Calcium-induced dehiscence of cortical granules in Xenopus laevis oocytes

Microinjection of 0.1 microgram of Ca++ into Xenopus laevis oocytes induces breakdown of the cortical granules. The cortical granules disappeared in both full grown (Stage VI) and small growing (Stage IV) oocytes. Microinjection of Mg++, K+, or Na+ had no effect on cortical granules in either Stage IV or Stage VI oocytes. Small quantities (0.03 microgram) of Ca++ induced dehiscence of the cortical granules only in proximity to the injection site.     

Cell cycle and developmental control of cortical excitability in Xenopus laevis

Interest in cortical excitability—the ability of the cell cortex to generate traveling waves of protein activity—has grown considerably over the past 20 years. Attributing biological functions to cortical excitability requires an understanding of the natural behavior of excitable waves and the ability to accurately quantify wave properties. Here we have investigated and quantified the onset of cortical excitability in Xenopus laevis eggs and embryos and the changes in cortical excitability throughout early development. We found that cortical excitability begins to manifest shortly after egg activation. Further, we identified a close relationship between wave properties—such as wave frequency and amplitude—and cell cycle progression as well as cell size. Finally, we identified quantitative differences between cortical excitability in the cleavage furrow relative to nonfurrow cortical excitability and showed that these wave regimes are mutually exclusive.     

Localization of cortical granule lectin ligand in Xenopus laevis egg jelly

The block to polyspermy in Xenopus laevis involves an interaction between a cortical granule lectin, released at fertilization, and a ligand located in the egg extracellular matrix. The egg extracellular matrix in X. laevis consists of a vitelline envelope and three distinct jelly layers, designated J1, J2 and J3. To localize cortical granule lectin ligand in the egg extracellular matrix, we used enzyme-linked lectin assays that showed that cortical granule lectin ligands were absent in J2, J3 and the vitelline envelope. Cortical granule lectin bound to a ligand(s) in J1 in a galactose-dependent fashion. In addition, we separated egg jelly macromolecules electrophoretically and, in conjunction with western blotting, have shown that J1 contains two major, high molecular weight ligands for cortical granule ligand. Finally, using confocal microscopy, we demonstrated that the ligand(s) for cortical granule lectin occupies a 20–30 μm thick band in a region of J1 just proximal to the vitelline envelope.     

The area octavo-lateralis in Xenopus laevis

Summary Central projections of afferents from the lateral line nerves and from the individual branches of the VIIIth cranial nerve in Xenopus laevis and Xenopus mülleri were studied by the application of HRP to the cut end of the nerves.Upon entering the rhombencephalon, the lateral line afferents form a longitudinal fascicle of ascending and descending branches in the ventro-lateral part of the lateral line neuropile. The fascicle exhibits a topographic organization, that is not reflected in the terminal field of the side branches. The terminal field can be subdivided into a rostral, a medial and a caudal part, each of which shows specific branching and terminal pattern of the lateral line afferents. These different patterns within the terminal field are interpreted as the reflection of functional subdivisions of the lateral line area. The study did not reveal a simple topographic relationship between peripheral neuromasts and their central projections.Two nuclei of the alar plate with significant lateral line input were delineated: the lateral line nucleus (LLN) and the medial part of the anterior nucleus (AN). An additional cell group, the intermediate nucleus (IN), is a zone of lateral line and eighth nerve overlap, although such zones also exist within the ventral part of the LLN and the dorsal part of the caudal nucleus (CN). Six nuclei which receive significant VIIIth nerve input are recognized: the cerebellar nucleus (CbN), the lateral part of the anterior nucleus, the dorsal medullary nucleus (DMN), the lateral octavus nucleus (LON), the medial vestibular nucleus (MVN) and the caudal nucleus (CN).All inner ear organs have more than one projection field. All organs project to the dorsal part of the LON and the lateral part of the AN. Lagena, amphibian papilla and basilar papilla project to separate regions of the dorsal medullary nucleus (DMN). There is evidence for a topographic relation between the hair cells of the amphibian papilla (AP) and the central projections of AP fibers. The sacculus projects extensively to a region between the DMN and the LON. Fibers from the sacculus and the lagena project directly to the superior olive. Fibers from the utriculus and the three crista organs terminate predominantly in the medial vestibular nucleus (MVN) and in the adjacent parts of the reticular formation, and their terminal structures appear to be organotopically organised. Octavus fiber projections to the cerebellum and to the spinal cord are also described.     

