Mitochondrial DNA synthesis in large and mature oocytes of Xenopus laevis

Deoxynucleosides are incorporated into mitochondrial DNA (mtDNA) of large oocytes; the rate of incorporation is about 2% of the mtDNA amount per 24 hr. When oocytes have been induced to mature in vitro with human chorionic gonadotropin (HCG), uptake and actual incorporation of thymidine decrease, although phosphorylation is enhanced. An examination of mtDNA replication shows that HCG treatment induces an increase in the relative synthesis of E-strands and an accumulation of D-loops. A similar effect is obtained by ethidium bromide treatment. Thus, gonadotropin appears to delay E-strand elongation and to synchronize mtDNA molecules at the begining of their replication cycle.     

Protein synthesis and phosphorylation patterns of bovine oocytes maturing in vivo

To investigate protein synthesis and phosphorylation during bovine oocyte maturation in vivo, oocytes were collected at consecutive times after the preovulatory luteinizing hormone (LH) peak. Therefore, heifers treated for superovulation were ovariectomized between 3 and 20 h after the maximum of the LH peak. Subsequently, cumulus-enclosed oocytes, selected from nonatretic follicles greater than 10 mm, were radiolabeled with 35S-methionine or 32P-orthophosphate for 3 h and individually prepared for gel electrophoresis. Changes in the protein synthesis patterns were observed coinciding with germinal vesicle breakdown (GVBD). No changes were detected during the ensuing maturation period or coinciding with the extrusion of the first polar body. In addition, the protein phosphorylation patterns exhibited striking differences around GVBD. In particular, a phosphoprotein band of 19 kDa and the two heavily phosphorylated proteins with apparent molecular weights between 50 and 60 kDa were present in patterns of oocytes in the germinal vesicle stage. The results are discussed in relation to previous data obtained during maturation in vitro.     

Protein kinase activities associated with actin cytoskeleton in the oocytes and eggs of African clawed frog

Protein phosphorylation with specific protein kinases plays the key role in the regulation of meiotic maturation of oocytes. However, little is known about the contribution of kinases to the temporal and positional regulation of the cytoskeleton rearrangement in maturing oocytes, including the actin cytoskeleton. In order to study a relationship between the kinase activities and actin cytoskeleton rearrangement, we analyzed protein phosphorylation in the isolated actin cytoskeleton of Xenopus laevis oocytes. Analysis of the full grown oocytes and eggs injected with [gamma-32P] "P has revealed phosphorylation of many proteins associated with the actin cytoskeleton and shown the appearance of three additional major phosphoproteins, 20, 43, and 69 kDa, during oocyte maturation. A significant number of these phosphoproteins were also found after incubation of the isolated cytoskeleton with [gamma-32P] "P in vitro, thus confirming that the kinases modifying these substrates are also specifically associated with actin. The in vivo and in vitro kinase activities were also stimulated during maturation. Analysis of kinase self-phosphorylation in situ and protein phosphorylation in solutions and substrate containing gels revealed a set of actin-associated kinases, including cAMP- and Ca(2+)-dependent kinases, as well as MAP, p34cdc2, and tyrosine kinase activities. Their level was the highest in the eggs. The involvement of kinases in the actin cytoskeleton rearrangement during oocyte maturation is discussed.     

Protein synthesis during hormonally induced meiotic maturation and fertilization in starfish oocytes

Uptake of radioactive amino acids and their incorporation into protein were examined during 1-methyladenine-induced maturation and subsequent fertilization of oocytes of the starfish Patiria miniata. The initial response to the hormone was a nearly immediate decrease in permeability to amino acids, indicating that the site of action of the hormone is on the cell surface. Protein synthesis began to increase starting about 12 min after 1-methyladenine stimulation and prior to germinal vesicle breakdown. It continued to rise throughout the first meiotic division. This protein synthesis was not required for assembly or initial functioning of the meiotic apparatus, although it was necessary for the completion of meiosis. Fertilization had no effect on the rate of protein synthesis.Oocytes of P. miniata provide an example of hormonal stimulation of protein synthesis in an invertebrate system.     

Hexokinase and not glycogen synthase controls the flux through the glycogen synthesis pathway in frog oocytes

Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycogen synthesis in vivo. We used metabolic control analysis (MCA) to determine the flux control coefficient for each of the enzymes involved in the pathway. Acute microinjection experiments in frog oocytes were specifically designed to change the endogenous activities of the enzymes, either by directly injecting increasing amounts of a given enzyme (HK, PGM and UGPase) or by microinjection of a positive allosteric effector (glc-6P for GS). Values of 0.61 ± 0.07, 0.19 ± 0.03, 0.13 ± 0.03, and −0.06 ± 0.08 were obtained for the flux control coefficients of hexokinase EC 2.7.1.1 (HK), phosphoglucomutase EC 5.4.2.1 (PGM), UDPglucose pyrophosphorylase EC 2.7.7.9 (UGPase) and glycogen synthase EC 2.4.1.11 (GS), respectively. These values satisfy the summation theorem since the sum of the control coefficients for all the enzymes of the pathway is 0.87. The results show that, in frog oocytes, glycogen synthesis through the direct pathway is under the control of hexokinase. Phosphoglucomutase and UDPG-pyrophosphorylase have a modest influence, while the control exerted by glycogen synthase is null.     

