Oviductal protease and trypsin treatment enhance sperm-envelope interaction in Bufo arenarum coelomic eggs

We describe the morphological and biochemical changes in Bufo arenarum coelomic egg envelopes (CE) following passage through the oviduct. In this species, the transformation of the CE into the vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein component. Electrophoretic patterns indicate that a pars recta oviductal protease selectively hydrolyzes in vitro the 84 and the 55 kDa glycoproteins of the CE. During the CE to VE transformation, the relative concentrations of gp48, 42 and 39 kDa also change. In in vitro tests, sperm binding to envelope glycoprotein occurs when they are exposed to VE but not when treated with CE, and VE labeled glycoproteins bind to the head and mid piece of the sperm. The gp39 VE component has 100% identity with internal domains of the sequence deduced from ovarian cDNA for the homologous zona pellucida glycoprotein type C (ZPC) protein precursor in B. arenarum. The effects of trypsin as a substitute for oviductal protease were also examined. Trypsin selectively attacks the 84 and the 55 kDa glycoproteins without hydrolyzing other components and renders coelomic eggs fertilizable in a jelly water preparation. Therefore, trypsin can mimic in vitro the biological action of the oviductal protease. However, it does not wholly mimic the biological action of the oviduct which, in B. arenarum at least, exceeds a mere proteolytic effect. This fact was verified by the lower fertility rates and the abnormal embryo development found when trypsin-treated coelomic eggs were fertilized in vitro.     

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.     

An oviducal enzyme isolated by affinity chromatography which acts upon the vitelline envelope of Bufo arenarum coelomic oocytes.

A trypsin-like oviducal proteinase acting upon the vitelline envelope of Bufo arenarum coelomic oocytes has been purified to apparent homogeneity by gel filtration on Sephadex G-200 and by affinity chromatography on a column of Sepharose 4-B containing covalently bound concanavlin A (Con A). The biologically active molecule migrated as a single band of protein upon SDS polyacrylamide gel electrophoresis.     

Effect of dehydroleucodine on meiosis reinitiation in Bufo arenarum denuded oocytes

In amphibian oocytes meiosis, the transition from G2 to M phase is regulated by the maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34/cdc2 and cyclin B. In immature oocytes there is an inactive complex (pre-MPF), in which cdc2 is phosphorylated on both Thr-161 and Thr-14/Tyr-15 residues. The dephosphorylation of Thr-14/Tyr-15 is necessary for the start of MPF activation and it is induced by the activation of cdc25 phosphatase. Late, to complete the activation, a small amount of active MPF induces an auto-amplification loop of MPF stimulation (MPF amplification). Dehydroleucodine (DhL) is a sesquiterpenic lactone that inhibits mammalian cell proliferation in G2. We asked whether DhL interferes with MPF activation. For this question, the effect of DhL (up to 30 microM) on the resumption of meiosis was evaluated, and visualized by germinal vesicle break down (GVBD), of Bufo arenarum oocytes induced in vitro by either: (i) removing follicle cells; (ii) progesterone stimulation; (iii) VG-content injection; or (iv) injection of mature cytoplasm. The results show that DhL induced GVBD inhibition, in a dose-dependent manner, in spontaneous and progesterone-induced oocyte maturation. Nevertheless, DhL at the doses assayed had no effect on GVBD induced by mature cytoplasm injection, but exerted an inhibitory effect on GVBD induced by GV content. On the basis of these results, we interpreted that DhL does not inhibit MPF amplification and that the target of DhL is any event in the early stages of the cdc25 activation cascade.     

Effect of follicle-stimulating hormone on metabolism and maturation in Bufo arenarum oocytes

In the fully grown Bufo arenarum oocyte, carbohydrate breakdown during the autumn-winter season is accomplished mainly through the glycolytic pathway followed by the krebs cycle. During the breeding season (spring-summer), carbohydrates are used mainly through the pentose phosphate cycle and through the variant of the Krebs cycle known as the glutamic aspartic cycle. The metabolism operating in the oocytes was determined using the following paramenters: 1) the capacity of isolated mitochondria to oxidize citrate and fumarate; 2) the enzymatic activities of phosphofructokinase (PEK) and glucose-6-phosphate dehydrogenase (G-6-PDH); and 3) cirate and ATP compartmentalization. The present paper shows that follicle-stimulating hormone (FSH) would be one of the factors responsible for summer metabolism, since ovary fractios obtained from winter specimens treated with the hormone acquired the metabolic characteristics corresponding to oocytes obtained from breeding-season animals from dose-response, and response in the function of time curves, it could be assumed that the optimum doses and times were 0.1 μg FSH/ml of incubation medium and 30 min treatment, respecitively. The metabolic effect of FSH upon oocytes could be mediaated by the adenylate cyclase cAMP system, since treatment of ovric fractions with cAMP 10-³ M reproduced the effects obtained with the hormone. In addition, 0.02 mg/ml tetracyline proved to block the effect of FSH. A direct metabolic action of FSH on body cavity oocytes (without follicle cells) was observed when submitting these oocytes to the same hormonal treatment.     