Protamine polymorphism in Xenopus laevis laevis

Protamines from individual frogs of the subspecies Xenopus laevis laevis were compared by electrophoresis on polyacrylamide gels containing acetic acid, urea, and Triton X-100 to determine if the expression of protamine genes differs among individuals. Two electrophoretic bands, SP2a and SP2b, appeared to be expressed as allelic variants. Of 33 frogs, 19 expressed only SP2a, 11 expressed both SP2a and SP2b, and three expressed only SP2b. Electrophoretic analysis of partial V8 protease digests could not distinguish the peptides released from SP2a and SP2b. Differences in sperm development between individuals were not detected by light or electron microscopy. The results suggest that protamine polymorphism can exist among individuals of a species without an apparent effect on sperm development or sperm function.     

On the contents of the cortical granules from Xenopus laevis eggs

The extruded contents of the cortical granules in eggs of Xenopus laevis were solubilized by exposure to divalent metal ion chelators. Chelator extraction of cortical granule (CG) material from intact fertilized or artificially activated eggs was quantitated by fluorescence spectroscopy. The isolated fertilization envelope, formed upon interaction between CG material and the preexisting vitelline envelope, was also subject to extraction. An ultrastructural analysis revealed that chelator exposure resulted in the disruption of the structural integrity of the CG-derived F-component of the fertilization envelope. CG material was isolated from Xenopus ova by three procedures: (1) extrusion from artificially activated, dejellied eggs; (2) extraction of intact, fertilized eggs; and (3) extraction of isolated fertilization envelopes. Only 4–5% of the CG protein recovered by extrusion or by extraction of the intact fertilized egg could be associated with the isolated fertilization envelopes. One predominant polypeptide fraction with an identical relative mobility was demonstrated in all CG preparations upon polyacrylamide gel electrophoresis in SDS. Polymeric forms of CG protein were detected in chelator extracted preparations. The presence of an intact jelly coat during CG breakdown was a prerequisite to the transformation of the vitelline envelope to a fertilization envelope with altered physicochemical characteristics. Further, the CG-derived F-component of the fertilization envelope did not appear to play a critical role in determining the physicochemical properties of the fertilization envelope.     

Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs.

Following fertilization, the Xenopus egg cortex rotates relative to the cytoplasm by 30 degrees about a horizontal axis. The direction of rotation, and as a result the orientation of the embryonic body axes, is normally specified by the position of sperm entry. The mechanism of rotation appears to involve an array of aligned microtubules in the vegetal cortex (Elinson and Rowning, 1988, Devl Biol. 128, 185-197). We performed anti-tubulin immunofluorescence on sections to follow the formation of this array. Microtubules disappear rapidly from the egg following fertilization, and reappear first in the sperm aster. Surprisingly, astral microtubules then extend radially through both the animal and vegetal cytoplasm. The cortical array arises as they reach the vegetal cell surface. The eccentric position of the sperm aster gives asymmetry to the formation of the array and may explain its alignment since microtubules reaching the cortex tend to bend away from the sperm entry side. The radial polymerization of cytoplasmic microtubules is not dependent on the sperm aster or on the female pronucleus: similar but more symmetric patterns arise in artificially activated and enucleate eggs, slightly later than in fertilized eggs. These observations suggest that the cortical microtubule array forms as a result of asymmetric microtubule growth outward from cytoplasm to cortex and, since cortical and cytoplasmic microtubules remain connected throughout the period of the rotation, that the microtubules of the array rotate with the cytoplasm.     

