Influence of long-term exposure to dietary cadmium on growth, maturation and reproduction of goldfish (subspecies: Prussian carp Carassius auratus gibelio B.).

The influence of long-term exposure of goldfish to dietary cadmium (Cd) on its accumulation in tissues, growth, ovarian development, luteinizing hormone (LH) secretion and a response to hormonal stimulation of spawning were evaluated. The study was conducted on four groups of females for the period of 3 years, from the age of 10 weeks to second spawning. Four doses of Cd were applied in the feed: 0 (control group), 0.1, 1 and 10 mg Cd g(-1) of feed (wet weight). The highest dose of Cd (10 mg g(-1)) inhibited growth and caused several behavioural effects. In contrast, lower dose of Cd (1 mg g(-1)) stimulated fish growth. The doses of Cd from 0.1 to 1 mg Cd g(-1) did not influence ovarian development. The gonado-somatic index (GSI) and histological analysis of ovaries showed no differences in ovarian development between the control group and the groups receiving these doses of Cd. However, in the group receiving the highest Cd dose, GSI decreased. This was associated with persistent, long-lasting elevation of plasma LH levels. Ovulation did not occur in this group. Injections of salmon GnRH-analogue (sGnRHa) alone or with domperidone (a dopamine receptor antagonist) in sexually mature fish caused an increase of LH levels in all groups, although in the group fed with the highest Cd dose the effect was weaker than in the other groups. After the first spawning season, a negative effect of lower Cd doses (0.1 and 1mg Cd g(-1)) on ovarian recrudescence (rebuilding of ovaries) and on the response to the consecutive hormonal stimulation of spawning was observed (lower number of ovulating females). There was a significantly higher content of Cd in the livers of fish than in their muscles. The results of hormonal stimulation of spawning and histological analysis of ovaries suggest that in goldfish cadmium acts mainly at the level of ovary rather than on the pituitary gland. We suppose that in the natural environment cadmium present in the feed can play an important role in the accumulation of this element in fish tissues and can influence vital physiological processes.

Transgenic rainbow trout expressed sGnRH-antisense RNA under the control of sGnRH promoter of Atlantic salmon.

A recombinant vector containing antisense DNA complementary to Atlantic salmon (Salmo salar) sGnRH cDNA driven by specific promoter Pab derived from a corresponding sGnRH gene was introduced into rainbow trout (Oncorhynchus mykiss) eggs. This resulted in transgenic animals that had integrated one copy of the transgene into their genome and transmitted it through the germline. Antisense-sGnRH mRNA (AS) was expressed mainly in the brain of transgenic AS(+) fish. Levels of sGnRH endogenous mRNA in the brain were lower in 11-month-old AS(+) fish compared with nontransgenic AS(-) individuals from the same F2 progeny. sGnRH levels significantly decreased in the pituitary of transgenic males and females around the maturation period and in the brain of AS(+) immature females compared with controls. No reliable statistical difference was found in the levels of FSH and LH between AS(+) and AS(-) groups either in immature or mature fish. The majority of transgenic fish reached maturity at the same time as did nontransgenic individuals, although the maturation of AS(+) animals seemed to be more asynchronous. For the first time, the influence of antisense messengers on endogenous mRNA in transgenic fish and the corresponding protein is described.

Post-ovulatory secretion of pituitary gonadotropins GtH I and GtH II in the rainbow trout (Oncorhynchus mykiss): regulation by steroids and possible role of non-steroidal gonadal factors.

In order to determine the factors of ovarian origin which can modulate the postovulatory secretion of the FSH-like gonadotropin (GtH I) and the LH-like gonadotropin (GtH II), freshly ovulated female rainbow trout were divided into two groups. In the first group the fish were stripped in order to eliminate the eggs and ovarian fluid from the body cavity, while in the second group the eggs were kept in the body cavity. Subsequently, fish from both groups were implanted with testosterone (10 mg/kg), 17beta-estradiol (10 mg/kg) or 17,20beta-ddihydroxy-4-regnen-3-one (17,20betaP) (1 mg/kg) or injected every 2 days with desteroidized ovarian fluid (1.5 ml/kg). The secretion of GtH I dramatically increased in stripped fish, reaching its maximum levels 2 weeks after ovulation. The preservation of eggs in the body cavity led to the suppression of this increase. The profiles of GtH II secretion were opposite to those encountered for GtH I because the increase of GtH II was observed only in unstripped fish. The administration of steroids showed that testosterone is able to inhibit GtH I release and stimulate that of GtH II in stripped fish, having no effect on the release of these gonadotropins in non-stripped animals. 17beta-Estradiol failed to modify GtH I secretion, however it decreased the release of GtH II in fish containing retained eggs in the body cavity. 17,20betaP had a delayed stimulating influence on GtH I release in unstripped fish. Finally, multiple injections of desteroidized ovarian fluid into stripped fish led to a significant decrease of GtH I release and to an increase of GtH II secretion. This study demonstrates that factors, which are present in ovarian fluid, modulate the post-ovulatory secretion of both gonadotropins--their net action is negative on GtH I and positive on GtH II. Among the steroids, testosterone is of major importance, being able to inhibit GtH I release and to stimulate that of GtH II. We also show that non-steroidal factors present in the ovarian fluid can influence the release of both gonadotropins, which indirectly supports the previous findings about the existence of inhibin/activin-like factors in fish.

