Radial Nerve Factor Research Articles

Starfish Gonadotropic Hormone: From Gamete-Shedding Substance to Relaxin-Like Gonad-Stimulating Peptide.

The first report of a gonadotropic substance in an invertebrate hot-water extract of radial nerve cords from starfish Asterias forbesi that induced the shedding of gametes when injected into the coelomic cavity in a ripe individual occurred in 1959. The active substance was named gamete-shedding substance (GSS) or radial nerve factor. GSS is the primary mediator of oocyte maturation and ovulation in starfish. However, the effect of GSS is indirect. Resumption of meiosis in immature oocytes and release from the ovary are induced by a second mediator, maturation-inducing hormone, identified as 1-methyladenine (1-MeAde) in starfish. The role of GSS is to induce 1-MeAde production by ovarian follicle cells. Thus, GSS was redesignated as gonad-stimulating substance (also GSS). Although GSS has been characterized biochemically as a peptide hormone, identification of the chemical structure had to wait until 2009. Fifty years after the initial finding, GSS was purified from the radial nerve cords of starfish Patiria pectinifera (P. pectinifera). The purified hormone was a heterodimer composed of A- and B-chains, with disulfide cross-linkages. Based on its cysteine motif, GSS is classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. More specifically, phylogenetic sequence analysis revealed that P. pectinifera GSS is a member of the relaxin-type peptide family. Therefore, GSS in starfish has been redesignated as relaxin-like gonad-stimulating peptide (RGP). Subsequently, orthologs of P. pectinifera RGP have been identified in other starfish species, including Asterias amurensis (A. amurensis), and Aphelasterias japonica (A. japonica).

Extrafollicular mediation of oocyte maturation by radial nerve factor in starfish Pisaster ochraceus

In starfish ovaries follicle cells that envelop each oocyte are thought to mediate the production of a maturation inducing substance (MIS), identified as 1-methyladenine, that induces maturation and spawning of oocytes after exposure to a gonadotropic substance secreted by the radial nerve (RNF). Studies were carried out to assess the possible role of extrafollicular cells within the ovarian wall in mediating this signal transduction process in the ovary of Pisaster ochraceus. Oocyte maturation and spawning occurred following the addition of RNF to intact ovarian tissue in vitro whereas no maturation occurred following the addition of RNF to germinal vesicle (GV) oocytes or GV oocytes surrounded by follicle cells. In contrast, oocyte maturation occurred when small ovarian wall fragments, lacking mature follicles, were incubated with GV oocytes and RNF. Neither actinomycin D nor cycloheximide altered RNF induction of oocyte maturation in the presence of the ovarian wall tissue whereas preheating (boiling water for 5 min) the tissue obliterated its response to RNF. Non-ovarian tissues failed to produce MIS in response to RNF. Results suggest that ovarian components other than the follicle cells that envelop fully grown immature oocyte are responsive to RNF and represent a significant and previously unrecognised intra-ovarian source of MIS.

Biosynthesis of 1-methyladenine by isolated segments of starfish ovaries.

