Mammalian sperm acquire fertilizing capacity after residing in the female tract, where physiological changes named capacitation take place. In animals with external fertilization as amphibians, gamete interactions are first established between sperm and molecules of the egg jelly coat released into the medium. Since dejellied oocytes are not normally fertilized, the aim of this study was to determine if the jelly coat of the toad Bufo arenarum promotes a “capacitating” activity on homologous sperm. We found that sperm incubation in diffusible substances of the jelly coat (Egg Water) for 90–180 sec is sufficient to render sperm transiently capable of fertilizing dejellied oocytes. The fertilizing state was correlated with an increase of protein tyrosine phosphorylation and a decrease of sperm cholesterol content. Inhibition of either the increase in tyrosine phosphorylation or cholesterol efflux affected the acquisition of fertilizing capacity. Phosphorylation and fertilization could be promoted with NaHCO3, and also by addition of beta cyclodextrin. Moreover, sperm could gain the ability to fertilize dejellied oocytes in the presence of these compounds. These data indicate that sperm should undergo a series of molecular changes to gain fertilizing capacity; these changes are reminiscent of mammalian sperm capacitation and take place before the acrosome reaction.
fertilization; spermatozoa; capacitation; jelly coat; amphibia; phosphorylation
Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of ∼30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS. Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure–function relationships and the role of glycans in the fertilization process within Xenopodinae.
exoglycosidase; MALDI-IRMPD mass spectrometry; O-glycan structure; tandem MS; X. borealis
1. The problem of the relation of the plasma membrane to the extraneous coats and cortex of the Nereis egg is discussed in the light of the observations of Lillie, Chambers, and Novikoff. 2. Evidence obtained from experiments with the centrifuge, and by treating eggs with alkaline sodium chloride, indicates that the plasma membrane of the unfertilized egg is external to the jelly precursor granules of the cortex. 3. Experiments with alkaline sodium chloride indicate that the perivitelline space of the fertilized egg is extraovular after jelly extrusion is complete. 4. The cortical behavior (membrane elevation) of the Nereis egg in alkaline sodium chloride and the cortical response (jelly extrusion) following activation of the egg in normal fertilization or parthenogenesis are attributed largely to the properties of the jelly, and presumably, to its reactions with calcium and hydroxyl ions.
The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.
The response of unfertilised Paracentrotus lividus eggs to γ-globulin fractions of antisera against isolated homologous jelly coat substance or homologous homogenates of jellyless eggs has been studied at the ultrastructural level. The antijelly γ-globulin caused precipitation of the jelly layer, the density of precipitation varying between different eggs and being proportional to the γ-globulin concentration. Agglutination of the jelly substance of adjacent eggs, which is species specific, occurred frequently with higher γ-globulin concentrations. Antiegg γ-globulins (from antiserum against total homogenates of jelly-free eggs or the heat-stable fraction thereof) did not produce these effects. Instead, these γ-globulins caused various structural alterations mostly representing stages in parthenogenetic activation. This species-specific activation was induced by the reaction of antibodies with some heat-stable egg antigens different from those involved in jelly precipitation. Surface alterations included the formation of small papillae, membrane blisters, hyaline layer, and activation membrane, the release of material from the cell surface, and the breakdown of cortical granules. These alterations were dependent on both γ-globulin concentration and the variable reactivity among different females. Aster formation, found intracellularly, verified that the surface responses represented real parthenogenetic activation and were not the result of immune lysis. No such alterations appeared in the controls.
Intracellular membrane networks including the endoplasmic reticulum (ER) and the Golgi apparatus experience dramatic reorganization upon entry into mitosis. However, the mechanisms driving these rearrangements and their importance for cell division are poorly understood. The GTPase Sar1 is a component of the secretory pathway and a key activator of anterograde transport of cargo from the ER to the Golgi. Here we show that Sar1 mutant proteins added to metaphase-arrested Xenopus laevis egg extracts cause dramatic effects on membrane organization. Live analysis of membrane structures in egg extract cytoplasm revealed a distinct network of sheets and tubules reflective of the organization of the ER in other systems. Addition of a constitutively active Sar1 GTPase mutant (H79G) increased membrane tubulation, while a dominant negative version Sar1 (T39N) impaired tubule organization. Although microtubule pelleting assays revealed that Sar1 associates with microtubules in the egg extract, and addition of Sar1 (H79G) mutant slightly destabilized spindle poles, bipolar spindle assembly was largely unaffected. Thus, spindles are stable to dramatic changes in mitotic membrane organization at metaphase, suggesting that mitotic membrane is not an upstream regulator of the mitotic spindle apparatus in Xenopus egg extracts.