The influence of gabaergic drugs upon LH secretion from the pituitary of the common carp: an in vitro study.

In the present study, the perifusions of whole pituitary glands of spermiating male common carp were performed in the presence of several GABAergic drugs. Muscimol (agonist of GABA(A) receptors) and bicuculline (the antagonist of the same type of GABA receptors) did not modify basal LH release. LH basal secretion was not modified when pituitaries were perifused with baclofen--an agonist of GABAB receptors. On the other hand, baclofen at doses of 10(-8) and 10(-4) M significantly decreased GnRH-A-induced LH release to about 86% and 88% of LH levels in control group, respectively. In our previous study we have shown that GABA decreased basal and GnRH-A-stimulated in vivo and in vitro LH release. In conclusion, it can be suggested that in the mature male carp GABA exerts an inhibitory influence on GnRH-stimulated LH release, probably through the inhibition of the GnRH action on gonadotropes. This inhibition seems to be mediated by the B type of GABA receptors.

Immunological cross-reactivity between rainbow trout GTH I and GTH II and their alpha and beta subunits: application to the development of specific radioimmunoassays.

Immunological cross-reactivities between rainbow trout GTH I and GTH II and their alpha and beta have been studied using highly purified rainbow trout gonadotropins and subunits and antibodies raised against beta subunits. From these observations radioimmunoassays have been developed for rainbow trout GTH I and GTH II. The GTH II RIA was highly specific and cross-reacted only with GTH II and its beta 1 subunits, with beta 2 being less potent than beta 1 in competing GTH II binding. There was no cross-reactivity with GTH I. Its sensitivity varied between 0.1 and 0.2 ng/ml, allowing GTH II measurement early in the reproductive cycle. Variations between and within assays were less than 10%. There was a lack of specificity of GTH I RIA (44% cross-reactivity with GTH II, when using labelled native GTH I). Reasons for this lack of sensitivity were studied. It cannot be attributed to beta subunits (less than 1.2% cross-reactivity). However, the cross-reactivity of alpha subunits was very important. This suggests that the presence of free alpha subunits in the medium can be responsible for the lack of specificity. Labelling native GTH I resulted in conformational change in molecular weight and dissociation of the hormone into subunits, whereas iodination did not induce GTH II dissociation. This dissociation can be avoided by labelling the stable form of GTH I. Using this radio-tracer, the specificity and the sensitivity of the assay were greatly improved (GTH II cross-reactivity was decreased to 3.7, mean sensitivity 0.87 +/- 0.072 ng/ml). The sensitivity of the assay diminished with ageing of labelled GTH I. The assay variation was 4.6% within an assay and 9.8% between assays. The use of labelled beta GTH I still increases the specificity (2.3% GTH II cross-reactivity), but with a 2.4-fold loss of sensitivity. In both GTH I and GTH II RIA plasma and spiked plasma with purified GTHs gave displacement curves parallel to standard. These assays were used to study pituitary responsiveness to a GnRH analogue in female rainbow trout prior to oocyte maturation. The effects of GnRH on GTH II secretion were confirmed. The peptide did not significantly stimulate GTH I secretion.

Involvement of gamma-aminobutyric acid in the control of GTH-1 and GTH-2 secretion in male and female rainbow trout.

The potential role of the neurotransmitter gamma-aminobutyric acid (GABA) in the control of the secretion of the two pituitary fish gonadotropins (GTH-1 and GTH-2) was investigated in male and female rainbow trout (Oncorhynchus mykiss). The presence of glutamate decarboxylase-positive fibers in the neurohypophyseal digitations adjacent to the gonadotropic cells was demonstrated by means of double immunohistochemistry, providing a morphofunctional support for potential GABA-gonadotropin interactions in both sexes. In spermiating males, in vivo treatment with GABA did not affect basal gonadotropin release, but stimulated GTH-1 release when coadministered with a gonadotropin-releasing hormone analogue (GnRHa), and potentiated GnRHa-stimulated GTH-2 release. In vitro, using dispersed pituitary cells, GABA stimulated basal GTH-1 and GTH-2 secretion, in a dose-dependent manner, and potentiated salmon GnRH effect on both hormones. In mature females, GABA induced in vivo a strong elevation of plasma GTH-2 levels after 2- 6 h of injection, but had no effect in vitro. GABA treatment in vivo was also stimulatory in recrudescent females, slightly increasing plasma GTH-2 levels in both saline- and GnRHa-treated fish (GnRHa alone has no effect at this stage). Immature fish were unresponsive to GABA/GnRHa treatments but, after steroid implantation [testosterone (T) or estradiol] for 13 days, injection of GABA stimulated GTH-2 release in vivo (also GTH-1 slightly in T-implanted fish). In conclusion, GABA has an overall stimulatory action on GTH-1 and GTH-2 secretion in rainbow trout, which depends on the sex and the reproductive stage of the fish. The stimulatory action of GABA might be exerted, at least in part, directly onto the gonadotropes, as it stimulates basal and GnRH-induced GTH-1 and GTH-2 secretion from dispersed pituitary cells.