Membrane Junctions in Xenopus Role in Calcium Regulation Eggs: Their Distribution Suggests a

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-nm gap within which electron-dense material is present. This material occurs in patches with an average center-to-center distance of ~30 nm. These junctions are rare in immature (but fully grown) oocytes (~2% 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% of the plasma membrane associated with junctions) as compared with the vegetal hemisphere (10-15%). 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 disappearance 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. Alterations of intracellular calcium activity have been demonstrated to be important regulators of early developmental processes in the eggs of a large number of organisms (26, 45, 47). Although the source of intracellular calcium is unknown, it has been suggested that the endoplasmic reticulum (ER) might be involved in the regulation of calcium activity in a manner analogous to that of the sarcoplasmic reticulum (SR) of muscle cells (5, 15). In muscle cells calcium is sequestered in and released from regions of the SR which form specialized junctions with regions of the plasma membrane (12, 44). Although the mechanism by which these junctions act is still unknown, there is evidence that they somehow respond to a depolarization of the plasma membrane by triggering release of Ca 2÷ from the SR (1, 14, 16, 32, 36, 43). We have been investigating the role of the cortical ER of Xenopus eggs in the regulation of calcium activity and report the first observation in an egg of membrane junctions that are morphologically similar to the plasma membrane-SR junctions of muscle ceils. We also report that these junctions in Xenopus eggs exhibit temporal and spatial patterns of distribution that are coincident with previously documented patterns of calcium-mediated events. MATERIALS AND METHODS Gravid, female Xenopus laevis were purchased from Nasco Science (Fort Atkinson, WI). Full-grown (Stage VI) oocytes (7) along with their encapsulating follicle cells were removed with watchmaker forceps from the ovaries of decapitated females and were incubated in O-R2 saline (48), pH 7.8. Oocytes were induced to undergo meiotic maturation (42) in vitro by incubating them continuously in 4 x 10 -6 M progesterone in O-R2 saline (20-22°C). Maturing oocytes were removed for fLxation at various times during the following 9-h period. Unfertilized, mature eggs were obtained by injecting gravid females with 100 IU human chorionic gonadotropin (Sigma Chemical Co., St. Louis, MO), followed 36-48 h later with an additional 800 IU. Eggs were stripped from the animals 6-8 h after the latter injection. For transmission electron microscopy, immature and maturing oocytes were fixed in ice-cold 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 90 min. In the case of mature, ovulated eggs, some were fixed immediately in icecold 6% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 90 rain, whereas with most, the jelly layers first were removed by incubating the eggs in 2% cysteine in 27% DeBoer's saline (53), pH 7.2, for 3-5 min. Dejellied eggs were rinsed three times and incubated 10-30 min in 50% O-R2 saline (pH 7.8) before fixation in ice-cold 3% glutaraldehyde in 0.1 ~1 phosphate buffer (pH 7.2) for 90 THE JOURNAL OF CELL BIOLOGY. VOLUME 96 APRIL 1983 1159-1163 © The Rockefeller U n iversity Press. 0021-9525/83/04/1159/05 $1.0