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.
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. The majority of these eggs exit meiotic arrest within 24 hours of hormone administration. Subsequently, post-meiotic eggs die in the frog genital tract by a well-defined apoptotic process. The hallmarks of egg degradation include prominent morphological changes, cytochrome c release, caspase activation, increase in ADP/ATP ratio, progressive intracellular acidification, egg swelling and all-out proteolysis of egg proteins. The sustained presence of post-apoptotic eggs in the genital tract of ageing frogs evidenced age-associated worsening of apoptotic clearance.
The direct observation of egg degradation in the Xenopus genital tract provides a clue to the physiological relevance of frog egg apoptosis. It works to eliminate the mature unlaid eggs retained in the animal body after ovulation. Our findings establish egg apoptosis as a major physiological process accompanying ovulation in frogs.
Apoptosis; Unlaid eggs; Maturation; Ovulation; Meiotic exit; Xenopus laevis; Genital tract
Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions including meiotic maturation and parthenogenetic activation of mammalian oocytes. We observed that nitric oxide donor SNAP was potent to induce parthenogenetic activation in Xenopus eggs. NO-scavenger CPTIO impaired the effects of SNAP, providing evidence for the effects of the latter to be specific upon NO release. In Xenopus eggs, SNAP treatment induced pigment rearrangement, pronucleus formation and exocytosis of cortical granules. At a biochemical level, SNAP exposure lead to MAPK and Rsk inactivation within 30 minutes whereas MPF remained active, in contrast to calcium ionophore control where MPF activity dropped rapidly. MAPK inactivation could be correlated to pronuclear envelope reformation observed. In SNAP-treated eggs, a strong increase in intracellular calcium level was observed. NO effects were impaired in calcium-free or calcium limited medium, suggesting that that parthenogenetic activation of Xenopus oocytes with a NO donor was mainly calcium-dependent.
The current model amphibian, Xenopus laevis, develops rapidly in water to a tadpole which metamorphoses into a frog. Many amphibians deviate from the X. laevis developmental pattern. Among other adaptations, their embryos develop in foam nests on land or in pouches on their mother’s back or on a leaf guarded by a parent. The diversity of developmental patterns includes multinucleated oogenesis, lack of RNA localization, huge non-pigmented eggs, and asynchronous, irregular early cleavages. Variations in patterns of gastrulation highlight the modularity of this critical developmental period. Many species have eliminated the larva or tadpole and directly develop to the adult. The wealth of developmental diversity among amphibians coupled with the wealth of mechanistic information from X. laevis permit comparisons that provide deeper insights into developmental processes.
One contributing factor in the worldwide decline in amphibian populations is thought to be exposure of eggs to UV light. Enrichment of pigment in the animal hemisphere of eggs laid in the sunlight defends against UV damage, but little is known about the cell biological mechanisms controlling such polarized pigment patterns. Even less is known about how such mechanisms were modified during evolution to achieve the array of amphibian egg pigment patterns. Here, we show that ectopic expression of the γ-tubulin regulator, Shroom2, is sufficient to induce co-accumulation of pigment granules, spectrin, and dynactin in Xenopus blastomeres. Shroom2 and spectrin are enriched and co-localize specifically in the pigmented animal hemisphere of Xenopus eggs and blastulae. Moreover, Shroom2 mRNA is expressed maternally at high levels in Xenopus. By contrast to Xenopus, eggs and blastulae of Physalaemus pustulosus have very little surface pigmentation. Rather, we find that pigment is enriched in the perinuclear region of these embryos, where it co-localizes with spectrin. Moreover, maternal Shroom2 mRNA was barely detectable in Physaleamus, though zygotic levels were comparable to Xenopus. We therefore suggest that a Shroom2/spectrin/dynactin-based mechanism controls pigment localization in amphibian eggs, and that variation in maternal Shroom2 mRNA levels accounts in part for variation in amphibian egg pigment patterns during evolution.