Acid-soluble 1-methyladenine (1-MeA) and an insoluble fraction containing 1-MeA were formed when radioactive L-methionine or adenine (A) were incubated with starfish ovary segments. Attempts to prepare free ribonucleic acid (RNA) from ovaries failed since it was strongly bonded to protein as ribonucleoprotein (RN-P) which was therefore used in studies involving RNA. Incubation of ovary segments with [8-14C]adenine-8 (A-8-14C) yielded both soluble 1-Me-A-8-14C and RN-P-8-14C, and similar incubation with L-[methyl-14C]methionine yielded soluble 1-MeA-14CH3 and RN-P-14CH3. Hydrolysis of RN-P-8-14C with 1 N HCl at 100 degrees yielded 91% of the initial radioactivity in the purine fraction, and of this 90% was in A, 9% in 1-MeA, and 1% in 4(5)-amino-5(4)-imidazole carboxamidine (AIMCAD). With RN-P-14CH3 the corresponding figures were 20, 45, 27, and 28%. Degradation of 1-MeA-8-14C or RN-P-8-14C with 6 N HCl at 110 degrees yielded radioactive AIMCAD which, on heating at pH 12, gave radioactive 4(5)-amino-5(4)-imidazole carboxamide (AICA). When 1-MeA-14CH3 or RN-P-14CH3 were similarly degraded, radioactive AIMCAD was formed, but the AICA possessed little or no radioactivity due to the loss of the radioactive methyl group. Addition of radial nerve factor (GSS) increased the yield of 1-MeA up to 19 times when radioactive L-methionine was substrate, but was ineffective with radioactive A. S-[8-14C]Adenosylmethionine was only about 3% as effective as L-[methyl-14C]methionine in supporting formation of 1-MeA, and its slight activity was not enhanced by GSS. Immature ovary segments were much more active than those from mature ovaries in synthesizing 1-MeA. The results support the contention that formation of free 1-MeA in starfish ovarian tissues involves methylation of adenine residues in a polynucleotide followed by liberation of 1-MeA by enzymatic hydrolysis.

HOLOTHURIAN OOCYTE MATURATION INDUCED BY RADIAL NERVE

Endogeneous substances responsible for maturation of holothurian oocytes were examined. Water-extracts of radial nerves from five species of sea cucumbers induced oocyte maturation. Cross-experiments on the maturation-inducing activity of radial nerve extracts indicated that the radial nerve extracts cross-react effectively among the sea cucumbers examined. From its heat-stability, protease-sensitivity, dialyzability, and elution-patterns through `Sephadex' G-15 and G-50 columns, the active factor appears to be a heat-stable peptide of several thousands' daltons. The active factor, termed radial nerve factor, acted on oocytes in isolated ovaries or isolated oocytes having follicle cells to induce maturation. It was ineffective on oocytes deprived of follicle cells. Isolated ovaries or testes incubated with radial nerve factor produced a secondary factor which directly induces maturation of the follicle cell-free oocyte. Follicle cells isolated from follicle-oocyte complexes also produced the secondary factor in the presence of radial nerve factor. These results show that the radial nerve factor stimulates the follicle cells to produce a secondary factor, and the latter, in turn, directly induces oocyte maturation.

In vitro Inhibition of Oocyte and Follicular Maturation and Spawning in Starfish ( Asterias forbesi) by 2-4-Dinitrophenol

The in vitro effects of 2-4-dinitrophenol (DNP) on spawning and follicular and oocyte maturation in starfish ovaries and its various cellular components were investigated. Spawning and oocyte and follicular maturation induced by starfish gonadotropin radial nerve factor (RNF) in isolated ovarian fragments were all inhibited by appropriate doses of DNP. DNP inhibits processes which occur shortly after addition of the gonadotropin; in ovarian fragments insensitivity to DNP inhibition occurred shortly after addition of RNF but prior to initiation of spawning. Spontaneous follicular and oocyte maturation which occurred following release of ovarian follicles into sea water was prevented by DNP. In non-spontaneously maturing follicles released from the ovary, DNP inhibited both follicle and oocyte maturation induced by the secondary stimulator of spawning and maturation, 1-methyladenine (1-MA). DNP also inhibited 1-MA induced meiotic maturation in isolated immature oocytes incubated in the absence of follicle cells. Inhibition of oocyte maturation was not associated with inhibition of 3H-1-MA incorporation by isolated oocytes. Immature oocytes incubated in the presence of DNP underwent maturation following washing and subsequent exposure to 1-MA. Immature oocytes initially exposed to both 1-MA and DNP, however, showed decreased maturation responsiveness following washing and re-exposure to 1-MA. The results suggest that the inhibitory effects of DNP on spawning and oocyte maturation are the result of direct effects on the oocytes and possibly other cells and tissues within the ovary.

