Developing major histocompatibility markers in a species of concern: the Sacramento perch Archoplites interruptus

Primers targeting two non‐neutral major histocompatibility complex (mhc) II β genes were developed and assayed across several disjoint Sacramento perch Archoplites interruptus sampling locations. Variability at the two mhc loci among sampling stocks strongly correlated to previous estimates with neutral markers, suggesting that the effect of genetic drift was not limited to neutrally evolving regions of the genome. The novel mhc primers will help develop admixture schemes in A. interruptus captive breeding programmes and will increase the success of future reintroductions of this species of concern.     

Primary culture of normal pituitary cells of carp (cyprinus carpio) for the study of gonadotropin release

Summary A method for preparing enzymaticlaly dispersed pituitary cell cultures of carp (Cyprinus carpio) is described. The cultures have been used to assay a synthetic analog of gonadotropin releasing hormone (GnRH) and to determine the specificity of steroids able to affect gonadotropin (GtH) release in vitro. Time course secretion studies indicated that by 48 h incubation, in the presence of 500 pM GnRH, cumulative secretion of gonadotropin (719 ng±90/2.5×10⁵ cells) had exceeded that of controls (446 ng±106/2.5×10⁵ cells). Estradiol-17β, progesterone, testosterone, and 11-ketotestosterone showed different inhibitory effects on pituitary basal GtH release. Based on the results, it was concluded that carp pituitary cell cultures can be applied to investigations of several aspects of the hypothalamo-hypophysial-gonadal system. This investigation was supported by the Deutsche Forschungsgemeinschaft, Bonn, FRG.     

Detection of a Single-Locus Gene on Channel Catfish Chromosomes by In-Situ Polymerase Chain Reaction

An in-situ polymerase chain reaction (ISPCR) procedure was applied to chromosomal localization of the gene, Ig H, encoding the immunoglobulin heavy chain of channel catfish (Ictalurus punctatus). Metaphase chromosomes were prepared by a replication banding procedure and subjected to ISPCR using biotin-labeled primers. The hybridization signals were detected with an avidin-fluorescein isothiocyanate (FITC)-based method, and chromosome bands revealed by simultaneous or sequential treatment methods. Standard fluorescent in-situ hybridization (FISH) was performed on chromosome preparations to compare with the ISPCR procedure. The Ig H gene was detected at the telomeric position of a chromosome with a relative length of 3.2 ± 0.2%. The Ig H-bearing chromosome detected by the FISH method was identical to that found by ISPCR procedure. Visibility of chromosome bands was reduced by heat and salt treatments and could not be analyzed after thermocycling. Therefore, specific identity of the chromosome bearing the Ig H gene remains unknown. Banding of fish chromosomes is difficult and poses a barrier for applying current molecular techniques to physical mapping of teleost genomes. Application of the ISPCR to chromosomal mapping is new for fish species and is only in initial stages of development for higher vertebrates.     

Female-specific target sites for both oestrogen and androgen in the teleost brain

To dissect the molecular and cellular basis of sexual differentiation of the teleost brain, which maintains marked sexual plasticity throughout life, we examined sex differences in neural expression of all subtypes of nuclear oestrogen and androgen receptors (ER and AR) in medaka. All receptors were differentially expressed between the sexes in specific nuclei in the forebrain. The most pronounced sex differences were found in several nuclei in the ventral telencephalic and preoptic areas, where ER and AR expression were prominent in females but almost completely absent in males, indicating that these nuclei represent female-specific target sites for both oestrogen and androgen in the brain. Subsequent analyses revealed that the female-specific expression of ER and AR is not under the direct control of sex-linked genes but is instead regulated positively by oestrogen and negatively by androgen in a transient and reversible manner. Taken together, the present study demonstrates that sex-specific target sites for both oestrogen and androgen occur in the brain as a result of the activational effects of gonadal steroids. The consequent sex-specific but reversible steroid sensitivity of the adult brain probably contributes substantially to the process of sexual differentiation and the persistent sexual plasticity of the teleost brain.     

Cyclic ADPR and calcium signaling in sea bream (Sparus aurata) egg fertilization.

The cell egg is in a state of quiescence and only after its fusion with the sperm, a series of pre-programmed metabolic processes will be activated, culminating with embryonic development. The egg/sperm fusion induces a transitory increase of Ca(2+) in the cytoplasm, which is responsible for the activation of both precocious and late reactions. The release of Ca(2+) occurs by stimulation of the ionic specific channels. In addition to IP(3), a new Ca-release inducer was recently evidenced, cyclic ADP ribose (cADPR), in some invertebrates and mammals. Here, we report the first evidence of the cADPR presence in fish. Our data also demonstrate that in the sea bream egg, cADPR is involved in the fertilization process; in fact, its level increases after the entrance of the sperm. By in vitro experiments, it was shown that cADPR induces a release of Ca(2+) in the egg homogenate, indicating that in sea bream, the increase of cADPR can induce an intracellular Ca(2+) release. Since cADPR is a product of NAD(+) metabolism, the activity of several enzymes involved in the NAD(+) metabolism was investigated. Sea bream eggs are pelagic and only floating eggs after insemination develop into viable embryos. In the present work, NAD(+) metabolism was studied in both types of egg. All the tested enzymes showed similar specific activity in both floating and sinking eggs. In the latter, cADPR was not detectable and the nucleotides content was significantly lower, evidencing a scarce energetic charge in sinking eggs.