Effect of gonadotropin-releasing hormone on phagocytic leucocytes of rainbow trout

To clarify the role of gonadotropin-releasing hormone (GnRH) in the fish immune system, in vitro effect of GnRH was examined in phagocytic leucocytes of rainbow trout (Oncorhynchus mykiss). Gene expression of GnRH-receptor was detected by RT-PCR in leucocytes from head kidney. Administration of sGnRH increased proliferation and mRNA levels of a proinflammatory cytokine, tumor necrosis factor (TNF)-α, in trout leucocytes. Superoxide production in zymosan-stimulated phagocytic leucocytes was also increased by sGnRH in a dose-related manner from 0.01 to 100 nM. There was no significant effect of sGnRH on mRNA levels of growth hormone (GH) expressed in trout phagocytic leucocytes. Immunoneutralization of GH by addition of anti-salmon GH serum into the medium could not block the stimulatory effect of sGnRH on superoxide production. These results indicate that GnRH stimulates phagocytosis in fish leucocytes through a GnRH-receptor-dependent pathway, and that the effect of GnRH is not mediated through paracrine GH in leucocytes.     

Lack of gonadotropin releasing-hormone action on in vivo and in vitro growth hormone release,in rainbow trout (Oncorhynchus mykiss)

In order to understand the mechanisms implicated at the hypothalamo-pituitary level in growth-reproduction interaction in salmonids, the gonadotropin-releasing hormone (GnRH) action on growth hormone (GH) release was studied, in rainbow trout (Oncorhynchus mykiss). In vivo, acute treatment with salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and an sGnRH analogue [(DArg6Pro9)sGnRH] was performed on catheterized fish. The different forms of GnRH have no effect on plasma GH levels of immature and mature fish, but induce a stimulation of gonadotropin (GtH) release in mature fish. In the present work we have adapted and validated a culture system for GH regulation studies. In this system, increasing doses of sGnRH, (DArg6Pro9)sGnRH and cGnRH-II are inactive on GH release (24 hr incubation) in immature or mature fish, but stimulate GtH release in a dose-dependent manner. sGnRH (10⁻⁶ M) has no action on GH release, whatever the incubation time (15 min–24 hr). In a perifusion system, sGnRH also has no action on GH release but stimulates GtH release. The present results obtained using in vivo and in vitro techniques adapted for GH regulation studies, show that GnRH does not function as a growth hormone-releasing factor in rainbow trout as it does in goldfish.     

Comparison of GNRH3 genes across salmonid genera

Genomic sequences of gonadotropin-releasing hormone genes were amplified and examined for sequence divergence among members of three different genera of the subfamily Salmoninae: rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and Arctic charr (Salvelinus alpinus). Sequences of GNRH3A and GNRH3B (formerly known as sGnRH1 and sGnRH2) were 97-99% similar in coding regions and 94-98% similar in non-coding regions among genera, but comparisons within species between GNRH3A and GNRH3B were only 90-92% similar in coding regions and 83-89% similar in non-coding regions. Polymorphisms in the parents of mapping families for each species allowed for linkage mapping of the GNRH3B gene in all three species and the GNRH3A gene in rainbow trout. GNRH3B maps to linkage group 6 in rainbow trout, linkage group 16 in Atlantic salmon and linkage group 25 in Arctic charr. GNRH3A mapped to linkage group 30 in rainbow trout.     

Time- and dose-related effects of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the goldfish pituitary

The goldfish brain contains two molecular forms of gonadotropin-releasing hormone (GnRH): salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In a preliminary report, we demonstrated the stimulation of gonadotropin hormone (GtH) subunit and growth hormone (GH) mRNA levels by a single dose of GnRH at a single time point in the goldfish pituitary. Here we extend the work and demonstrate time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH gene expression in vivo and in vitro. The present study demonstrates important differences between the time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH mRNA levels. Using primary cultures of dispersed pituitary cells, the minimal effective dose of cGnRH-II required to stimulate GtH subunit mRNA levels was found to be 10-fold lower than that of sGnRH. In addition, the magnitudes of the increases in GtH subunit and GH mRNA levels stimulated by cGnRH-II were found to be higher than the sGnRH-induced responses. However, no significant difference was observed between sGnRH and cGnRH-II-induced responses in vivo. Time-related studies also revealed significant differences between sGnRH- and cGnRH-II-induced production of GtH subunit and GH mRNA in the goldfish pituitary. In general, the present study provides novel information on time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH mRNA levels and provides a framework for further investigation of GnRH mechanisms of action in the goldfish pituitary.     

鲤鱼sGnRH基因克隆及其在成熟个体的表达分析

采用RACE方法,从鲤鱼脑组织克隆了两个差异的sGnRH(salmon GnRH[Trp^7Leu^8]GnRH)cDNAs,即cDNA1和cDNA2,其长度分别为393和478bp。两个cDNAs都包括一个285bp开放阅读框,编码的sGnRH前体为94个氨基酸残基,由一个信号肽、sGnRH十肽和一个由蛋白水解位点(Gly-Lys-Arg)连接的促性腺激素释放激素相关肽共3部分组成。用内含子捕获得到相应的两个差异sGnRH基因,即sGnRH genel和gene2,其基本结构都包括4个外显子和3个内含子,3个内含子的核苷酸相似性分别为71．1％、76．1％和88．0％。鲤鱼sGnRH cDNAs及基因的基本结构和编码特点与已报道的不同形式GnRH cDNAs和GnRH基因相似,由此推测所有类型的GnRH可能来自一个共同的祖分子。Southern杂交进一步证实鲤鱼基因组存在两个不同的sGnRH基因座位。相对定量RT-PCR检测发现,两个sGnRH基因除在精巢的表达存在差异外,在脑区、垂体和成熟卵巢共表达。其中两个sGnRH基因在端脑和下丘脑的表达水平明显高于后脑区。根据sGnRH mRNAs在多个脑区、性腺和垂体的共存推测,sGnRH可能对鲤鱼下丘脑-垂体-性腺轴的调节有至关重要作用,同时可能起神经调节剂或自分泌和旁分泌调节因子的作用。     

