Markers of Genetic Variation in Blue Gourami (Trichogaster trichopterus) as a Model for Labyrinth Fish.

Markers of genetic variation between species are important for both applied and basic research. Here, various genes of the blue gourami (Trichogaster trichopterus, suborder Anabantoidei, a model labyrinth fish), many of them involved in growth and reproduction, are reviewed as markers of genetic variation. The genes encoding the following hormones are described: kisspeptins 1 and 2, gonadotropin-releasing hormones 1, 2, and 3, growth hormone, somatolactin, prolactin, follicle- stimulating hormone and luteinizing hormone, as well as mitochondrial genes encoding cytochrome b and 12S rRNA. Genetic markers in blue gourami, representing the suborder Anabantoidei, differ from those in other bony fishes. The sequence of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene of blue gourami is often used to study the Anabantoidei suborder. Among the genes involved in controlling growth and reproduction, the most suitable genetic markers for distinguishing between species of the Anabantoidei have functions in the hypothalamic-pituitary-somatotropic axis: pituitary adenylate cyclase-activating polypeptide and growth hormone, and the 12S rRNA gene.

Molecular characterization of kisspeptin receptors and gene expression analysis during oogenesis in the Russian sturgeon (Acipenser gueldenstaedtii).

Sturgeons belong to a subclass of fishes that derived from ray-finned fish ancestors preceding the emergence of teleosts. The Russian sturgeon (Acipenser gueldenstaedtii) is a late-maturing fish with the females reaching puberty under aquaculture conditions at 6-10 years of age. Since kisspeptin has been shown to be a key hormone involved in regulation of major reproductive processes of many vertebrate species, this study was conducted to better understand the kisspeptin receptor (KissR) in sturgeon. In this study we have cloned Russian sturgeon KissR1 from brain mRNA and observed the ontogeny of rsKissR1 mRNA expression in ovarian follicles. Multiple sequence alignment of KissR1, KissR4, and their orthologs revealed that the Russian sturgeon (rs) KissR1 sequence shares 64%-77% identity with elephant shark, coelacanth, and gar and 44-58% identity with tetrapod and teleost KissR1 sequences, while KissR4 seemed to share <65% identity to eel KissR2 and ~57% identity to Perciformes and Cypriniformes. Further rsKissR4 showed <97% identity to reed fish KissR4, <63% with Squamata (Reptiles) and gar KissR4. A phylogenetic analysis revealed that rsKissR1 is more closely related to coelacanth and gar KissR1 than teleost, while rsKissR4 was part of the KissR4 clade and shared higher similarity with Actinopterygiian sequences. We have further predicted homology models for both rsKiss receptors and performed in-silico analyses of their binding to a kiss-10 peptide. Both sturgeon and zebrafish Kiss1 and Kiss2 activated rsKissR1 via both PKC/Ca2+ and PKA/cAMP signal-transduction pathways, while rsKissR2 was found to be less effective and was not activated by stKiss peptides. Ovarian rsKissR transcript levels for 10 fishes were determined by real-time PCR and significantly increased concomitantly with oogenesis, where the highest level of expression was evident in black follicles. These data suggest that extra-neuronal expression of the kisspeptin receptor may be involved in sturgeon reproduction in a manner dependent on reproductive development.

Brain Control Reproduction by the Endocrine System of Female Blue Gourami (Trichogaster trichopterus).

