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.     

Lipids during Bufo arenarum oogenesis

The content and composition of phospholipids and triacylglycerols (TAGs) in Bufo arenarum oocytes in stages III and IV of their oogenesis were studied. The total amount of phospholipids in stage IV oocytes is 0.5-fold higher than in stage III oocytes. In both cases, the main phospholipids are phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A striking observation concerns the high level of diphosphatidylglycerol (DPG) in stage III oocytes, which could be indicative of a relatively larger mitochondrial population with respect to other oogenetic stages. A net increase in sphingomyelin content was found during oogenesis. This fact could be related to the role of this phospholipid in the signal transductional pathways. In PC, palmitic (16:0), linoleic (18:2) and oleic (18:1) are the major fatty acids for both types of oocytes, while in PE the main acyl groups are 18:1, 16:0, arachidonic acid (20:4n6) and 18:2. PE is more unsaturated than PC and both phospholipids are more unsaturated in stage III oocytes than in stage IV oocytes. The amount of triacylglycerols is 0.3-fold higher in stage IV oocytes than in stage III oocytes. In both stages, the main fatty acids are 18:2, 18:1 and 16:0. During oogenesis, a significant increase in 18:1 and 18:3n3, and a decrease in 18:2 of TAG were found. The unsaturation index of TAGs from stage IV oocytes is higher than that from stage III oocytes. The TAG increase during oogenesis is consistent with the putative use of these lipids as a source of energy in embryo development.     

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.     

Ultrastructural changes during nuclear maturation in Bufo arenarum oocytes.

During progesterone-induced nuclear maturation the oocytes of Bufo arenarum undergo a series of nuclear and cytoplasmic changes. The breakdown of heterocellular communications between the follicular cell projections and the oocyte microvilli, and the consequent enlargement of the perivitelline space, were observed at the animal pole. The more evident cytoplasmic feature during nuclear maturation comprised the gathering of glycogen granules in clusters, some phagocytosed by empty vesicles. With respect to the location of these vesicles, some were observed in close proximity to the oolemma and others were freely suspended in the perivitelline space, extruded from the oocyte. Other visible events were the disruption of the annulate lamellae, the formation of an elaborate cortical endoplasmic reticulum and the rearrangement of the cortical granules in a monolayer immediately beneath the oolemma together with aggregates of endoplasmic reticulum cisternae. Our results show that during nuclear maturation the nuclear oocyte changes include a flattening of the spherical oocyte nucleus, its migration towards the surface of the animal pole, the disappearance of the nucleoli and the dissolution of the nuclear envelope.     

Immunoidentification of CRF-like material in the intermaxillary glands during Bufo arenarum development

The presence of corticotropin-releasing factor-like material in the intermaxillary glands was studied by immunocytochemical techniques during the metamorphosis of Bufo arenarum. The intermaxillary glands appeared at stage XV (midprometamorphosis) with CRF-like material slightly immunoreactive. These glands are located posterior to the premaxillae and between the nasal capsules in the roof of the mouth and are formed of alveoli or tubules. During metamorphic climax, corticotropin-releasing factor-like material was identified strongly immunostained at the apices of the secretory cells. It was observed that collecting ducts of the gland open to the anterior palatal surface suggesting that the secretion could be ingested by tadpoles. Our results clearly showed that ir-CRF-like material present in the intermaxillary glands is ingested by tadpoles during metamorphosis and could play an important role during amphibian development.     

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.     

Adenylic nucleotides and energy charge during the embryonic development of Bufo arenarum

The contents of ATP, ADP and AMP were determined by HPLC and adenylic energy charge (AEC) was estimated during different stages of the embryonic development of Bufo arenarum up to the tailbud stage. All the developmental stages studied showed a high ATP content (about 1.04-1.48 nmol/emb.). The concentration of ADP was low (0.025-0.041 nmol/emb.) but rose slightly at the neural tube stage. AMP was undetectable before the tailbud stage. AEC values were almost constant (about 0.987-0.992) throughout the period studied. Only a fall at the tailbud stage could be detected which can be related to this more advanced cellular differentiation stage.     

The absence of DNA from the blood serum of Bufo arenarum

The absence of DNA from the blood serum of the toad Bufo arenarum was confirmed by classical colorimetric techniques and by a new method which allows differentiation between DNA and the glycosaminoglycans (GAG). A component of serum which had some of the properties of DNA was identified as a GAG. This component gave positive reactions in colorimetric assays for DNA, but the absorption spectra were completely different from those of authentic DNA. The compound was also not hydrolyzed at all by treatment with deoxyribonuclease.     

Effect of meiotic maturation on yolk platelet lipids from Bufo arenarum oocytes

Progesterone induces the resumption of meiosis in Bufo arenarum full-grown arrested oocytes through a nongenomic mechanism called meiotic maturation. Growing evidence indicates that lipids are involved in the maturation process. They are mainly located in yolk platelets, the principal organelles of amphibian oocytes. The aim of the present study was to analyze the effect of progesterone-induced maturation on lipids from B. arenarum yolk platelets. Ovarian oocytes, manually obtained, were incubated with progesterone to induce maturation. Yolk platelets were isolated by centrifugation at low velocity. Lipids were separated by thin-layer chromatography. For compositional analysis, they were derivatized by methanolysis, and were identified and quantified in a gas-liquid chromatograph. Phospholipid content decreased in progesterone-treated oocytes, mainly as a result of a decrease at the level of phosphatidylcholine (PC). The turnover of this lipid is considered crucial for the completion of meiosis. Sphingomyelin also underwent a decrease that could be related to the important role of ceramide as an inducer of germinal vesicle breakdown. Maturation effect on fatty acid composition registered significant changes in PC whose saturated fatty acids increased. A net increase in arachidonic acid was observed in phosphatidylserine after progesterone treatment. The contents of total triacylglycerols and diacylglycerols were not significantly modified by hormone effect while free fatty acids underwent a significant increase as a result of polyunsaturated fatty acids increase. Altogether, our results demonstrate that yolk platelet lipids are involved in the resumption of the meiotic cell cycle, thus suggesting that these organelles participate in a dynamic role during amphibian development.     

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     

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.     

Purification and biological characterization of N-acetyl beta-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-beta-D-glucosaminidase, beta-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, and alpha-D-glucosidase activities are measured in spermatozoa. N-acetyl-beta-D-glucosaminidase is the major sperm glycosidase activity assayed. However, N-acetyl-beta-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-beta-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-beta-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-beta-D-glucosaminidase plays an important role in toad fertilization.     

The Differentiation of the Vitelline Envelope of Xenopus Oocytes*

We have studied the differentiation of the vitelline envelope (V.E.) of the oocyte of the anuran Xenopus laevis. The V.E. precursor material is synthesized by the oocyte concomitantly with the onset of vitellogenesis, and its extrusion reaches a maximum at late vitellogenesis. Oocytes at different stages of growth were incubated in L-[³H]fucose and the progress of incorporation was followed by kinetic and histoautoradiographic analysis. We found that the highest overall rate of incorporation was exhibited by the vitellogenic oocytes. These oocytes showed clusters of grains in the perinuclear and in the cortical areas. The highest accumulation of grains in the V.E. was associated with the late vitellogenic stage, when the differentiation of the V.E. was almost complete. L-[³H]Fucose labelled glycoproteins have been identified by electrophoretic analysis of V.E. prepared from late vitellogenic stages.     

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.     

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.