calcimycin and 1-methyladenine

Starfish oocytes can be fertilized after germinal vesicle breakdown (GVBD) and artificial parthenogenesis can be induced by activating the oocytes after GVBD (post-GVBD activation). In the present study, parthenogenotes were obtained by the activation of immature oocytes with caffeine before treatment with 1-methyladenine (1-MeAde) to induce oocyte maturation. Most of the caffeine-treated eggs developed as tetraploids, as parthenogenotes produced by the post-GVBD activation. The parthengenotes were derived only from eggs that failed to extrude polar bodies, mostly from eggs failing to extrude a second polar body. Eggs derived from immature oocytes activated by A23187, treated with 1-MeAde and post-treated with cytochalasin B failed to extrude polar bodies, and eventually developed into parthenogenetic embryos. These results indicate that the present parthenogenesis mechanism shares the same characteristics as that achieved by post-GVBD activation in the suppression of polar body formation as a key means for successful starfish parthenogenesis.

Topics: Adenine; Animals; Caffeine; Calcimycin; Cytochalasin B; Female; Ionophores; Oocytes; Parthenogenesis; Polyploidy; Starfish

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 approximately 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 mu m 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 mu m, 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.

Topics: Adenine; Animals; Calcimycin; Cell Membrane; Exocytosis; Fertilization; Meiosis; Microscopy, Electron; Microvilli; Oocytes; Starfish; Vitelline Membrane

An increased phosphorylation of ribosomal protein S6 has been shown to be correlated with an increase of intracellular pH (pHi) and with stimulation of protein synthesis in many systems. In this research changes in ribosome phosphorylation following hormone-induced meiotic maturation and fertilization or activation by ionophore A23187 were investigated in starfish oocytes. The hormone was found to stimulate, even in the absence of external Na+, the phosphorylation on serine residues of an Mr 31,000 protein identified as S6, as well as that of an acidic Mr 47,000 protein, presumably S1, on threonine residues. Phosphorylation of ribosomes was an early consequence of hormonal stimulation and did not decrease after completion of meiotic maturation. Fertilization or activation by ionophore of prophase-arrested oocytes also stimulated ribosome phosphorylation. Only S6 was labeled in this case, but to a lesser extent than upon hormone-induced meiotic maturation. Changes in pHi were monitored with ion-specific microelectrodes throughout meiotic maturation and following either fertilization or activation. The pHi did not change before germinal vesicle breakdown (GVBD) following hormone addition, but it increased before first polar body emission. It also increased following fertilization or activation by ionophore or the microinjection of Ca-EGTA. In all cases, alkalinization did not depend on activation of an amiloride-sensitive Na+/H+ exchanger. Microinjection of an alkaline Hepes buffer or external application of ammonia, both of which increased pHi, prevented unfertilized oocytes from arresting after formation of the female pronucleus and induced chromosome cycling. Phosphorylation of S6 was still observed following fertilization or induction of maturation when pHi was decreased by external application of acetate, a treatment which suppressed the emission of polar bodies. Protein synthesis increased in prophase-arrested oocytes after fertilization or activation. It also increased after ammonia addition, although this treatment did not stimulate S6 phosphorylation.

Topics: Adenine; Amino Acids; Animals; Calcimycin; Female; Fertilization; Hydrogen-Ion Concentration; Ionophores; Meiosis; Oocytes; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Ribosomal Proteins; Starfish

Microinjection of EGTA into prophase-blocked oocytes does not inhibit hormone-induced meiosis reinitiation, although it prevents oocyte activation by fertilization, by ionophore A23187, or by subsequent microinjection of otherwise efficient Ca2+ buffers. In contrast microinjection of Ca2+ buffers inhibits 1-methyladenine-induced meiosis reinitiation. Oocytes can be released from Ca2+ inhibition by raising hormone concentration or by the subsequent transfer of cytoplasm taken from maturing oocytes. Ca2+-microinjected oocytes remain inhibited up to 1 h after microinjection, although free Ca2+ concentration comes back to its resting value less than 30 sec after microinjection. Cyanide, which decreases ATP content and depresses Ca2+-pumping activity, reversibly inhibits 1-methyladenine-induced meiosis reinitiation. These results do not support the hypothesis that Ca2+ is the second messenger of the hormone in meiosis reinitiation of starfish oocytes, although they support the view that elimination of Ca2+ from some component of the oocyte cortex (perhaps the plasma membrane) might be a compulsory event for transduction of the hormonal message.

Topics: Adenine; Animals; Calcimycin; Calcium; Egtazic Acid; Female; Meiosis; Oocytes; Ovum; Potassium Cyanide; Starfish