Shroom2; Spectrin; pigmentation; melanosome; Physalaemus
Echo Planar Imaging (EPI), often utilized in functional MRI (fMRI) experiments, is well known for its vulnerability to inconsistencies in the static magnetic field (B0). Correction for these field inhomogeneities usually involves measuring the magnetic field at a single time point, and using this static information to correct a series of images collected over the course of one or multiple experiments. However, common phenomena, such as respiration and motion, change the characteristics of the B0 field homogeneity in a time-dependent and often unpredictable manner, rendering previous field measurements invalid. The effects of these changes are particularly large in the image phase, due to its direct and sensitive relationship to the magnetic field, and methods utilizing complex information can suffer enormously. This dependence can be exploited to estimate the temporal dynamics of the B0 field. Use of this information to correct fMRI data can provide more effective motion correction, reduce temporal “noise,” and can substantially restore statistically significant power to complex fMRI data analysis. All of the necessary information is embedded in complex EPI images, and results indicate this is a robust way to improve the quality of fMRI data, especially when used with complex analysis.
Exposure of rats to high strength static magnetic fields of 7 T or above has behavioral effects such as the induction of locomotor circling, the suppression of rearing, and the acquisition of conditioned taste aversion (CTA). To determine if habituation occurs across magnetic field exposures, rats were pre-exposed two times to a 14 T static magnetic field for 30 min on two consecutive days; on the third day, rats were given access to a novel 0.125% saccharin prior to a third 30-min exposure to the 14 T magnetic field. Compared to sham-exposed rats, pre-exposed rats showed less locomotor circling and an attenuated CTA. Rearing was suppressed in all magnet-exposed groups regardless of pre-exposure, suggesting that the suppression of rearing is more sensitive than other behavioral responses to magnet exposure. Habituation was also observed when rats under went pre-exposures at 2–3 hour intervals on a single day. Components of the habituation were also long lasting; a diminished circling response was observed when rats were exposed to magnetic field 36 days after 2 pre-exposures. To control for possible effects of unconditioned stimulus pre-exposure, rats were also tested in a similar experimental design with two injections of LiCl prior to the pairing of saccharin with a third injection of LiCl. Pre-exposure to LiCl did not attenuate the LiCl-induced CTA, suggesting that 2 pre-exposures to an unconditioned stimulus are not sufficient to explain the habituation to magnet exposure. Because the effects of magnetic field exposure are dependent on an intact vestibular apparatus, and because the vestibular system can habituate to many forms of perturbation, habituation to magnetic field exposure is consistent with mediation of magnetic field effects by the vestibular system.
vestibular; locomotor circling; rearing; conditioned taste aversion; lithium chloride
We observed a slight drop in the growth of Xenopus laevis and Pseudacris triseriata larvae following acute exposure (24–48 h) during egg development to three concentrations of TCDD (0.3, 3.0, 30.0 μg/l). Our exposure protocol was modeled on a previous investigation that was designed to mimic the effects of maternal deposition of TCDD. The doses selected were consistent with known rates of maternal transfer between mother and egg using actual adult body burdens from contaminated habitats. Egg and embryonic mortality immediately following exposure increased only among 48 h X. laevis treatments. Control P. triseriata and X. laevis completed metamorphosis more quickly than TCDD-treated animals. The snout-vent length of recently transformed P. triseriata did not differ between treatments although controls were heavier than high-dosed animals. Likewise, the snout-vent length and weight of transformed X. laevis did not differ between control and TCDD treatments. These findings provide additional evidence that amphibians, including P. triseriata and X. laevis are relatively insensitive to acute exposure to TCDD during egg and embryonic development. Although the concentrations selected for this study were relatively high, they were not inconsistent with our current understanding of bioaccumulation via maternal transfer.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); polyhalogenated aromatic hydrocarbons (PHAHs); tadpole development
Prophase I oocytes, free of follicle cells, and metaphase II eggs of the amphibian Xenopus laevis were subjected to transient treatments with the protein kinase C activators, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 1-olyeoyl-2-acetyl-sn-glycerol. In both oocytes and eggs, these treatments triggered early events of amphibian development: cortical granule exocytosis, cortical contraction, and cleavage furrow formation. Surprisingly, activation of oocytes occurred in the absence of meiotic resumption, resulting in cells with an oocytelike nucleus and interior cytoplasm, but with a zygotelike cortex. PMA-induced activation of oocytes and eggs did not require external calcium, a prerequisite for normal activation of eggs. PMA-induced activation of eggs was inhibited by retinoic acid, a known inhibitor of protein kinase C. In addition, pretreatment of eggs with retinoic acid prevented activation by mechanical stimulation and inhibited activation by calcium ionophore A23187. The results suggest that protein kinase C activation is an integral component of the Xenopus fertilization pathway.
