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Cortical changes in starfish (Asterina pectinifera) oocytes during 1-methyladenine-induced maturation and fertilisation/activation
- Frank J. Longo, Mark Woerner, Kazuyoshi Chiba, Motonori Hoshi
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Maturation of the starfish oocyte cortex to produce an effective cortical granule reaction and fertilisation envelope is believed to develop in three phases: (1) pre-methyladenine (1-MA) stimulation; (2) post-1-MA stimulation, pregerminal vesicle breakdown; and (3) post-germinal vesicle breakdown. The present study was initiated to identify what each of these phases may encompass, specifically with respect to structures associated with the oocyte cortex, including cortical granules, microvilli and vitelline layer. 1-MA treatment brought about an orientation of cortical granules such that they became positioned perpendicular to the oocyte surface, and an ∼ 4-fold decrease in microvillar length. A-23187 activation of immature oocytes treated with (10 min; pregerminal vesicle breakdown) or without 1-MA resulted in a reduction in cortical granule number of 21% and 41%, respectively (mature oocytes underwent a 96% reduction in cortical granules). Elevation of the fertilisation envelope in both cases was significantly retarded compared with activated mature oocytes. In activated mature oocytes, the vitelline layer elevated 20.0 ± 5.4 μm from the egg's surface, whereas in immature oocytes treated with just A-23187 or with 1-MA (10 min) and A-23187, it lifted 0.35 ± 0.1 and 0.17 ± 0.04 μm, respectively. The fertilisation envelopes of activated (or fertilised) immature oocytes also differed morphologically from those of mature oocytes. In activated, immature oocytes, the fertilisation envelope was not uniform in its thickness and possessed thick and thin regions as well as fenestrations. Additionally, it lacked a complete electron-dense stratum that characterised the fertilisation envelopes of mature oocytes. The nascent perivitelline space of immature oocytes was also distinguished by the presence of numerous vesicles which appeared to be derived from microvilli. Differences in the morphology of cortices from activated (fertilised) and non-activated, immature and mature oocytes substantiate previous investigations demonstrating three phases of cortical maturation, and are consistent with physiological changes that occur during oocyte maturation, involving ionic conductance of the plasma membrane, establishment of slow and fast blocks to polyspermy and elevation of a fertilisation envelope.
Stages leading to and following fusion of sperm and egg plasma membranes
- Frank J. Longo, Susan Cook, David H. McCulloh, Pedro I. Ivonnet, Edward L. Chambers
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The site of gamete interaction of electrophysiologically recorded Lytechinus variegatus eggs, fixed with osmium tetroxide (O5O4) and/or glutaraldehyde (GTA) at varying intervals after the onset of the increase in membrane conductance induced by an attached sperm, has been examined by high-voltage and conventional transmission electron microscopy. Although GTA and a GTA-O5O4 mixture iduced different electrical responses, specimens prepared with the two fixatives were ultrastructurally similar. In specimens observed within 5 s of the change in conductance, the acrosomal process projected through the vitelline layer and abutted the egg plasma membrane. A conspicuous layer of bindin surrounded the acrosomal process and connected the sperm to the egg's vitelline layer. In a fortuitous specimen fixed within 4 s following the change in conductance, the area of contact between the gamete plasma membranes possessed a trilaminar structure that separated the egg's and sperm's cytoplasms. The morphology of this area of contact was consistent with previously proposed intermediates of membrane fusion. Five to six seconds after the change in conductance, the sperm was connected to the egg via a narrow cytoplasmic bridge that consisted of the former acrosomal process and a projection of the egg cortex. The region of the bridge midway between the fused gametes was encircled by dense material that marked the site of sperm-egg fusion. Gamete interactions in which the activation potential was recorded (unclamped egg) were comparable in time and ultrastructure to events taking place in voltage-clamped eggs except for one major difference. Intact cortical granules (one to three) were observed beneath the tip of the incorporating sperm in unclamped eggs fixed following the onset of the activation potential, whereas all cortical granules dehisced in clamped eggs.
Inositol triphosphate receptors in sea urchin sperm
- Otilia Zapata, James Ralston, Carmen Beltraán, Jan B. Parys, Ji Long Chen, Frank J. Longo, Alberto Darszon
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Inositol 1,4,5-triphosphate (Ins(1,4,5)P3) is a second messenger that regulates Ca2+ channels in many important cell signalling pathways. In sea urchin sperm the outer investment of the egg triggers the acrosome reaction (AR) that involves Ins(1,4,5)P3 production and the opening of two Ca2+ channels. Here we have sought to identify a high-affinity Ins(1,4,5)P3 receptor in Strongylocentrotus purpuratus sperm. An Ins(1,4,5)P3 binding component was affinity-purified 12-fold from sperm extracts. It displayed similar characteristics to the Ins(1,4,5)P3 receptor from other sources: pH-dependent high affinity for Ins(1,4,5)3(KD=261 nM), a τ1/2 of association and dissociation of 50 and 40s, respectively, specificity (IC)50>5μM for Ins (1)P1, Ins(1,4)P2 and Ins(1,3,4,5 P4), and pharmacological sensitivity 10 and 100μ heparin/ml inhibited 75% and 100% binding respectively). An antibody against the carboxy-terminal of the type I Ins(1,4,5)P3 receptor of somatic cells recognised a Plasma membrane component in the sperm head and less intensely in the flagella. This antibody also recongnised a 240 kDa band from isolated head plasma membranes, and weakly in flagellar membrane. This IP3 receptor-like protein may mediate the sustained uptake of Ca2+ through the second Ca2+ chanel opened during the AR.