Ultrastructure of human mature oocytes after vitrification

Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morpho-functional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microsco py has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.Key words: oocyte, MII, vitrification, ultrastructure, TEM, human.     

Hormone-induced parthenogenetic activation of mature starfish oocytes

1-Methyladenine, which has been previously shown to be the hormone responsible for meiosis reinitiation in starfish oocytes, triggers parthenogenetic activation when applied to matured starfish oocytes after emission of the second polar body and formation of the pronucleus. In Marthasterias glacialis and Asterias rubens oocytes parthenogenetic activation includes elevation of a fertilization membrane, cleavage and the formation of normal bipinnaria larvae. Activation is likely to result from 1-methyladenine interaction with the category of stereospecific membrane receptors involved in meiosis reinitiation, since structural requirements of this compound are identical for both biological responses. Appearance of oocyte responsiveness to 1-MeAde after, but not before emission of the second polar body cannot be accounted for by their increased sensitivity to intracellular Ca²⁺ at that time, although it is shown that Ca²⁺ mediates hormone effect in inducing parthenogenetic activation. Pretreatment of immature oocytes with the free hormone in excess strongly inhibits the 1-methyladenine-induced parthenogenetic activation of the oocytes when they have completed maturation.It is suggested that reappearance of 1-MeAde sensitivity when oocytes form a pronucleus depends either upon recruitment or new receptor units or on the reactivation of pre-existing inactivated receptors at this stage of oocyte maturation.     

Active maturation-promoting factor is present in mature mouse oocytes

Cytoplasmic extracts of meiotically mature mouse oocytes were injected into immature Xenopus laevis oocytes, which underwent germinal vesicle breakdown within 2 h. Germinal vesicle breakdown was not inhibited by incubation of the Xenopus oocytes in cycloheximide (20 micrograms/ml). Identically prepared extracts of meiotically immature mouse oocytes, arrested at the germinal vesicle stage by dibutyryl cyclic AMP (100 micrograms/ml), did not induce germinal vesicle breakdown in Xenopus oocytes. The results show that maturation-promoting factor activity appears during the course of oocyte maturation in the mouse.     

Insect protein synthesis in frog cells: the translation of honey bee promelittin messenger RNA in Xenopus oocytes

Venom glands of young queen bees (Apis mellifera) synthesize the toxic peptide melittin as their main product. Melittin is formed by proteolytic cleavage of a precursor, promelittin. Unfractionated RNA prepared from venom glands was injected into Xenopus oocytes and was shown to direct the synthesis of a promelittin-like substance. About half of the peptide chain made in oocytes has been sequenced; the 17 amino acid residues identified correspond exactly with sequences found in promelittin from venom gland cells. These results yield final proof that injected messenger RNAs can be read with great fidelity. The translation of a messenger from an insect gland shows that at least some of the translational systems within the oocyte are neither cell-type nor phylum specific. It seems likely that the oocyte can be used to assay any kind of eukaryotic mRNA.The conversion of promelittin to melittin could not be detected in oocytes. Moreover, the promelittin synthesized in oocytes differs at the carboxyl end from the product made in gland cells, for the latter terminates with glutamine amide while the oocyte material probably ends with an amino acid with a free α-carboxyl group. Some of the post-translational modifications characteristic of gland cells thus do not seem to take place in oocytes.     

Pathways involved in RGD-mediated calcium transients in mature bovine oocytes

An arginine-glycine-aspartic acid (RGD)-containing peptide has been reported to generate calcium transients in bovine oocytes similar to those observed at fertilization. The research objective herein was to evaluate the response of bovine oocytes to an RGD peptide after injection with known antagonists of calcium releasing mechanisms in order to determine the initial calcium releasing pathway. Oocytes were injected with either heparin, an inhibitor of inositol 1,4,5-trisphosphate (IP3) induced calcium response, or procaine, which inhibits calcium release through the ryanodine receptor. Oocytes injected with heparin prior to RGD exposure did not display a calcium response. Oocytes injected with procaine prior to RGD exposure did exhibit a calcium response. Electroporation of IP3, caffeine, or exposure to RGD alone elicited a calcium response for each treatment group. Injection of heparin, procaine, vehicle medium (VM), or exposure to a non-RGD-containing peptide alone failed to elicit a calcium response. The data indicates that the RGD peptide is able to induce calcium transients in oocytes inhibited with procaine, but not those inhibited with heparin. These data suggest the pathway whereby the RGD peptide induces the first intracellular calcium transient in bovine oocytes is through IP3-mediated stores.     

Protein synthesis inhibitors prevent both spontaneous and hormone-dependent maturation of isolated mouse oocytes

The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors [CX, emetine (EM), and puromycin (PUR)] each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the controls (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP.(ABSTRACT TRUNCATED AT 250 WORDS)