Interactions between a micro-magnet array and living cells may guide the establishment of cell networks due to the cellular response to a magnetic field. To manipulate mesenchymal stem cells free of magnetic nanoparticles by a high magnetic field gradient, we used high quality micro-patterned NdFeB films around which the stray field’s value and direction drastically change across the cell body. Such micro-magnet arrays coated with parylene produce high magnetic field gradients that affect the cells in two main ways: i) causing cell migration and adherence to a covered magnetic surface and ii) elongating the cells in the directions parallel to the edges of the micro-magnet. To explain these effects, three putative mechanisms that incorporate both physical and biological factors influencing the cells are suggested. It is shown that the static high magnetic field gradient generated by the micro-magnet arrays are capable of assisting cell migration to those areas with the strongest magnetic field gradient, thereby allowing the build up of tunable interconnected stem cell networks, which is an elegant route for tissue engineering and regenerative medicine.
We have purified the major DNA ligase from Xenopus laevis eggs and raised antibodies against it. Estimates from SDS PAGE indicate that this DNA ligase is a 180 kDa protein. This enzyme is similar to the mammalian type I DNA ligase which is presumed to be involved in DNA replication. We have also analysed DNA ligase activity during X. laevis early development. Unfertilized eggs contain the highest level of activity reflecting the requirement for a large amount of DNA replicative enzymes for the period of intense replication following fertilization. In contrast with previous studies on the amphibians axolotl and Pleurodeles, the major DNA ligase activity detected during X. laevis early development is catalysed by a single enzyme: DNA ligase I. And the presence of this DNA ligase I in Xenopus egg before fertilization clearly demonstrates that the exclusion process of two forms of DNA ligase does not occur during X. laevis early development.
To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Domínguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289–1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5–10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985–993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation.
The literature on biological effects of magnetic and electromagnetic fields commonly utilized in magnetic resonance imaging systems is surveyed here. After an introduction on the basic principles of magnetic resonance imaging and the electric and magnetic properties of biological tissues, the basic phenomena to understand the bio-effects are described in classical terms. Values of field strengths and frequencies commonly utilized in these diagnostic systems are reported in order to allow the integration of the specific literature on the bio-effects produced by magnetic resonance systems with the vast literature concerning the bio-effects produced by electromagnetic fields. This work gives an overview of the findings about the safety concerns of exposure to static magnetic fields, radio-frequency fields, and time varying magnetic field gradients, focusing primarily on the physics of the interactions between these electromagnetic fields and biological matter. The scientific literature is summarized, integrated, and critically analyzed with the help of authoritative reviews by recognized experts, international safety guidelines are also cited.
We have observed the presence of membrane junctions formed between the plasma membrane and cortical endoplasmic reticulum of mature, unactivated eggs of xenopus laevis. The parallel, paired membranes of the junction are separated by a 10-mn gap within which electron-dense material is present. This material occurs in patches with an average center-to-center distance of approximately 30 nm. These junctions are rare in immature (but fully grown) oocytes (approximately 2 percent of the plasma membrane is associated with junctions) and increase dramatically during progesterone-induced maturation. Junctions in the mature, unactivated egg are two to three times more abundant in the animal hemisphere (25-30 percent of the plasma membrane associated with junction) as compared with the vegetal hemisphere (10-15 percent). Junction density decreases rapidly to values characteristic of immature oocytes in response to egg activation. The plasma membrane-ER junctions of xenopus eggs are strikingly similar in structure to membrane junctions in muscle cells thought to be essential in the triggering of intracellular calcium release from the sarcoplasmic reticulum. In addition, the junctions’ distinctive, animal-vegetal polarity of distribution, their dramatic appearance during maturation, and their disapperance during activation are correlated with previously documented patterns of calcium-mediated events in anuran eggs. We discuss several lines of evidence supporting the hypothesis that these junctions in xenopus eggs are sites that transduce extracellular events into intracellular calcium release during fertilization and activation of development.
Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.
The homogeneity and stability of the static magnetic field are of paramount importance to the accuracy of MR procedures that are sensitive to phase errors and magnetic field inhomogeneity. It is shown that intense gradient utilization in clinical horizontal-bore superconducting MR scanners of three different vendors results in main magnetic fields that vary on a long time scale both spatially and temporally by amounts of order 0.8–2.5 ppm. The observed spatial changes have linear and quadratic variations that are strongest along the z direction. It is shown that the effect of such variations is of sufficient magnitude to completely obfuscate thermal phase shifts measured by proton-resonance frequency-shift MR thermometry and certainly affect accuracy. In addition, field variations cause signal loss and line-broadening in MR spectroscopy, as exemplified by a fourfold line-broadening of metabolites over the course of a 45 min human brain study. The field variations are consistent with resistive heating of the magnet structures. It is concluded that correction strategies are required to compensate for these spatial and temporal field drifts for phase-sensitive MR protocols. It is demonstrated that serial field mapping and phased difference imaging correction protocols can substantially compensate for the drift effects observed in the MR thermometry and spectroscopy experiments.
Magnetic field; Field homogeneity; Magnet stability; Field mapping; MR thermometry
A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.
Exposure to anthropogenic chemicals during development can disrupt the morphogenesis of organ systems. Use of the herbicide atrazine has been debated in recent years because of its implicated, but poorly characterized, effects on vertebrates. Previous studies primarily examined the effects of atrazine exposure during metamorphosis or early developmental stages of amphibians.
We sought to identify and characterize the susceptibility during the often-overlooked developmental stage of organ morphogenesis.
We used a static renewal experimental treatment to investigate the effects of 10, 25, and 35 mg/L atrazine from early organ morphogenesis through the onset of tadpole feeding in the aquatic amphibian model system, Xenopus laevis. We quantified malformations of the body axis, heart, and intestine, as well as apoptosis in the midbrain and pronephric kidney.
We found a significant dose-dependent increase in the percentage of atrazine-exposed tadpoles with malformations of multiple tissues including the main body axis, circulatory system, kidney, and digestive system. Incidence of apoptotic cells also increased in the both midbrain and kidney of atrazine-exposed tadpoles.
Our results demonstrate that acute atrazine exposure (10–35 mg/L for ≤ 48 hr) during early organ morphogenesis disrupts proper organ development in an amphibian model system. The concurrent atrazine-induced apoptosis in the pronephric kidney and midbrain begins to elucidate a mechanism by which atrazine may disrupt developmental processes in nontarget organisms.
atrazine; development; morphogenesis; organogenesis; teratogens; Xenopus laevis
Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.
We have examined the effects of Xenopus pp60c-src with constitutive kinase activity on the morphology and maturation of Xenopus laevis oocytes. When RNA encoding this deregulated variant was injected into stage VI oocytes, we observed a gross alteration in the cortex of the oocyte. This alteration involved aggregation of pigment and invagination of the cortex in a large area proximal to the site of injection. This phenomenon was not seen in oocytes injected with RNA encoding wild-type pp60c-src. We have correlated this phenomenon with the tyrosine phosphorylation of 84- and 100-kDa proteins. These phosphorylated proteins colocalized with the alteration in the oocyte cortex when assayed by both biochemical and immunocytochemical methods. Neither the pigment aggregation nor phosphorylation of the 84- and 100-kDa proteins was observed in oocytes expressing a nonmyristoylated version of the deregulated pp60c-src. Expression of deregulated Xenopus fyn, a src-family member, resulted in a phenotype similar to that seen with deregulated src. However, in the fyn-injected oocytes, many more proteins were phosphorylated on tyrosine than in the src-injected oocytes. Progesterone stimulation of oocytes expressing deregulated pp60c-src resulted in an increase in the number of tyrosine-phosphorylated proteins. This change may represent the response of pp60src to the resumption of the cell cycle in maturing oocytes. These data suggest that the oocyte may be a particularly useful system for investigating the role of pp60c-src in the regulation of cytoskeletal structure and in the regulation of events associated with the cell cycle.