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.     

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.     

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.     

Spontaneous and LH-induced maturation in Bufo arenarum oocytes: importance of gap junctions

It has been demonstrated in Bufo arenarum that fully grown oocytes are capable of meiotic resumption in the absence of a hormonal stimulus if they are deprived of their follicular envelopes. This event, called spontaneous maturation, only takes place in oocytes collected during the reproductive period, which have a metabolically mature cytoplasm. In Bufo arenarum, progesterone acts on the oocyte surface and causes modifications in the activities of important enzymes, such as a decrease in the activity of adenylate cyclase (AC) and the activation of phospholipase C (PLC). PLC activation leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP(3)), second messengers that activate protein kinase C (PKC) and cause an increase in intracellular Ca(2+). Recent data obtained from Bufo arenarum show that progesterone-induced maturation causes significant modifications in the level and composition of neutral lipids and phospholipids of whole fully grown ovarian oocytes and of enriched fractions in the plasma membrane. In amphibians, the luteinizing hormone (LH) is responsible for meiosis resumption through the induction of progesterone production by follicular cells. The aim of this work was to study the importance of gap junctions in the spontaneous and LH-induced maturation in Bufo arenarum oocytes. During the reproductive period, Bufo arenarum oocytes are capable of undergoing spontaneous maturation in a similar way to mammalian oocytes while, during the non-reproductive period, they exhibit the behaviour that is characteristic of amphibian oocytes, requiring progesterone stimulation for meiotic resumption (incapable oocytes). This different ability to mature spontaneously is coincident with differences in the amount and composition of the phospholipids in the oocyte membranes. Capable oocytes exhibit in their membranes higher quantities of phospholipids than incapable oocytes, especially of PC and PI, which are precursors of second messengers such as DAG and IP(3). The uncoupling of the gap junctions with 1-octanol or halothane fails to induce maturation in follicles from the non-reproductive period, whose oocytes are incapable of maturing spontaneously. However, if the treatment is performed during the reproductive period, with oocytes capable of undergoing spontaneous maturation, meiosis resumption occurs in high percentages, similar to those obtained by manual defolliculation. Interestingly, results show that LH is capable of inducing GVBD in both incapable oocytes and in oocytes capable of maturing spontaneously as long as follicle cells are present, which would imply the need for a communication pathway between the oocyte and the follicle cells. This possibility was analysed by combining LH treatment with uncoupling agents such as 1-octanol or halothane. Results show that maturation induction with LH requires a cell-cell coupling, as the uncoupling of the gap junctions decreases GVBD percentages. Experiments with LH in the presence of heparin, BAPTA/AM and theophylline suggest that the hormone could induce GVBD by means of the passage of IP(3) or Ca(2+) through the gap junctions, which would increase the Ca(2+) level in the oocyte cytoplasm and activate phosphodiesterase (PDE), thus contributing to the decrease in cAMP levels and allowing meiosis resumption.     

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.     

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.     

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.     

Involvement of purines and phosphoinositides in spontaneous and progesterone-induced nuclear maturation of Bufo arenarum oocytes

Although progesterone is the established maturation inducer in amphibia, it has been demonstrated that Bufo arenarum oocytes resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called "spontaneous maturation." The present studies were designed to evaluate the participation of purines and phosphoinositides in the spontaneous and progesterone-induced maturation in Bufo arenarum full-grown oocytes. The presented data demonstrate that high intracellular levels of purines such as cAMP or guanosine can inhibit both spontaneous and progesterone-induced maturation in full-grown denuded Bufo arenarum oocytes. Moreover, the fact that the mycophenolic acid was able to induce maturation in denuded oocytes obtained during the nonreproductive period in a manner similar to that of the progesterone and also to increase the percentages of spontaneous maturation suggests that in Bufo arenarum, inosine monophosphate dehydrogenase inhibition is an important step in the resumption of meiosis. Inhibition of the phosphatidylinositol 4,5 bisphosphate hydrolysis by treatment of denuded oocytes with neomycin totally blocks spontaneous and progesterone-induced maturation, suggesting that the products of this hydrolysis (1,2 diacylglycerol and inositol 1,4,5 trisphosphate) may be involved in the maturation process of Bufo. In addition, our results indicate that the activation of protein kinase C is also involved in both types of maturation.     