Characterization of spawn-inducing factors in the sea urchin, Strongylocentrotus purpuratus

The spawn-inducing radial nerve factor (RNF) of the sea urchin, Strongylocentrotus purpuratus, is a heat-stable polypeptide. The molecular weight of this RNF, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is 5600 ± 800. The RNF induces sea urchin gonads to produce a nonprotein gonad factor. This gonad factor (GF), as well as 1-methyladenine (the starfish gonad factor), causes sea urchin testis fragments to spawn. GF co-chromatographs with 1-methyladenine on thin-layer chromatography plates.

Echinoid Spawning Induced by a Radial Nerve Factor

An extract of radial nerves of the sea urchin. Strongylocentrotus purpuratus, induces spawning within 1 minute in this species as well as in other echinoids, but a delayed spawning in several asteroid species. The concentration of the spawn-inducing factor in the radial nerves fluctuates annually correlated with the reproductive season of this species along the coast of southern California.

Activation of 1-methyladenosine by starfish oocytes

Meiotic maturation in starfish oocytes is initiated by the purine base 1-methyladenine but not the nucleoside (1-methyladenosine) of this compound. The extent to which this may be the result of inaccessibility of 1-methyladenosine to cellular sites was investigated. Oocytes were homogenized at various stages of maturation, incubated with 1-methyladenosine and the effects of these homogenates on oocyte maturation were subsequently tested. Oocyte homogenates incubated with 1-methyladenosine were active in producing oocyte maturation, whereas homogenates not incubated with the nucleoside were inactive. Thus, activation of 1-methyl-adenosine occurred in response to oocyte homogenization. Specificity studies indicated that only compounds containing 1-methyladenine were activated in response homogenization. Activation of the starfish gonadotrophin (radial nerve factor) was not observed. These studies suggest that the starfish oocytes contain biochemical mechanism(s) for rendering 1-methyl-adenosine, an effective stimulator of oocyte maturation. Under normal conditions the integrity of the cell surface makes this interaction impossible. Activation of 1-methyladenosine, therefore, provides a simple and sensitive indicator of oocyte cellular integrity or damage.

Induction of oocyte shedding and meiotic maturation in Pisaster ochraceus: kinetic aspects of radial nerve factor and ovarian factor induced changes.

The addition of a radial nerve factor (RNF) to starfish ovarian tissue in vitro results in the release into the incubation media of a substance (ovarian factor) with different chemical properties but many biological properties similar to those of the RNF. A series of experiments were conducted to assess and compare the time course during which these substances initiate their biological effects. The effects of these substances on the process of oocyte shedding and germinal vesicle breakdown were assessed under in vitro conditions.1. Within approximately a 1 hour period following the addition of RNF, ovarian factor activity was found in the incubation media. Activity in some instances was found prior to induction of shedding.2. Oocyte shedding induced by the addition of both RNF amid ovarian factor occurred folowing a lag period of approximately 1 hour. The rate of oocyte shedding in response to RNF and the highest dose of ovarian factor were indistinguishable.3. Free oocytes exhibited germinal vesicle breakdown in response to ovarian factor but not to RNF. Germinal vesicle breakdown in response to ovarian factor occurred within 30 minutes of the addition of ovarian factor to free oocytes. Germinal vesicle breakdown in free oocytes occurred prior to either ovarian factor or RNF induced shedding from ovarian fragments.4. The continual presence of RNF in the incubation media was not required for the induction of shedding from ovarian fragments.5. Free oocytes did not require the continual presence of the ovarian factor for germinal vesicle breakdown to occur. An exposure time of approximately 15 minutes insured meiotic maturation in free oocytes.These data support the hypothesis that the ovarian factor is a normal intermediary substance in RNF-induced shedding and in oocyte germinal vesicle breakdown or meiotic maturation.