Blue gourami belongs to the Labyrinithici fish and the Anabantiform order. It is characterized by a specific organ located above its gills for the respiration of atmospheric oxygen. This specific adaptation to low oxygen levels affects reproduction that is controlled by the brain, which integrates different effects on reproduction mainly through two axes-the gonadotropic brain pituitary gonad axis (BPG) and the hypothalamic-pituitary-somatotropic axis (HPS axis), including the interactions between them. This brain control reproduction of the Anabantoidei suborder summarizes information that has been published on the hormones involved in controlling the reproduction system of a model female blue gourami fish (Trichogaster trichopterus), including unpublished data. In the whole-brain transcriptome of blue gourami, 17 transcription genes change during vitellogenesis in the brain. The hormones involved in reproduction in blue gourami described in the present paper include: Kisspeptin 2 (Kiss 2) and its receptors 1 and 2 (KissR 1 and 2); gonadotropin-releasing hormone 1, 2 and 3 (GnRH1, 2 and 3); GnRH receptor; pituitary adenylate cyclase-activating polypeptide (PACAP) and its related peptide (PRP); somatolactin (SL); follicle-stimulating hormone (FSH); luteinizing hormone (LH); growth hormone (GH); prolactin (PRL), 17ß-estradiol (E2); testosterone (T); vitellogenesis (VTL); and 17α,20ß- dihydroxy-4-pregnen-3-one (17,20P). A proposed quality model is presented regarding the brain control oogenesis in blue gourami that has a Labyrinth organ about which relatively little information has been published. This paper summarizes the complex various factors involved in the interactions between external and internal elements affecting the brain of fish reproduction in the Anabantiform order. It is suggested to study in the future the involvement of receptors of hormones, pheromones, and genome changes in various organs belonging to the reproduction system during the reproduction cycles about which little is known.

Gonadotropins in the Russian Sturgeon: Their Role in Steroid Secretion and the Effect of Hormonal Treatment on Their Secretion.

In the reproduction process of male and female fish, pituitary derived gonadotropins (GTHs) play a key role. To be able to specifically investigate certain functions of Luteinizing (LH) and Follicle stimulating hormone (FSH) in Russian sturgeon (Acipenser gueldenstaedtii; st), we produced recombinant variants of the hormones using the yeast Pichia pastoris as a protein production system. We accomplished to create in vitro biologically active heterodimeric glycoproteins consisting of two associated α- and ß-subunits in sufficient quantities. Three dimensional modelling of both GTHs was conducted in order to study the differences between the two GTHs. Antibodies were produced against the unique ß-subunit of each of the GTHs, in order to be used for immunohistochemical analysis and to develop an ELISA for blood and pituitary hormone quantification. This detection technique revealed the specific localization of the LH and FSH cells in the sturgeon pituitary and pointed out that both cell types are present in substantially higher numbers in mature males and females, compared to immature fish. With the newly attained option to prevent cross-contamination when investigating on the effects of GTH administration, we compared the steroidogeneic response (estradiol and 11-Keto testosterone (11-KT) in female and males, respectively) of recombinant stLH, stFSH, and carp pituitary extract in male and female sturgeon gonads at different developmental stages. Finally, we injected commercially available gonadotropin releasing hormones analog (GnRH) to mature females, and found a moderate effect on the development of ovarian follicles. Application of only testosterone (T) resulted in a significant increase in circulating levels of 11-KT whereas the combination of GnRH + T did not affect steroid levels at all. The response pattern for estradiol demonstrated a similar situation. FSH levels showed significant increases when GnRH + T was administered, while no changes were present in LH levels.

The role of brain peptides in the reproduction of blue gourami males (Trichogaster trichopterus).

In all vertebrates, reproduction and growth are closely linked and both are controlled by complex hormonal interactions at the brain-pituitary level. In this study, we focused on the reciprocal interactions between brain peptides that regulate growth and reproductive functions in a teleostei fish (blue gourami Trichogaster trichopterus). An increase in gonadotropin-releasing hormone 1 (GnRH1) gene expression was detected during ontogeny, and this peptide increased growth hormone (GH) and ß follicle-stimulating hormone (ßFSH) gene expression in pituitary cell culture. However, although no change in gonadotropin-releasing hormone 2 (GnRH2) gene expression during the reproductive cycle or sexual behavior was detected, a stimulatory effect of this peptide on ß gonadotropins (ßGtH) gene expression was observed. In addition, pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38) inhibited GnRH-analog-induced ßFSH gene expression, and co-treatment of cells with GnRH-analog and PACAP-38 inhibited GnRH-analog-stimulatory and PACAP-38-inhibitory effects on GH gene expression. These findings together with previous studies were used to create a model summarizing the mechanism of brain peptides (GnRH, PACAP and its related peptide) and the relationship to reproduction and growth through pituitary hormone gene expression during ontogenesis and reproductive stages in blue gourami.