Cysteine string proteins are associated with cortical granules of Xenopus laevis oocytes

Cysteine string proteins (csps) are associated with secretory organelles in a wide range of eukaryotic cells. Functional studies of these proteins indicate that they subserve one or more vital steps in the pathway of regulated exocytosis. Here, we document the presence of csps in fully grown (stage VI) oocytes of the frog, Xenopus laevis. Both Northern and immunoblot data support the conclusion that csps are expressed in these cells. In addition, immunoreactive csp is seen even at the earliest stage of oocyte development, namely, in stage I oocytes. Finally, immunoblot and immunocytochemical results indicate that csps are associated with cortical granules of stage II-VI oocytes. These observations suggest that csps participate in the cortical reaction that underlies the sustained block to polyspermy in Xenopus eggs. Moreover, because of the relative ease of manipulating cells as large as Xenopus oocytes, this system harbors considerable promise as a model for studying the role of csps and other proteins in exocytotic events.     

Angiotensin II stimulates an endogenous response in Xenopus laevis ovarian follicles

While responses to angiotensin II have previously been induced in Xenopus laevis oocytes after injection of messenger RNA extracted from mammalian tissue, no endogenous responses of ovarian tissue to this hormone have been reported. Here we describe such an endogenous dose-dependent response to angiotensin II, detected by conventional electrophysiological techniques, in follicular oocytes. The ED50 of the response was estimated to be 0.15 +/- 0.07 microM (S.E.M.). Maximal depolarization, obtained at 1 microM angiotensin II, was 18.3 +/- 1.4 mV (n = 18, three experiments using oocytes from two toads, mean resting membrane potential = -42 +/- 2 mV). The response was absent from collagenase-treated oocytes or follicular oocytes treated with octanol, suggesting that the receptors are predominantly in the follicular layer surrounding the oocytes.     

Association of Rous sarcoma virus DNA with Xenopus laevis spermatozoa and its transfer to ova through fertilization

Mature Xenopus laevis spermatozoa are capable of binding plasmid pAPrC carrying the complete Rous sarcoma virus (RSV) DNA. Each sperm cell associates, on an average, with 70–160 molecules of the plasmid DNA in a DNase resistant form, if the spermatozoa were exposed to the DNA at a concentration of 1.0–1.4 μg/10⁷ sperm cells. Fertilization with pAPrC-treated spermatozoa induced developmental malformations in 25–30% of embryos. Immunohistochemical analysis of tissue sections from defective animals revealed aberrations in myotomal structures, and increased expression of pp60^src protein in myoblasts, neuronal tube, and epidermis. The presence of characteristic v-src and RSV-long terminal repeat (LTR) sequences in X. laevis DNA was detected by PCR analysis. Embryonic RNA hybridized with a src-specific and an RSV-LTR specific probes indicating expression of the viral DNA. Plasmid DNAs without the v-src gene (pATV9) or completely free of any RSV sequences (pBR322) did not induce any changes in embryonic development. Our results provide evidence that the pBR322-cloned DNA form of the RSV genome associates with frog sperm cells in a DNase-resistant manner suggesting internalization and may be subsequently carried into eggs during the process of artificial fertilization. Correlation between the defective morphogenesis of X. laevis and increased expression of the src gene as well as an interference of RSV DNA with the developmental programs of frog embryos are discussed. © 1996 Wiley-Liss, Inc.     

Decay of the oocyte-type heat shock response of Xenopus laevis

Xenopus oocytes have a complex heat shock response. During transition of the oocyte into fertilized egg, the heat shock response undergoes several qualitative and quantitative changes culminating in its complete extinction. Heat shock induces oocytes to synthesize four heat shock proteins (hsps): 83, 76, 70, and 57. After ovulation, two additional proteins (hsps 22 and 16) are inducible. The heat shock response of spawned eggs can be modified by changing the ionic configuration of the external medium and by adding pyruvate and oxaloacetate to the media. Since Xenopus eggs do not synthesize mRNA, these modifications to the external medium apparently alter the utilization of preexisting messenger RNAs in protein synthesis. Artificial activation terminates inducibility of hsps 76, 57, and 16 and diminishes the hsp 70 response. Two new heat shock proteins-66 and 48-are also inducible in artificially activated eggs. Fertilization, on the other hand, terminates the heat shock response; no hsps can be induced. However, hsp 70 appears to be made constitutively in fertilized eggs. RNA blot analyses reveal that oogenic hsp 70 messenger RNA is retained in eggs and early embryos. This messenger is apparently used for heat-induced synthesis of hsp 70 before fertilization and for constitutive synthesis of hsp 70 in zygotes.