Involvement of cyclic adenosine 3′,5′-monophosphate in methylation during 1-methyladenine production by starfish ovarian follicle cells

Summary

Resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeAde) produced by ovarian follicle cells under the influence of a gonad-stimulating substance (GSS). With respect to 1-MeAde production by follicle cells of the starfish, Asterina pectinifera, (1) the action of GSS is initiated by a receptor mediated activation of G-proteins, resulting in the activation of adenylate cyclase and cyclic AMP (cAMP) formation; (2) 1-MeAde produced under the influence of GSS is not prestored within the follicle cells but is newly synthesized from a 1-MeAde precursor; (3) AMP plays an important role in the process of methylation during 1-MeAde biosynthesis induced by GSS.     

Induction and development of exogastrulae in the starfish,Pisaster ochraceus

The process of mouth and coelom formation in exogastrulae of the starfish, Pisaster ochraceus, induced by LiCl, has been studied with the light microscope, scanning and transmission electron microscopes. Bending and segmentation of the exogastrulated archenteron with the formation of either single or double coelomic pouches follows the same schedule as the control. In addition, a region of the exogastrular ectoderm, which corresponds to the area of the mouth in controls, undergoes invagination. Early morphogenesis of the archenteron and invagination of the ectoderm during mouth formation appear to be intrinsic properties of these structures.

At the time of mouth formation in the controls, a discrete region adjacent to the distal end of the exogastrulated archenteron becomes sticky. Examination of this region shows that the surfaces of the archenteron cells are relatively smooth and that processes of the mesenchyme cells extend between them. The evidence suggests that the mesenchyme cells are responsible for the stickiness, and that they may guide the archenteron and ectoderm into contact and maintain the contact during normal mouth formation.     

Adhesive papillae on the brachiolar arms of brachiolaria larvae in two starfishes, Asterina pectinifera and Asterias amurensis, are sensors for metamorphic inducing factor(s)

It has been hypothesized by Barker that starfish brachiolaria larvae initiate metamorphosis by sensing of metamorphic inducing factor(s) with neural cells within the adhesive papillae on their brachiolar arms. We present evidence supporting Barker's hypothesis using brachiolaria larvae of the two species, Asterina pectinifera and Asterias amurensis. Brachiolaria larvae of these two species underwent metamorphosis in response to pebbles from aquaria in which adults were kept. Time-lapse analysis of A. pectinifera indicated that the pebbles were explored with adhesive papillae prior to establishment of a stable attachment for metamorphosis. Microsurgical dissections, which removed adhesive papillae, resulted in failure of the brachiolaria larvae to respond to the pebbles, but other organs such as the lateral ganglia, the oral ganglion, the adhesive disk or the adult rudiment were not required. Immunohistochemical analysis with a neuron-specific monoclonal antibody and transmission electron microscopy revealed that the adhesive papillae contained neural cells that project their processes towards the external surface of the adhesive papillae and they therefore qualify as sensory neural cells.     

Centrifugal bisection of starfish oocytes

Immature oocytes or mature eggs of starfish were centrifuged in a sucrose density gradient. They were then separated into two fractions of fragments, nucleate light fragments and anucleate heavy fragments. Vital-staining experiments showed that the oocytes were elongated along the animal-vegetal (AV) axis during the centrifugation in a contrast to centrifuged eggs whose centrifugal axis was not related to the AV axis. The light and heavy oocyte fragments were comprised of animal and vegetal halves of oocytes, respectively. When matured and fertilized, most of the light oocyte fragment-derived embryos failed gastrulation and developed into Dauerblastulae. Two-dimensional gel electrophoretic analysis of fragments revealed that three basic proteins were predominantly enriched in the heavy oocyte fragments but scarcely detected in the light oocyte fragments. One of these proteins, App20, was identified as a homologue of cyclophilin (peptidyl-prolyl cis-trans isomerase). The present study provides a simple means of separating a population of starfish oocytes into animal and vegetal halves, thereby enabling us to analyze any difference of components between animal and vegetal cytoplasm of the oocytes.     