Involvement of GnRH, PACAP and PRP in the reproduction of blue gourami females (Trichogaster trichopterus).

In vertebrates, gonadotropin-releasing hormone (GnRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) are key hormones regulating growth and reproduction in the brain-pituitary axis. The regulating hormonal interactions are of great interest, therefore, the aim of this study is to provide novel insights into the involvement of brain GnRH and PACAP in oogensis and spermatogenesis in a fish model, the blue gourami (Trichogaster trichopterus). cDNA cloning of two GnRH forms combined with phylogenetic analysis revealed that three paralogous GnRH forms exist in blue gourami and evolve as a result of genome duplication. GnRH1 mRNA levels are related to final oocyte maturation (FOM), and this peptide stimulated ß follicle-stimulating hormone (ßFSH) and growth hormone (GH) gene expression; GnRH2 stimulated ß gonadotropins (GtH) gene expression and GnRH analog combined with PACAP-38 synergistically upregulate GH and ßFSH gene expression. The data presented, together with previous studies in our lab, enable suggesting mechanisms explaining the physiological relevance of these peptides in the regulation of gametogenesis and steroidogenesis in blue gourami females. These findings support the biological importance of the GnRH and PACAP hormones family, enabling them to stimulate differential biological functions in the regulation of growth and reproduction.

Effect of environmental temperature on growth- and reproduction-related hormones gene expression in the female blue gourami (Trichogaster trichopterus).

Fish are ectothermic vertebrates, and their gonadal development and spawning are affected by changes in environmental temperature. Recent global temperature changes have increased the importance of studying the effect of temperature on reproduction. The aim of this paper was to study the effect of temperature on oogenesis and hormone gene expression related to reproduction and growth in the blue gourami female maintained under non-reproductive and reproductive conditions. In females under non-reproductive conditions, vitellogenic oocytes, gonadotropin-releasing hormone 3 (GnRH3), ß luteinizing hormone (ßLH) and growth hormone (GH) mRNA levels were affected by temperature changes. In females maintained under reproductive conditions with non-reproductively active males, a percentage of females in the final oocyte maturation (FOM) stage, pituitary adenylyl cyclase activating polypeptide (PACAP and PRP-PACAP), gonadotropins and GH mRNA levels were affected due to temperature changes. In females maintained under reproductive conditions with reproductively active males, also GnRH3 and insulin-like growth factor 1 (IGF-1) were affected by temperature changes. In conclusion, in blue gourami females, changes in environmental temperature affect oogenesis through changes in brain and pituitary hormone mRNA levels.

Temperature affects brain and pituitary gene expression related to reproduction and growth in the male blue gouramis, Trichogaster trichopterus.

This study examined the effect of temperature on reproduction and growth-related factors in blue gourami males under nonreproductive and reproductive conditions. Males that were maintained under nonreproductive conditions did not build nest and the gonado-somatic index (% GSI) was significantly higher in fish maintained at 27°C compared with fish maintained at 23°C. The relative mRNA levels of brain gonadotropin-releasing hormone 3 (GnRH3), pituitary adenylate cyclase-activating polypeptide (PACAP), insulin-like growth factor-1(IGF-1), pituitary ß-luteinizing hormone (ßLH), and prolactin were significantly higher when the fish were maintained at 27°C than at 23°C or 31°C. ß-Follicle-stimulating hormone (ßFSH) mRNA levels were significantly lower when maintained at 31°C than at the other temperatures. Nests were observed only in males under reproductive conditions. In these fish, higher mRNA levels of GnRH3, PACAP, ßFSH, ßLH and prolactin were detected at 27°C, and higher mRNA levels of IGF-1 were detected at 23°C, when compared with other temperature of maintenance or with fish that did not build nest. In conclusion, we propose that temperature has more effect on the transcription of genes, associated with reproduction, than on those pertaining to growth.