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.     

The cytoplasm of mouse germinal vesicle stage oocytes can enhance somatic cell nuclear reprogramming

In mammalian cloning, evidence suggests that genomic reprogramming factors are located in the nucleus rather than the cytoplasm of oocytes or zygotes. However, little is known about the mechanisms of reprogramming, and new methods using nuclear factors have not succeeded in producing cloned mice from differentiated somatic cell nuclei. We aimed to determine whether there are functional reprogramming factors present in the cytoplasm of germinal vesicle stage (GV) oocytes. We found that the GV oocyte cytoplasm could remodel somatic cell nuclei, completely demethylate histone H3 at lysine 9 and partially deacetylate histone H3 at lysines 9 and 14. Moreover, cytoplasmic lysates of GV oocytes promoted somatic cell reprogramming and cloned embryo development, when assessed by measuring histone H3-K9 hypomethylation, Oct4 and Cdx2 expression in blastocysts, and the production of cloned offspring. Thus, genomic reprogramming factors are present in the cytoplasm of the GV oocyte and could facilitate cloning technology. This finding is also useful for research on the mechanisms involved in histone deacetylation and demethylation, even though histone methylation is thought to be epigenetically stable.     

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.     

Kinetic and temporal factors influence chilling injury to germinal vesicle and mature bovine oocytes

In this study we examined the effects of low, above freezing temperatures on the viability and functionality of bovine oocytes. Germinal vesicle (GV) stage and in vitro matured oocytes (MII) were exposed to various combinations of time (15 and 60 min) and temperature (4, 16, 23, and 39 degrees C). After being treated, the ability of oocytes to undergo maturation and fertilization in vitro was examined, as well as their viability assayed by two fluorescent probes, fluorescein diacetate (FDA) and 5-carboxyfluorescein diacetate (cFDA). Cooling GV oocytes to 16 degrees C for 15 min reduced the fertilization rate by more than 40%, compared with those left at 39 degrees C. Surprisingly, cooling oocytes to 4 degrees C reduced the fertilization rate by only 10% compared with control. Exposing GV oocytes to temperatures below 23 degrees C reduced their viability. Similar to the reduction in fertilization, the viability of GV oocytes after exposure to 16 degrees C was reduced by more than 50%, whereas exposure to 4 degrees C reduced it by only 9%. Viability measurements using FDA and cFDA gave comparable results and showed a similar trend. The viability of MII oocytes and of GV oocytes pretreated with butylated hydroxytoluene, following exposure to low temperatures, was higher compared with that of GV controls. We interpret these results as indicating chilling effects on membrane integrity. Improving the chilling resistance of bovine oocytes may facilitate their short- and long-term preservation.     

The role of germinal vesicle in maturation of Pleurodeles waltlii oocytes induced by steroids

The maturation of oocytes from unstimulated Pleurodeles waltlii females can be induced in vitro. All full-grown oocytes covered by follicular envelopes mature under the influence of progesterone, testosterone, and a synthetic steroid (CDMT). Enucleation of oocytes prior to maturation induction resulted in a significantly lower percentage of maturation. Irradiation, or the treatment of oocytes with actinomycin D, did not affect their maturation. Therefore the lower percentage of maturation after enucleation must be due to the lack of karyoplasm. Karyoplasm appears to be necessary for the production of the maturation-promoting factor in P. waltlii oocytes.     

