.     

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, IP?Rs 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 IP?Rs 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 IP?Rs 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 IP?Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP?Rs, probably through a CICR mechanism. Both receptors play a role in Ca2+ release mechanisms although their relative contribution to the activation process is unclear.     

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.     

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.     

Modulators of Bufo arenarum ovulation

In this study we investigated ovulation in vitro using ovary samples from Bufo arenarum with respect to their response to stimulation with homologous pituitary homogenate (HPH) or with progesterone and prostaglandins (PGF2alpha and PGE1) as intermediates of pituitary action. Ovary samples were obtained from animals captured during the breeding period. Our results demonstrate that the ovulatory response to all different inducers was dose dependent, the highest percentage of ovulated oocytes being obtained with HPH treatment. An important increase in the ovulatory response was obtained by the association of PGF2alpha with either HPH or progesterone at suboptimal doses, indicating that this prostaglandin induced a synergistic potentiating effect. Incubation with cyclooxygenase inhibitors (indomethacin or diclofenac sodium) produced a significant decrease in the ovulation induced by HPH, demonstrating that prostaglandins are involved in the action of the pituitary gland in this process. According to our results, PGE1 not only had no participation in the ovulatory process, but also produced an inhibitory effect on ovulation induced by HPH treatment.     

Blood lead concentration and delta-aminolevulinic acid dehydratase activity in adult Bufo arenarum

The effects of sublethal doses of lead (as acetate) on blood parameters of adult male Bufo arenarum were studied. Toads received one single injection with 10, 25, 50 or 100 mg/kg of body weight, equivalent to approximately 1/90-1/10 of the 120 h-LD50; seven days after the injections, the hematocrit and the blood delta-aminolevulinic acid dehydratase (ALAD) activity were measured. Hematocrit of lead-injected animals did not exhibit significant changes respective to controls that received sodium acetate (range 29.8-38.8%). Blood lead concentrations were positively and significantly correlated with the injected metal doses. Blood ALAD activity declined proportionately to the doses of the metal as well as to its whole blood concentration. Because of its sensitivity and specificity, it was concluded that the activity of delta-ALAD may be adopted as a reliable biomarker of Bufo arenarum experimental lead intoxication.     

Mitochondrial lipids in Bufo arenarum full-grown oocytes

Both the content and composition of polar and neutral lipids from the mitochondrial fraction of ovarian full-grown Bufo arenarum oocytes were analysed in the present study. Triacylglycerols (TAG) represent 33% of the total lipids, followed by phosphatidylcholine (PC), free fatty acids (FFA) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) or cardiolipin, a specific component of the inner mitochondrial membrane, represents about 4% of the total lipid content. Palmitic (16:0) and arachidonic (20:4n6) acids are the most abundant fatty acids in PC and PE, respectively. DPG is enriched in fatty acids with carbon chain lengths of 18, the principal component being linoleic acid. In phosphatidylinositol (PI), 20:4n6 and stearic acid (18:0) represent about 72 mol% of the total acyl group level. The main fatty acids in TAG are linoleic (18:2), oleic (18:1), and palmitic acids. The fatty acid composition of FFA and diacylglycerols (DAG) is similar, 16:0 being the most abundant acyl group. PE is the most unsaturated lipid and sphingomyelin (SM) has the lowest unsaturation index.     

Nuclear maturation inducers in Bufo arenarum oocytes.

The present study analyses the effect of dihydrotestosterone (DHT) and mammalian insulin on the nuclear maturation of Bufo arenarum oocytes under in vitro conditions. The response of fully grown follicle oocytes to DHT, shown by germinal vesicle breakdown (GVBD), occurred in a manner dependent on dose, time and sexual cycle period. The highest oocyte sensitivity to the hormone appeared during the breeding period, a fact evinced by high GVBD percentages after short incubation periods and at a low hormone concentrations. Insulin also proved effective in inducing nuclear maturation, although its action was only visible at high concentrations and after a long incubation period. The combination of insulin and steroid hormones (DHT or progesterone), both at subliminal doses, caused a noticeable potentiating synergism, resulting in a rapid and important increase in GVBD. Another effect of insulin was the acquisition by oocytes of steroid sensitivity during folliculogenesis.     

Characterization of urea transport in Bufo arenarum oocytes

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water.     

Angiotensin converting enzyme in the toad Bufo arenarum

Angiotensin converting enzyme activity (ACEA) was determined in serum, kidney, whole skin and isolated epithelia homogenates of the South American toad Bufo arenarum. ACEA was present in the tissues and serum of the toad. The activity was higher in the kidney, as compared to that of the whole skin or isolated epithelium. Captopril, teprotide and EDTA, caused a significant decrease in the ACEA. Possible physiological roles for the presence of ACEA in the toad are discussed.     

Chloride Currents in Skeletal Muscles of Bufo arenarum

Cl⁻ currents (I _Cl) were measured in short fibers (1–2 mm) from the lumbricalis muscle of toads (Bufo arenarum) with two microelectrodes (15°C). Initially the fibers were equilibrated in a high K⁺-containing solution: (mm) K₂SO₄ 68; Na₂SO₄ 20; KCl 60; CaSO₄ 8; MgSO₄ 1; HEPES 2.5. Constant pulses were applied when all the external K⁺ was replaced by Cs⁺: Cs₂SO₄ 68; Na₂SO₄ 20; CsCl 60; CaSO₄ 8; HEPES 2.5 (pH 7.5). Under these conditions about 80–90% of the current is carried by Cl⁻. The current-voltage relation is almost linear implying constant conductance and hence voltage-independent permeability. The voltage dependence of the net Cl⁻ current could be fitted by constant field equation with a P _Cl of 3.3 × 10⁻⁶ cm/sec. In a separate group of experiments a two-pulse technique was used to estimate the availability and the inactivation of the initial I _Cl during a test pulse. After returning the potential to the holding potential for various times, test pulses of the same amplitude and duration of the prepulses were applied. The initial current during the test pulse was 70% of the initial current during the prepulse and the recovery was complete in less than 300 msec with a linear relationship between the current during the test pulse and the amplitude of the preceding prepulse. When the test pulses were preceded by a positive prepulse, the initial current for any given test pulse was larger than with a negative prepulse. If we assumed that the initial current during the test pulse is a measure of the number of channels open at the end of the prepulse, these results suggest that hyperpolarizing pulses inactivate and depolarizing prepulses activate the I _Cl. Received: 31 March 1995/Revised: 27 October 1995     

Vitelline envelope of Bufo arenarum: biochemical and biological characterization

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 microg/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.     

Sex steroid binding protein from Bufo arenarum: further characterization

The effects of temperature, pH, divalent cations, 2-mercaptoethanol (Et-SH), N-ethylmaleimide (NEM), and phenylmethylsulfonyl fluoride (PMSF) on the dihydrotestosterone (DHT) binding to sex steroid binding protein from Bufo arenarum (baSBP) were examined. The temperature curve indicated that the binding remained stable up to 50 degrees C and the pH curve showed maximum binding between pH 7 and 9. The incubations of baSBP with divalent cations, NEM and Et-SH demonstrated that baSBP require disulfides and sulfhydryl groups for steroid binding or to maintain an adequate protein conformation. On the other hand, PMSF had no effect on the binding, consequently, serine residues appear not to be involved in DHT binding to baSBP. These results indicate that baSBP has a behavior resembling that of human SBP.