Association between pituitary adenylate cyclase-activating polypeptide and reproduction in the blue gourami.

In order to gain a better understanding of the roles of pituitary adenylate cyclase-activating polypeptide (PACAP) in reproduction and growth, the expression of the PACAP gene during the reproduction cycle and its potential role in regulating gonadotropin and growth hormone (GH) gene transcription in blue gourami were investigated. The cDNA sequences of the full-length blue gourami brain PACAP and that of its related peptide (PRP) were acquired. PACAP cDNA had two variants, obtainable by alternative splicing: a long form encoding for both PRP and PACAP and a short form encoding only for PACAP. In females, mRNA levels of PACAP were very high only in individuals with oocytes in the maturation stage, as compared to levels in unpaired vitellogenic and non-vitellogenic fish. The PACAP mRNA levels in males were high only in nest builders, as opposed to in non-nest building males and juveniles. In pituitary culture cells from high vitellogenic females, PACAP38 (the 38 amino acid form) only brought about an increase in betaFSH levels, without altering GH and betaLH mRNA levels. On the other hand, in adult non-reproductive male pituitary cells, PACAP38 decreased the GH mRNA level. Based on these results, we propose that in the blue gourami, PACAP is involved in the final oocyte maturation stage in females, whereas in males, it is associated with sexual behavior. In addition, the effect of PACAP38 on pituitary hormone gene expression is different in females and males, indicating that PACAP38 is potentially a hypophysiotropic regulator of reproduction, which mediates pituitary hormone expression.

Anatomical, hormonal and histological descriptions of captive Russian sturgeon (Acipenser gueldenstaedtii) with intersex gonads.

Sturgeons are known throughout the world as the source of black caviar. Their declining populations in their native habitats, mainly the Caspian Sea, due to over-fishing for meat and caviar production, destruction of their spawning grounds and water pollution, have led to their introduction into aquaculture in areas with suitable conditions, including Israel. Recently, we noticed an unusual phenomenon in these normally gonochoristic species. Several 5-year-old female sturgeons were found to have one or more testicular sections in each of their two gonads, forming an intersexual gender. Further examination of other fish from the same age group revealed 14% fish with intersex gonads among a population of 5000 fish that had been pre-selected as females. This phenomenon has not been found however in other age groups of Russian sturgeons, cultured at the same facility. Sturgeons are a generally gonochoristic species, and hermaphroditism is only very infrequently observed under natural or normal breeding conditions. Moreover, these rare cases have all been from polluted habitats. The present work is the first description of fish containing intersex gonads in Russian sturgeon (Acipenser gueldenstaedtii). We describe the phenomenon anatomically and histologically, and examine plasma steroid levels and pituitary gonadotropin gene expression by comparing fish with intersex gonads with normal females and males of the same age group. Intersex gonads were typical female ovaries with one or more white testicular components embedded in each. The testis components were not uniform in size or location among the two gonads of each fish or among different fish, and they showed marked differences in distribution. The ovarian component of the intersex gonad was at the pre-vitellogenic stage as in normal females, and the testis component contained spermatids and mature spermatozoa as in normal males of the same age. However, in terms of estradiol and 11-ketotestosterone plasma levels, as well as of pituitary gonadotropin (betaLH and betaFSH) gene-expression levels, the fish with intersex gonads were more similar to the normal males than to the normal females, even though the testis part of the intersex gonad was smaller than the ovarian part. To examine the possibility that the fish containing intersex gonads were hybrids, phylogenetic trees were constructed from the consensus sequences of Cytochrome b and control region (D-loop) genes. Results indicated no differences between the fish with intersex gonads and normal males or females of the same age group. However, statistically significant differences were found between different age groups of Russian sturgeon, as well as of white sturgeons (A. transmontanus), grown under the same culture conditions.

