Spontaneous Overactivation of Frog Eggs Triggers Necrotic Cell Death.

The excessive activation of frog eggs, referred to as overactivation, can be initiated by strong oxidative stress, leading to expedited calcium-dependent non-apoptotic cell death. Overactivation also occurs spontaneously, albeit at a low frequency, in natural populations of spawned frog eggs. Currently, the cytological and biochemical events of the spontaneous process have not been characterized.

Oxidative Stress-Induced Overactivation of Frog Eggs Triggers Calcium-Dependent Non-Apoptotic Cell Death.

Excessive activation of frog eggs (overactivation) is a pathological process that renders eggs unfertilizable. Its physiological inducers are unknown. Previously, oxidative stress was shown to cause time- and dose-dependent overactivation of frog eggs.

Protein pI and Intracellular Localization.

The protein isoelectric point (pI) can be calculated from an amino acid sequence using computational analysis in a good agreement with experimental data. Availability of whole-genome sequences empowers comparative studies of proteome-wide pI distributions. It was found that the whole-proteome distributions of protein pI values are multimodal in different species.

Modulation of Intracellular ROS and Senescence-Associated Phenotypes of Oocytes and Eggs by Selective Antioxidants.

Aging of oocytes and eggs diminishes their reproductive and developmental potential. It has been demonstrated previously that reactive oxygen species (ROS) contribute to accelerated aging of various cells. In the present study, we measured intracellular levels of ROS and investigated effects of several selective antioxidants (AOXs) on the viability and functional activity of aging oocytes and eggs of the African clawed frog .

Toward the understanding of biology of oocyte life cycle in : No oocytes left behind.

Background: For the past more than 25 years, we have been focusing on the developmental and reproductive biology of the female gametes, oocytes, and eggs, of the African clawed frog .

Methods: The events associated with the life cycle of these cells can be classified into the four main categories: first, oogenesis and cell growth in the ovary during the first meiotic arrest; second, maturation and ovulation that occur simultaneously and result in the acquisition of fertilization competence and the second meiotic arrest; third, fertilization, that is sperm-induced transition from egg to zygote; and fourth, egg death after spontaneous activation in the absence of fertilizing sperm.

Main Findings: Our studies have demonstrated that signal transduction system involving tyrosine kinase Src and other oocyte/egg membrane-associated molecules such as uroplakin III and some other cytoplasmic proteins such as mitogen-activated protein kinase (MAPK) play important roles for successful ovulation, maturation, fertilization, and initiation of embryonic development.

In Vitro Reconstruction of Oocyte Ovulation.

Progesterone is widely used to induce maturation of isolated fully grown oocytes of the African clawed frog, . However, the hormone fails to release oocytes from the layer of surrounding follicle cells. Here, we report that maturation and follicle rupture can be recapitulated in vitro by treating isolated follicular oocytes with progesterone and low doses of the matrix metalloproteinase (MMP), collagenase, which are ineffective in the absence of the steroid.

Localization-specific distributions of protein pI in human proteome are governed by local pH and membrane charge.

Background: Whole-proteome distributions of protein isoelectric point (pI) values in different organisms are bi- or trimodal with some variations. It was suggested that the observed multimodality of the proteome-wide pI distributions is associated with subcellular localization-specific differences in the local pI distributions. However, the factors responsible for variation of the intracellular localization-specific pI profiles have not been investigated in detail.

Biochemical Hallmarks of Oxidative Stress-Induced Overactivation of Eggs.

Egg overactivation occurs with a low frequency in the populations of naturally ovulated frog eggs. At present, its natural inducers, molecular mechanisms, and intracellular events remain unknown. Using microscopic and biochemical analyses, we demonstrate here that high levels of hydrogen peroxide-induced oxidative stress can cause time- and dose-dependent overactivation of eggs.

Autophosphorylation of MAP kinase disables the MAPK pathway in apoptotic Xenopus eggs.

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of various cellular processes, including cell survival and apoptosis. Here, we report that Xenopus p42 MAPK becomes phosphorylated in apoptotic eggs, however this modification does not activate the enzyme. Using phosphorylation residue-specific antibodies, we demonstrate that this modification occurs on the Tyr residue in the MAPK activation segment, pinpointing the autophosphorylation mechanism.

Activity and intracellular localization of senescence-associated β-galactosidase in aging Xenopus oocytes and eggs.

Senescence-associated β-galactosidase (SA-β-gal) serves as a marker of senescence in aging somatic cells. However, little is known about SA-β-gal dynamics in aging gamete cells. To address this issue, here we investigated activity and intracellular localization of SA-β-gal in freshly obtained and aging oocytes and eggs of the African clawed frog Xenopus laevis.