Endogenous and heavy-metal-ion-induced metallothionein gene expression in salmonid tissues and cell lines

Endogenous levels of metallothionein (MT) mRNA were detected by RNA probes in several somatic and germ-line tissues of rainbow trout, such as eggs, ovaries and immature testis. These levels may be related to metal-ion homeostasis in the observed tissues. The induction kinetics of trout MT isoform B (MT-B) mRNA were studied after single intraperitoneal injections of CdCl2, CuCl2 and ZnCl2. MT-B mRNA was induced within 12 h in liver, kidney, spleen and gills. However, over the 48-h experimental period, the kinetics of MT-B mRNA accumulation differed in response to the three metal salts, possibly due to differential handling of the salts by these tissues. Multiple metal-salt injections induced high levels of MT-B mRNA in the four tissues studied. In the rainbow trout hepatoma cell line, ZnCl2 was a better inducer of the MT-B gene, as compared to CdCl2 and CuCl2. The expression of the exogenous trout MT-B promoter in Chinook salmon embryonic cell line indicates the presence of MT regulatory factors. In contrast, the endogenous MT genes in these cells are quiescent, possibly due to the methylation of their promoter region.     

Neurosecretory neurons of the nucleus preopticus (NPO) express salmon GnRH mRNA and show reproduction phase-related variation in the female Indian major carp, Cirrhinus cirrhosus

We studied the expression of sGnRH mRNA in the neurons of the nucleus preopticus (NPO) of the Indian major carp, Cirrhinus cirrhosus, and their correlation with the reproductive status of the fish. Non-radioisotopic in situ hybridization histochemistry protocol employing biotinylated-oligonucleotide probes complementary to salmon GnRH, cichlid GnRH I, catfish GnRH, chicken GnRH II (from cichlid and catfish), and mammalian GnRH, were applied to the sections through the POA of the female Indian major carp Cirrhinus cirrhosus. Incubation with the probe complimentary to salmon GnRH (sGnRH) mRNA from salmon, produced distinct hybridization signal in the cytosol of several neurosecretory neurons of the magnocellular and parvocellular subdivisions of the NPO of the fish collected during February-April (preparatory phase) and May-June (prespawning phase). However, no signal was detected in the NPO of fish collected during July-August (spawning phase). Application of other antisense probes, or sense probe for salmon GnRH mRNA, produced no signal. We suggest that NPO neurons in C. cirrhosus may express sGnRH mRNA, produce GnRH peptide, and play a role in regulation of pituitary-ovary axis.     

Effects of progesterone and estradiol on the reproductive axis in immature diploid and triploid rainbow trout

In fish species, many studies demonstrated the crucial role of estradiol (E2) in the development of the reproductive axis, but progesterone (P) has been described mainly as a precursor steroid and no clear role by itself has been reported. Moreover, a cooperative effect of P (or another progestin) and E2 in fish has never been reported to our knowledge. In the present work, we investigated the effects of P, alone or in combination with E2, on the reproductive-axis of immature rainbow trout (Oncorhynchus mykiss). Liver vitellogenin and estradiol receptor (rtER) mRNA levels increased after E2 treatment, but were unchanged by P treatments as a reflection of peripheral action of steroids. In contrast, at the pituitary level, LH contents increased after E2 and/or P treatments. Focusing on the brain level, we confirmed a clear up regulation of rtER expression by E2 in sterile triploid females, and we also demonstrated a similar stimulating effect of P alone but no cooperative effect together with E2. In conclusion, our data demonstrate that in immature trout, prior to the beginning of the first reproductive cycle, unlike E2, P is able to stimulate the reproductive brain-pituitary axis without affecting vitellogenin synthesis in the liver.     

Changes in brain GnRH mRNA and pituitary GnRH peptide during testicular maturation in barfin flounder

The pleuronectid barfin flounder (Verasper moseri) expresses three forms of gonadotropin-releasing hormone (GnRH) in the brain. To clarify the physiological roles of the respective forms during testicular maturation, changes in brain GnRH mRNA levels and pituitary GnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. Fish hatched in April 2000. The gonadosomatic index remained low until October 2001 and then rapidly increased in January 2002. Fish continued to grow from hatching through testicular maturation. Fish spermiated in March 2002. The amount of seabream GnRH (sbGnRH) mRNA per brain significantly increased in January 2002 and remained at high levels in March 2002. The amounts of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) mRNA per brain did not show significant changes during the experimental periods. Pituitary sbGnRH peptide content significantly increased in March 2002. Pituitary sGnRH peptide and cGnRH-II peptide contents were extremely low compared to sbGnRH peptide levels and showed no significant changes during the experiment. These results indicate that sbGnRH is involved in the testicular maturation of barfin flounder.