cDNA cloning of blue gourami (Trichogaster trichopterus) prolactin and its expression during the gonadal cycles of males and females.

BACKGROUND: The blue gourami fish (Trichogaster trichopterus) provides a unique model for the study of reproduction endocrinology in teleost fish. Its oocyte development may be controlled easily, and the vitellogenic and final maturation phases may be separated artificially in the laboratory. Moreover, this gourami exhibits exclusive parental behavior. AIM: The aim of the present study was to clone and sequence the blue gourami PRL (bgPRL) cDNA in order to enable the determination of its mRNA levels in the male and female blue gourami during the gonadal cycles. MATERIALS AND METHODS: bgPRL was cloned by extracting total RNA from freshly excised pituitaries of gourami fish, followed by cDNA synthesis, rapid amplification of cDNA ends (RACE)-PCR and finally, sequencing. bgPRL mRNA expression was determined by realtime PCR, and results were normalized with 18S RNA. RESULTS: When bgPRL was compared to PRLs of other fish, it had the most homology with PRL of Perciformes and the least with those of Anguilliformes. bgPRL was expressed during the entire gonadal cycle in males and females. The average levels of PRL mRNA in juvenile and low vitellogenetic females were lower than in mature females (at high vitellogenesis and maturation), but the differences were not significant. On the other hand, the PRL mRNA levels in mature reproductive males (nestbuilders) and non-reproductive (non-nest-builders) were significantly higher in comparison to young males. CONCLUSIONS: The results of this study imply that PRL has a possible role in the endocrine control of gonadal development in fish, in addition to its role in reproductive behavior.

Cloning of Russian sturgeon (Acipenser gueldenstaedtii) growth hormone and insulin-like growth factor I and their expression in male and female fish during the first period of growth.

In this study, the GH and IGF-I of the Russian sturgeon (rs), Acipenser gueldenstaedtii, were cloned and sequenced, and their mRNA gene expression determined. In addition, to improve our understanding of the GH function, the expression of this hormone was assessed in young males and females. Moreover, IGF-I expression was quantified in young males and compared to that in older ones. The nucleotide sequence of the rsGH cDNA was 980 bp long and had an open reading frame of 642 bp, beginning with the first ATG codon at position 39 and ending with the stop codon at position 683. A putative polyadenylation signal, AATAAA, was recognized 42 bp upstream of the poly (A) tail. The position of the signal- peptide cleavage site was predicted to be at position 111, yielding a signal peptide of 24 amino-acids (aa) and a mature peptide of 190 aa. When the rsGH aa sequence was compared with other species, the highest degree of identity was found to be with mammalians (66-70% identity), followed by anguilliformes and amphibia (61%) and other fish (39-47%). The level of rsGH mRNA was discovered to be similar in pituitaries of females and males of 5 age groups (1, 2, 3, 4, and 5- yr-old). In females and males, the levels did not change dramatically during the first 5 yr of growth. The partial nucleotide sequence of the rsIGF-I was 445 bp long and had an open reading frame of 396 bp, beginning with the ATG codon at position 50. The position of the signal-peptide cleavage site was predicted to be at position 187, yielding a signal peptide of 44 aa. The highest level of IGF-I mRNA expression was recorded in the kidney of adult sturgeons. The IGF-I mRNA expression levels in the intestine, pituitary gland, and liver were not significantly different. Low levels of expression were found in the brain, heart, and muscle. In most tissues, there was no significant difference between mRNA levels of one and 5-yr-old fish. In conclusion, based on the GH-sequence analysis, A. gueldenstaedtii is genetically distant from other teleosts. The expression of the GH mRNA was similar in males and females, and its level remained constant during the first 5 yr of growth. While the IGF-I mRNA expression differed amongst various tissues, the level in each tissue was similar in 1 and 5-yr-old fish.

Cloning of European eel (Anguilla anguilla) FSH-beta subunit, and expression of FSH-beta and LH-beta in males and females after sex determination.

The European eel (Anguilla anguilla) is a catadromic teleost species with a complex life cycle, both in sea and freshwater environments. The sex determination phase of gonadal development occurs in a freshwater environment. Polymorphism occurs in increasing rates with respect to gender. While males stop growing at approximately 150 g, females continue to grow to being much larger. In this study, we cloned the cDNA FSH-beta subunit of the European eel (A. anguilla), and measured the mRNA levels of FSH-beta and LH-beta in males and females after sex determination. The FSH-beta subunit cDNA consisted of 1068 bp, encoding a 127 amino acid peptide. A comparison between European and Japanese eels of the FSH-beta amino acid sequence showed 98% similarity.

Growth differences and growth hormone expression in male and female European eels [Anguilla anguilla (L.)].

In this study, we examined the growth differences of males and females following a sex reversion, and the growth hormone (GH) expression variation between sexes of European eels [Anguilla anguilla (L.)]. A high percentage of females (88%) was found in the group fed with estradiol 17beta compared to the control group (comprised of only 6% female eels), which was defined as the male population. Significant differences between growth rate and size were found following 480 days of growth, whereby the males reached 60+/-4.3 g (means+/-SE) in size and the females 73.4+/-5.9 (g+/-SE); after 600 days, the males reached 114.1+/-4.3 and the females 171+/-11.7 (g+/-SE). A cDNA coding for the complete growth hormone of the European eel A. anguilla (eeGH) was cloned by RACE PCR using several sets of degenerate oligonucleotides. The eeGH cDNA coding region is 627 bp long. A sequence comparison of eeGH with Anguilla japonica GH (jeGH) cDNA showed a 98% identical base. Comparison of the deduced amino acid sequence revealed 99% identical residues, meaning that a difference exists in only two of the 209 residues. In both cases, the differing residues in the eeGH amino acid sequence are lysine. We measured the mRNA levels of growth hormone in the pituitaries of male and female eels growing at different rates. A significantly higher expression of eeGH was found in the female eels in comparison to the males. These results show that different levels of GH transcription eeGH can explain the growth rate differences between male and female European eels.