Membrane Microdomains as Platform to Study Membrane-Associated Events During Oogenesis, Meiotic Maturation, and Fertilization in Xenopus laevis.

Studies on the egg plasma membrane-associated tyrosine kinase Src have shed light on the identity of the molecular machinery that is responsible for gamete interaction and possibly fusion in African clawed frog Xenopus laevis. Here we describe our protocol for identifying and analyzing molecular and cellular machinery that contributes to a variety of biological processes in the course of oogenesis, oocyte maturation, egg fertilization, and early embryogenesis in Xenopus. Our current special interest is to evaluate the hypothesis that the oocyte/egg membrane microdomain (MD)-associated uroplakin III-Src system is responsible for mediating sperm-egg membrane interaction/fusion signal to the oocyte/egg cytoplasm to initiate embryonic and zygotic development in this species.

Reconstitution of Intracellular Calcium Signaling in Xenopus Egg Extracts.

Generation of calcium signal in the cytoplasm of fertilized or parthenogenetically activated eggs has been extensively studied in the intact eggs of several biological species. Calcium transient was found to elicit a plethora of biochemical and cellular events in these cells. Remarkably, intracellular calcium signaling can also be reconstituted in cell-free environment.

Electrostatic Switch Function in the Mechanism of Protein Kinase A I Activation: Results of the Molecular Dynamics Simulation.

We used molecular dynamics to find the average path of the A-domain H → B conformational transition in protein kinase A I. We obtained thirteen productive trajectories and processed them sequentially using factor and cross-correlation analyses. The conformational transition is presented as partly deterministic sequence of six events.

Global decay of mRNA is a hallmark of apoptosis in aging Xenopus eggs.

Cytoplasmic mRNAs are specifically degraded in somatic cells as a part of early apoptotic response. However, no reports have been presented so far concerning mRNA fate in apoptotic gametes. In the present study, we analyzed the content of various cytoplasmic mRNAs in aging oocytes and eggs of the African clawed frog, Xenopus laevis.

Content of intrinsic disorder influences the outcome of cell-free protein synthesis.

Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now.

Organization of the multiaminoacyl-tRNA synthetase complex and the cotranslational protein folding.

Aminoacyl-tRNA synthetases (ARSs) play an essential role in the protein synthesis by catalyzing an attachment of their cognate amino acids to tRNAs. Unlike their prokaryotic counterparts, ARSs in higher eukaryotes form a multiaminoacyl-tRNA synthetase complex (MARS), consisting of the subset of ARS polypeptides and three auxiliary proteins. The intriguing feature of MARS complex is the presence of only nine out of twenty ARSs, specific for Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met, and Pro, regardless of the organism, cell, or tissue types.

Calcium signaling and meiotic exit at fertilization in Xenopus egg.

Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis.

Identification of multiple physicochemical and structural properties associated with soluble expression of eukaryotic proteins in cell-free bacterial extracts.

Bacterial extracts are widely used to synthesize recombinant proteins. Vast data volumes have been accumulated in cell-free expression databases, covering a whole range of existing proteins. It makes possible comprehensive bioinformatics analysis and identification of multiple features associated with protein solubility and aggregation.

Correlations between predicted protein disorder and post-translational modifications in plants.

Motivation: Protein structural research in plants lags behind that in animal and bacterial species. This lag concerns both the structural analysis of individual proteins and the proteome-wide characterization of structure-related properties. Until now, no systematic study concerning the relationships between protein disorder and multiple post-translational modifications (PTMs) in plants has been presented.

Bioinformatics analysis and optimization of cell-free protein synthesis.

Cell-free protein synthesis offers substantial advantages over cell-based expression, allowing direct access to the protein synthetic reaction and meticulous control over the reaction conditions. Recently, we identified a number of statistically significant correlations between calculated and predicted properties of amino acid sequences and their amenability to heterologous cell-free expression. These correlations can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

Unlaid Xenopus eggs degrade by apoptosis in the genital tract.

Background: In several species with external fertilization, including frogs, laid unfertilized eggs were found to die by apoptosis outside of the animal body. However, there is no apparent reason for the externally laid eggs to degrade by this process, considering that apoptosis developed as a mechanism to reduce the damaging effect of individual cell death to the whole organism.

Results: Here, we demonstrate that a number of eggs are retained in the genital tract of the African clawed frog Xenopus laevis after gonadotropin-induced ovulation.