In vitro maturation and fertilization of goat oocytes frozen at the germinal vesicle stage

The ability of frozen immature goat oocytes to undergo in vitro maturation (IVM) and fertilization (IVF) was investigated. Fully grown germinal vesicle stage (GV-stage) goat oocytes were submitted to different variables of cryopreservation: 1) exposure to propanediol before maturation but without freezing to detect the level of damage attributable to propanediol alone, 2) removal of cumulus cells to mimic damage attributable to osmotic stress during cryoprotectant exposure or freezing procedure, and 3) rapid freezing with propanediol. Maturation and fertilization rates were 82.1, 71, 65.3 and 23.7% and 71.2, 40, 58.4 and 23.1% for control, exposed, denuded and frozen oocytes, respectively. These results indicate that freezing sticto sensu (i.e., cooling and warming phases) have detrimental effects on IVM of GV-stage oocytes, whereas the reduced IVF rates of post-thaw matured oocytes are imputable to a cryoprotectant effect.     

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.     

Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes

Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes were studied using a new method that allows a clearer visualization of both nucleolus and chromatin after Hoechst staining. The GV chromatin of porcine oocytes was classified into five configurations, based on the degree of chromatin condensation, and on nucleolus and nuclear membrane disappearance. While the GV1 to 4 configurations were similar to those reported by previous studies, the GV0 configuration was distinct by the diffuse, filamentous pattern of chromatin in the whole nuclear area. Most of the oocytes were at the GV0 stage in the <1 and 1-1.9 mm follicles, but the GV0 pattern disappeared completely in the 2-2.9 and 3-6 mm follicles. As follicles grew, the number of oocytes with GV1 configurations increased and reached a maximum in the preovulatory follicles 4 hr post-hCG injection. During maturation in vivo, the number of GV1 oocytes decreased while oocytes undergoing GVBD increased. The percentage of oocytes with GV3 and GV4 configurations was constant during oocyte growth except at the 2-2.9 mm follicle stage, but these configurations disappeared completely after hCG injection. On the contrary, the in vitro maturing oocytes showed a large proportion of GV3 and GV4 configurations. There was no significant difference in distribution of chromatin configurations between the nonatretic and atretic follicles, and between oocytes with more than two layers of cumulus cells and those with less than one layer or no cumulus cells. Overall, our results suggested that (i) the GV0 configuration in porcine oocytes corresponded to the "nonsurrounded nucleolus" pattern in mice and other species; (ii) all the oocytes were synchronized at the GV1 stage before GVBD and this pattern might, therefore, represent a nonatretic state; (iii) the GV3 and GV4 configurations might represent stages toward atresia, or transient events prior to GVBD that could be switched toward either ovulation or atresia, depending upon circumstances; (iv) the in vitro systems currently used were not favorable for oocytes to switch toward ovulation (or final maturation); (v) the number of cumulus cells was not correlated with the chromatin configuration of oocytes, indicating that the beneficial effect of cumulus cells on oocyte maturation and development may simply be attributed to their presence during in vitro culture.     

The changes in sperm nuclei after penetrating fish oocytes matured without germinal vesicle material in their cytoplasm

The processes occurring from sperm penetration to chromosome formation in the cytoplasm of Oocytes matured in vitro, after removal of the germinal vesicle (GV) and before hormonal stimulation, were observed with electron microscope. The dechorionated oocytes, matured without the participation of the GV material, responded to sperm penetration by initiating a cortical reaction within 20 seconds after insemination. The pentrating sperm nuclei transformed to male pronuclei with vesiculation of the nuclear membrane, chromatin decondensation, and formation of a pronuclear membrane. Before cleavage, however, no chromosome formation was observed in these oocytes. Instead, the fully grown pronuclei change to a picnotic chromatin mass without or with an only fragmented nuclear membrane, then disappeared. On the contrary, sperm nuclei that penetrated into the cytoplasm of naked eggs containing GV material during maturation underwent pronuclear and chromosomal formation. Judging from these observation in Oryzias oocytes, the GV material seems to be unnecessary for the formation of pronucleus from the compact sperm nucleus, but is essential for the process of chromosomal formation.