Chemical properties and physiological actions of a starfish radial nerve factor and ovarian factor

Incubation of starfish ovarian tissues with radial nerve factor (RNF) results in the appearance of a second biologically active substance (s) (ovarian factor) in the incubation media. Studies were conducted to eluciate the chemical and physiological properties of these two substances. The two substances could be physically separated by gel filtration following elution from columns of Sephadex G-25. The RNF and ovarian factor were both relatively heat stable. RNF but not ovarian factor was inactivated following enzymatic digestion with pronase. The ovarian factor appears to be a small molecular weight, heat stable, non proteinaceous material. The physiological effects of a starfish radial nerve factor (RNF) and the ovarian factor were compared. The actions of these substances were evaluated on the basis of changes in various components and responses in starfish ovarian fragments maintained in vitro. Both RNF and ovarian factor induced shedding of oocytes and a concomitant decrease in ovarian fragment weight. The magnitude of weight decrease was related to the dose of ovarian factor. The extent of oocyte shedding was similar in fragments treated with RNF and the highest doses of ovarian factor. RNF and ovarian factors produce oocyte germinal vesicle breakdown and dispersion of follicular cells from around oocytes within ligated and unligated ovarian fragments. Increased levels of calcium in seawater did not produce any of these effects; however, washing ovarian fragments in calcium-free seawater partially inhibited the action of RNF and ovarian factor. Oocytes matured within ligated ovarian fragments, following exposure to RNF or ovarian factor, and subsequently released, formed polar bodies. Upon addition of sperm, these eggs formed fertilization membranes and developed in a normal manner to gastrula. These experiments demonstrate that the ovarian factor has several biological effects or initiates a series of interrelated events. All biological activities previously attributed only to the RNF are apparently shared by this ovarian factor.

Increased germinal vesicle breakdown in oocytes of the sea cucumber Parastichopus californicus induced by starfish radial nerve extract

Starfish radial nerve factor (RNF) increased the percent of germinal vesicle breakdown in oocytes of the sea cucumber Parastichopus californicus. Sea cucumbers can now be used in studies requiring over 90% germinal vesicle breakdown or development. Oocytes of P. californicus treated with RNF are excellent material for the experimental cytology of spindles.

Effect of calcium on the structure and functional response of the starfish ovary to radial nerve factor.

AbstractThe effects of calcium‐free sea water (CaFSW) on the starfish ovarian structure and on ovarian responsiveness to radial nerve factor (RNF) were studied following in vitro incubation. Incubation of ovaries in CaFSW resulted in a progressive elongation of the entire ovary and the dispersion of follicular cells from around the oocytes contained within the ovary. The changes in ovarian length but not the follicular cell detachment were reversible when ovaries were placed in sea water containing calcium. Addition of Mg++ or Na+ to CaFSW did not prevent ovarian elongation or follicular cell dispersal. Strontium ions prevented and reversed ovarian elongation when added to CaFSW; however, follicular cell dispersion was not inhibited. The oocyte shedding, normally induced by RNF, was prevented by maintaining the ovary in CaFSW. The degree of shedding inhibition was dependent upon the duration of washing in CaFSW. RNF did not prevent or reverse the ovarian elongation induced by CaFSW. The inhibitory action of CaFSW on RNF‐induced oocyte shedding could be reversed by the addition of Ca++ or Sr++ but not by the introduction of Mg++ or Na+. The degree of reversibility was dependent upon the concentration of Ca++ ions. The data strongly indicate that contractile tissue in the ovarian wall is important in mediating oocyte shedding in response to RNF.

Variable sensitivity of starfish ovarian tissue to radial nerve factor

Incubation of starfish ovarian tissue with RNF does not result in complete inactivation of RNF. Ovarian tissue contains unshed oocytes with intact germinal vesicles even in the presence of RNF. The limited action of RNF ovarian tissue appears to be a result of ovarian insensitivity rather than insufficient RNF.