A receptor linked to a Gi-family G-protein functions in initiating oocyte maturation in starfish but not frogs

The stimulation of oocyte maturation by 1-methyladenine in starfish, and by a steroid in frogs, has been proposed to involve G-protein-coupled receptors. To examine whether activation of receptors linked to G(i) or G(z) was sufficient to cause oocyte maturation, we expressed mammalian G(i)- and G(z)-linked receptors in starfish and frog oocytes. Application of the corresponding agonists caused meiosis to resume in the starfish but not the frog oocytes. We confirmed that the receptors were effectively expressed in the frog oocytes by using a chimeric G-protein, G(qi), that converts input from G(i)- and G(z)-linked receptors to a G(q) output and results in a contraction of the oocyte's pigment. These results argue against G(i) or G(z) functioning to cause maturation in frog oocytes. Consistently, maturation-inducing steroids did not cause pigment contraction in frog oocytes expressing G(qi), and G(z) protein was not detectable in frog oocytes. For starfish oocytes, however, our results support the conclusion that G(i) functions in 1-methyladenine signaling and suggest the possibility of using frog oocyte pigment contraction as an assay to identify the 1-methyladenine receptor. To test this concept, we coexpressed G(qi) and a starfish adenosine receptor in frog oocytes and showed that applying adenosine caused pigment contraction.     

Heteroplasmic conjugates formed by the fusion of starfish oocyte pairs with a 12 minute time difference in the maturation phase

Two starfish oocytes with a 12 min time difference in the maturation phase were fused together with electric pulses to make a heteroplasmic conjugate. The starfish used were Asterina pectinifera. The emergence of the first meiotic spindle and the extrusion of the polar bodies in the conjugate were timed. Under polarization microscopy two meiotic spindles emerged with a time difference of 10-11 min, which is close to the time difference in the maturation phase between the original oocytes before fusion. In contrast, subsequent formation of the first two polar bodies occurred successively with a short time lag of 1-3 min between them. Times for the formation of both polar bodies were midway between the anticipated times for polar body formation in respective non-fused control oocytes. Thus, in one nucleus the meiotic division was delayed, while in another nucleus it was accelerated, in a single heteroplasmic conjugate. These two sets of observations indicate the presence of a certain control system that regulates progression of the cell cycle at a point during the period from the entry into metaphase through to late anaphase of meiosis I in starfish oocytes. This type of cell cycle control in starfish oocytes is obviously distinct from the currently accepted view of the cell cycle control by the spindle assembly checkpoint that monitors unattached kinetochores of mitotic chromosomes.     

Formation of polar-body-like structures induced in polyspermic starfish oocytes that had been inseminated at the germinal vesicle stage

Immature starfish oocytes, which are arrested at the first meiotic prophase and contain a large nucleus called the germinal vesicle (GV), are known to accept multiple sperm on insemination. We found that if these polyspermic starfish oocytes are induced to mature, they often form small protrusion(s) adjacent to the first polar body emitted shortly earlier. We refer to these protrusion(s) as 'polar-body-like structures (PLS).' Fluorescent staining of PLS indicated that they were not merely cytoplasmic protrusions, but contained some chromatin. Maturing process of these polyspermic oocytes was examined by immnofluorescent staining, which showed that: (i) numerous sperm asters were observed after the onset of GV breakdown; (ii) before the first polar body (PB1) emission, a complex microtubular structure resembling a multipolar spindle was formed; and (iii) several isolated asters were observed after PB1 emission. These results indicate that PLS formation may be induced by interaction of meiosis-I spindle with paternal centrosomes incorporated at GV stage.     

Development of the starfish <Emphasis Type="Italic">Asterias amurensis</Emphasis> under laboratory conditions

Under laboratory conditions the development of the starfish Asterias amurensis Lütken from Vostok Bay (Sea of Japan) was studied at 14 and 17°C. At 14°C and a salinity of 31.6–32.6, ciliated coeloblastulae hatched from egg envelopes 19 h after fertilization. At this temperature the development proceeded slowly and stopped at the stage of bipinnaria. At 17°C and normal salinity of seawater, the development of A. amurensis was successful. The swimming blastula appeared in 14 h. It took 30.5 h for the embryos to reach the gastrula stage. The larvae began swimming in a horizontal position with the apical tip ahead. The dipleurula appeared at 60 h. These larvae began feeding. At 71 h after the beginning of development, the early bipinnaria has developed. In the larva, the edged ciliated band, the preoral plate, and the anal plate were already formed. At the age of 4.2 days, the larvae reached the stage of bipinnaria and the brachiolaria stage developed by 26–28 days after fertilization. The larvae had three identical brachiolar arms with attachment papillae on their tips and an attachment disk. In 37–44 days (at 17°C) the pelagic phase of A. amurensis development was completed by the attachment of larvae to the bottom plates and termination of metamorphosis. Most likely, the specificity to a substrate is not expressed in the brachiolaria of A. amurensis. They can settle on almost any hard substrate which is coated with a bacterial film. The newly settled juvenile starfish had five well-developed arms and moved using their ambulacral podia.Original Russian Text Copyright © 2005 by Biologiya Morya, Kashenko.