Effect of insulin on spontaneous and progesterone-induced GVBD on Bufo arenarum denuded oocytes

Progesterone is considered as the physiological steroid hormone that triggers meiosis reinitiation in amphibian oocytes. Nevertheless, isolated oocytes can be induced to undergo germinal vesicle breakdown (GVBD) in a saline medium by means of treatment with various hormones or inducing agents such as other steroid hormones, insulin or an insulin-like growth factor. It has been demonstrated that Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. This study was undertaken to evaluate the participation of the purine and phosphoinositide pathway in the insulin-induced maturation of oocytes competent and incompetent to mature spontaneously, as well as to determine whether the activation of the maturation promoting factor (MPF) involved the activation of cdc25 phosphatase in Bufo arenarum denuded oocytes. Our results indicate that insulin was able to induce GBVD in oocytes incompetent to mature spontaneously and to enhance spontaneous and progesterone-induced maturation. In addition, high intracellular levels of purines such as cAMP or guanosine can reversibly inhibit the progesterone and insulin-induced maturation process in Bufo arenarum as well as spontaneous maturation. Assays of the inhibition of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis and its turnover by neomycin and lithium chloride respectively exhibited a different response in insulin- or progesterone-treated oocytes, suggesting that phosphoinositide turnover or hydrolysis of PIP2 is involved in progesterone- but not in insulin-induced maturation. In addition, the inhibitory effect of vanadate suggests that an inactive pre-maturation promoting factor (pre-MPF), activated by dephosphorylation of Thr-14 and Tyr-15 on p34cdc2, is present in Bufo arenarum full-grown oocytes; this step would be common to both spontaneous and hormone-induced maturation. The data presented here strongly suggest that insulin initiates at the cell surface a chain of events leading to GVBD. However, our studies point to the existence of certain differences between the steroid and the peptide hormone pathways, although both involve the decrease in intracellular levels of cAMP, the activation of phosphodiesterase (PDE) and the activation of pre-MPF.     

Lectin binding sites in the vitelline envelope of Bufo arenarum oocytes: Role in fertilization

Incubation of dejellied spawned oocytes from Bufo arenarum with different lectins results in a decrease of oocyte fertility. Concanavalin A was the most effective lectin; phytohem-agglutinin P and wheat germ lectin were less effective. Agglutinin from soybean was scarcely active. These lectin effects could be ascribed to a hindering of specific sites for some proteases, since the same treatment renders the oocyte vitelline envelope insensitive to spermatolysin (an essential requisite for fertilization) and to trypsin. Also in this case concanavalin A was the most effective lectin. Univalent concanavalin A was also effective in blocking the fertility of dejellied oocytes. These results indicate that the residues of α-D-glucose and α-D-mannose present in the vitelline envelope are involved in gamete interactions in Bufo arenarum. This idea is also supported by the finding that dejellied oocytes (fertilizables) have a number of binding sites for concanavalin A that is three or four orders of magnitude higher than coelomic or fertilized oocytes (both not penetrable by spermatozoa).     

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.     

Effect of the isolated germinal vesicle of Bufo arenarum oocytes upon spermatozoa

Bufo arenarum sperm treated with isolated germinal vesicle stopped motility in a few minutes and lost fertilization capacity. Treated with egg water, it recovered motility and was able to fertilize.     

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.     

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.     

Nucleotides and sugar nucleotides in early development of Bufo arenarum. Coelomic oocytes.

Nucleotides and sugar nucleotides from coelomic oocytes of Bufo arenarum were extracted with trichloroacetic acid and analyzed by ion-exchange chromatography. The hypoxanthine and guanine were sequencially eluted from the column with water. Nucleotides and sugar nucleotides were eluted with a linear gradient of ammonium chloride. The first peak of ultraviolet adsorption eluted from the resin was a complex mixture of at least three substances. The main component was identified as cytidine diphosphocholine by chemical, enzymatic, and chromatographic analyses. Preliminary experiments suggest a possible role for this compound during oogenesis, since immature oocytes incubated in vitro with [¹⁴C]choline showed an active metabolism of this substance with rapid incorporation in choline phosphate, cytidine diphosphocholine, and lecithin.     

Absence of DNA in yolk platelets of Bufo arenarum oocytes

The absence of DNA in the yolk platelets of Bufo arenarum oocytes was verified. Oocytes from the coelomic cavity were used in order to avoid possible contamination by the ovarian tissue. Four methods for DNA analysis were employed, and the possibility of interference was carefully checked. The presence of three possible contaminants, which produced positive reactions from the DNA analysis, was detected. These were the glycosaminoglycan from the superficial layer of the platelets, sucrose, and polyvinylpyrrolidone from the buffer commonly used during the isolation of platelets. The present evidence points to the necessity of using at least two different methods for DNA determination with due regard for their sensitivity and specificity before any firm conclusion can be reached on the presence or absence of DNA in nonnuclear and mitochondrial parts of the oocyte.     

