Goldfish Embryos Research Articles

The Mos/MAPK pathway is involved in metaphase II arrest as a cytostatic factor but is neither necessary nor sufficient for initiating oocyte maturation in goldfish.

Mos plays a crucial role in meiotic cell division in vertebrates. In Xenopus, Mos is involved in the initiation of oocyte maturation as an initiator and in the arrest at the metaphase II stage (MII) as a component of the cytostatic factor (CSF). The function of Mos is mediated by MAP kinase (MAPK). We investigated the function of the Mos/MAPK pathway during goldfish oocyte maturation induced by 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), a natural maturation-inducing hormone in fishes. Mos was absent in immature goldfish oocytes. It appeared before the onset of germinal vesicle breakdown (GVBD), increased to a maximum in mature oocytes arrested at MII and disappeared after fertilization. MAPK was activated after Mos synthesis but before maturation-promoting factor (MPF) activation, and its activity reached maximum at MII. Injection of either Xenopus or goldfish c-mos mRNA into one blastomere of 2-cell-stage Xenopus and goldfish embryos induced metaphase arrest, suggesting that goldfish Mos has a CSF activity. Injection of constitutively active Xenopus c-mos mRNA into immature goldfish oocytes induced MAPK activation, but neither MPF activation nor GVBD occurred. Conversely, the injection of goldfish c-mos antisense RNA inhibited both Mos synthesis and MAPK activation in the 17alpha,20beta-DP-treated oocytes, but these oocytes underwent GVBD. These results indicate that the Mos/MAPK pathway is not essential for initiating goldfish oocyte maturation despite its general function as a CSF. We discuss the general role of Mos/MAPK during oocyte maturation, with reference to the difference in contents of inactive MPF (pre-MPF) stored in immature oocytes.

Vsx-1 and Vsx-2: differential expression of two paired-like homeobox genes during zebrafish and goldfish retinogenesis.

Vsx-1 and Vsx-2 are two homeobox genes that were cloned originally from an adult goldfish retinal library. They are members of the paired-like:CVC gene family, which is characterized by the presence of a paired homeodomain and an additional conserved region, termed the CVC domain. To analyze the possible roles for Vsx-1 and Vsx-2 in eye development, we used in situ hybridization to examine their expression patterns in zebrafish and goldfish embryos. Vsx-2 is initially expressed by proliferating neuroepithelial cells of the presumptive neural retina, then it is down-regulated as differentiation begins, and it is finally reexpressed at later stages of differentiation in a subset of cells, presumed to be bipolar cells, in the inner nuclear layer. In contrast, Vsx-1 is expressed only weakly in undifferentiated, presumptive neural retina and is then up-regulated selectively in presumptive bipolar cells at early stages of differentiation (when Vsx-2 is turned off), before decreasing to an intermediate level, which is maintained in the differentiated (adult) retina. The restricted expression patterns of Vsx-2 correspond to the observed phenotypes in mice with the ocular retardation mutation (orJ), further supporting the notion that Vsx-2 and Chx10 are homologues. The sequential complimentary and then corresponding expression patterns of Vsx-1 and Vsx-2 suggest that these similar transcription factors may be recruited for partially overlapping, but distinct, functions during the development of the retina.

Utilization of yolk fatty acids by goldfish embryos and larvae

In each of two separate experiments, eggs from a single female goldfish were fertilized, incubated at 22°C and sampled regularly up to day 6 when the larvae could be expected to commence feeding. Hatching normally occurred on Day 4. Lipids were extracted from the eggs and larvae and the neutral lipid and neutral phospholipids were isolated on aminopropyl columns. Fatty acid analysis of the eggs revealed the typical situation in fish where the phospholipids were rich in polyunsaturated fatty acids (PUFA) and the neutral lipids were rich in monounsaturated fatty acids (MUFA). Assay of lipid masses revealed that little depletion of lipid occurred until after hatch and that the neutral phospholipids were the principal fraction consumed. Although the neutral lipid mass did not change substantially during development, its fatty acid profile did. The proportions of several PUFA in the neutral lipids, especially 22∶6(n-3), 20∶5(n-3) and 20∶4(n-6), increased substantially during development while proportions of MUFA and 18∶2(n-6) declined. This appears to be a mechanism by which the larva can retain essential fatty acid released on hydrolysis of phospholipid while deriving the benefits of catabolism of phospholipid as fuel, namely the provision of phosphate and choline for intermediary metabolism and for the synthesis of macromolecules and neurotransmitter.

Cryosurvival of goldfish embryo after subzero freezing

Goldfish embryos of pre-morula, somite formation, optic vesicle, heart formation and pigment formation stages were tested for cryoprotectant toxicity, using DMSO and glycerol at 10, 12 and 15% concentration. Those that showed 50% or more survival were employed for cold shock test, from 24 to 4 °C at the rate of –1 °C/min. Only embryos at the heart formation stage protected with 12% DMSO showed 60% survival and 35% hatching. A group of embryos of that stage went through a freezing and thawing test to temperatures of –15, –25 and –35 °C. They showed 30, 5 and 2% survival and 12, 3 and 1% hatching respectively.

Neurolin, a cell surface glycoprotein on growing retinal axons in the goldfish visual system, is reexpressed during retinal axonal regeneration

The mAb E 21 recognizes a cell surface glycoprotein selectively associated with fish retinal ganglion cell axons that are in a state of growth. All retinal axons and ganglion cells in goldfish embryos stained for E 21. In adult fish, however, E 21 immunoreactivity exhibited a patterned distribution in ganglion cells in the marginal growth zone of the continuously enlarging fish retina and the new axons emerging from these cells in the retina, optic nerve, and optic tract. The E 21 antigen was absent from older axons, except the terminal arbor layer in the tectum, the Stratum fibrosum et griseum superficiale where it was uniformly distributed. Upon optic nerve transection, the previously unlabeled axons reacquired E 21 positivity as they regenerated throughout their path to the tectum. Several months after ONS, however, E 21 staining disappeared from the regenerated axons over most of their lengths but reappeared as in normal fish in the terminal arbor layer. The immunoaffinity-purified E 21 antigen, called Neurolin, has an apparent molecular mass of 86 kD and contains the HNK1/L2 carbohydrate moiety, like several members of the class of cell adhesion molecules of the Ig superfamily. The NH2-terminal amino acid sequence has homologies to the cell adhesion molecule DM-Grasp recently described in the chicken. Thus, retinal ganglion cell axons express Neurolin during their development and are able to reexpress this candidate cell adhesion molecule during axonal regeneration, suggesting that Neurolin is functionally important for fish retinal axon growth.

Induction of developmental abnormalities in larval goldfish, Carassius auratus L., under cool incubation conditions

Compared with incubation at a constant 22° C, exposure of goldfish embryos and larvae to 13° C, under a variety of thermal protocols, caused increased frequencies of abnormal development and, in some cases, reduced survival to hatching. The low‐temperature incubation conditions were particularly deleterious when eggs were incubated at 13° C from the outset, regardless of the temperature at which the donor female ovulated and the eggs were fertilized. Significantly higher frequencies of developmental abnormalities were also noted when embryos were transferred from 22°C to 13°C at 6, 24, 128 and, in one case, 175 h after fertilization. In three of five experiments, subjecting embryos and larvae to diel fluctuations between 22 and 13° C, with a 5‐h hold at the lower temperature, caused an increase in development abnormalities. These results demonstrate that the thermal requirements of goldfish embryos and larvae necessitate a delay in ovulation and spawning until water is sufficiently warm. Developmental abnormalities can be induced by exposure to cool (13° C) conditions, at least up to the time that swimbladder inflation occurs.