Effects of ionomycin and thapsigargin on ion currents in oocytes of Bufo arenarum

In this study, two electrode voltage clamp technique was used to assess the ionic current of oocytes of the South American toad Bufo arenarum and to study the dependence of these currents on the extracellular and intracellular Ca2+ concentrations. Ca2+ chelators, ionomycin -a calcium ionophore- and thapsigargin, a blocker of the Ca2+ pump of the sarcoplasmic reticulum, were used. The main results were the following: Most oocytes showed a voltage activated rectifying conductance. Ionomycin (1 microM) increased inward and outward currents in control solution. The effect of ionomycin was blocked partially at negative potentials and was blocked completely at positive potentials in absence of extracellular Ca2+. When the oocytes were treated with thapsigargin (2 microM) or BAPTA-am, a membrane-permeant intracellular chelator in control solution (10 microM), ionomycin did not increased either inward nor outward currents. The conclusion of our experiments is that there are two sources of Ca2+ for activation of the current induced by ionomycin, the cytoplasmic stores and the extracellular space. We believe ionomycin directly translocates Ca2+ from the SER into the cytoplasm but not from the extracellular medium. Ca2+ entry probably occurs through store-operated-Ca-channels.     

The role of calcium in the nuclear maturation of Bufo arenarum oocytes

In Bufo arenarum, progesterone is the physiological maturation inducer. However, in this species, oocytes reinitiate meiosis with no need of an exogenous hormonal stimulus when deprived of their enveloping cell, a phenomenon called spontaneous maturation. We demonstrated that in Bufo arenarum spontaneous maturation occurs only in oocytes obtained during the reproductive period, which can be considered competent to mature spontaneously, in contrast to those in the non-reproductive period, which are incompetent. Interestingly, full-grown Bufo arenarum oocytes always respond to progesterone regardless of the season in which they are obtained. There is a general consensus that both a transient increase in intracellular calcium and a decrease in cAMP-dependent protein kinase activity are the first steps in the mechanisms by which progesterone induces maturation in amphibians. In the present work we analysed the role of calcium in the spontaneous and progesterone-induced maturation of Bufo arenarum oocytes. Results demonstrated that the absence of calcium in the incubation medium or the prevention of Ca(2+) influx by channel blockers such as CdCl2 or NiCl2 did not prevent meiosis reinitiation in either type of maturation. The inhibition of the Ca(2+)-calmodulin complex in no case affected the maturation of the treated oocytes. However, when the oocytes were deprived of calcium by incubation in Ca(2+)-free AR + A23187, meiosis resumption was inhibited. In brief, we demonstrated that in Bufo arenarum the reinitiation of meiosis is a process independent of extracellular calcium at any period of the year and that oocytes require adequate levels of intracellular calcium for germinal vesicle breakdown to occur.     

Polyphosphoinositide synthesis and protein phosphorylation in the plasma membrane from full-grown Bufo arenarum oocytes.

1. Polyphosphoinositide content and phosphorylation of lipids and proteins were analyzed in oocytes of the toad Bufo arenarum Hensel. 2. Plasma membrane-enriched fractions obtained from full-grown, prophase-arrested oocytes incorporated 32P into both phospholipids and proteins after incubation with [gamma-32P]ATP in an Mg(2+)-containing medium. Phosphatidylinositol 4-phosphate (PIP), phosphatidate (PA) and phosphatidylinositol-4,5-bisphosphate (PIP2) were the only labelled lipids. The 32P incorporation depended on incubation time, the amount of protein, and the ATP concentration. 3. Autoradiography of polyacrylamide gel electropherograms and scintillation counting showed that the radioactivity was mainly associated with a group of membrane proteins having an M(r) of 87,000. 4. This paper provides evidence for the capacity of prophase-arrested oocytes from Bufo arenarum to synthesize polyphosphoinositides and to phosphorylate distinct membrane proteins.     

Nucleotides and nucleotide sugars in early development of Bufo arenarum. I. Mature oocytes

A study of nucleotides and nucleotide sugars in mature oocytes of Bufo arenarum has been performed. Two samples of oocytes from ovulations of ten females each were analyzed by ion-exchange chromatography and the following nucleotides and nucleotide sugars found²: NAD, UDPGNAc, UDPGalNAc, UDPG, UDP, NADP, CTP, UTP, UDPGA, ADP, GDP, GTP, and ATP. The nucleotides and sugar-nucleotides were identified on the basis of chemical, enzymic, and chromatographic analysis. To minimize the decomposition of unstable substances, the extraction was performed with trichloroacetic acid in the cold, the contact of the sample with the acid was minimal and ammonium chloride was used for the elution from the Dowex 1-resin. In order to detect compounds present in minute amounts, very large samples were used (60,000 to 120,000 oocytes). A nucleotide of complex structure, which binds weakly to the column, was detected in the two samples analyzed.     

Tyrosination-detyrosination of the COOH-terminus of alpha-tubulin in oocytes and embryos of Bufo arenarum

Soluble tubulin from Bufo arenarum oocytes and early embryos was shown to be composed mainly of the non-tyrosinable species. The low proportion of tyrosinable tubulin was almost exclusively constituted by the tyrosinated form. Compared with oocytes and embryos, toad brain contained a higher proportion of tyrosinable tubulin constituted mainly by the non-tyrosinated form. Tubulin carboxypeptidase was detected in toad brain but not in oocytes and embryos.