Cloning of FSHbeta, LHbeta, and glycoprotein alpha subunits from the Russian sturgeon (Acipenser gueldenstaedtii), beta-subunit mRNA expression, gonad development, and steroid levels in immature fish.

The Russian sturgeon, Acipenser gueldenstaedtii, is a late-maturing Acipenseriformes. To elucidate the role of FSH and LH in its reproduction, we cloned its glycoprotein alpha-subunit (GPalpha) and gonadotropin beta-subunits (FSHbeta and LHbeta) using 5' and 3' RACE-PCR. The nucleotide sequences of the Russian sturgeon (st) GPalpha, FSHbeta, and LHbeta are 345, 384, and 411 bp long, encoding peptides of 91, 115, and 114 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity with those of other teleosts. Sequence analysis showed that stFSHbeta is more similar to higher vertebrate FSHbetas (35-37%) than to higher vertebrate LHbetas (26-30%). The next objective of this work was to compare the development of sturgeon gonads at the very first stages of their growth with the expression of their gonadotropins. Sturgeons at ages 1, 2, 3 or 4 years were sacrificed. The expression of their gonadotropin beta-subunits was determined using quantitative real-time PCR, and their gonads were examined histologically, followed by a determination of the plasma levels of estradiol in females and 11-ketotestosterone (11-KT) in males. The expression levels of stFSHbeta subunit was found to be higher in fish at 3 and 4 years of age than in 1-year olds. mRNA levels of stLHbeta were higher than those of stFSHbeta in both genders. Moreover mRNA levels of stFSHbeta detected in females were significantly higher than those found in males. Even at age 4 years, all female Russian sturgeons tested contained gonads at the pre-vitellogenic stage, with small oocytes and very low levels of estradiol in the plasma. However, among the males, at ages 3 and 4 years, we found testes that contained spermatids and spermatozoa. Those males were found to have significantly high GSI (gonadosomatic index; gonadal weight as a percentage of BW) levels, stLHbeta expression and 11-KT levels.

Cloning and developmental expression of the cytochrome P450 aromatase gene (CYP19) in the European eel (Anguilla anguilla).

To characterize the involvement of the aromatase gene during the process of sex determination in the European eel (Anguilla anguilla), the expression of its gonadal form was determined during various developmental stages. The cloned cDNA from the European eel gonad (EeCYP19) contains an open reading frame of 1539 bp, encoding a deduced protein of 513 residues. The predicted amino acid sequence shows 97% identity with that of the Japanese eel, and 59-69% of identity with those of the CYP19 gonadal and brain forms of other teleost fish. Two potential initiation sites (ATG) were found downstream of the first ATG codon. A fluorescent-based method of real-time PCR was developed to quantify EeCYP19 expression. The expression levels of EeCYP19 in the gonads of adult males were approximately 12- and 30-fold lower than the levels in adult females and juvenile eels previously treated with E2, respectively. Expression of aromatase was found only in a single specimen in the control group. In contrast, no difference was found among sexes in the aromatase expression in the brain. Treatment with aromatase inhibitor (AI) of juvenile eel resulted in the total loss of aromatase expression in the gonads and brains. The results of this work revealed that AI treatment not only reduces the synthesis of estradiol, but reduces the expression levels of EeCYP19 as well. No evidence for the presence of a distinct extra-gonadal (brain) form of aromatase in the European eel could be provided.

betaFSH, betaLH and growth hormone gene expression in blue gourami (Trichogaster trichopterus, Pallas 1770) during spermatogenesis and male sexual behavior.

The relationship between gonadal development (histological evidence for spermiogenesis and/or spermatogenesis), sexual behavior (nest-building) and mRNA levels of gonadotropins (betaFSH and betaLH) and growth hormone (GH) in the male pituitary was investigated. Amplification of betaFSH cDNA showed a significantly higher mRNA level in mature males (whether sexually active or not) than in juveniles. However, following PCR amplification of betaLH cDNA, a significantly higher mRNA level was found in the sexually active group compared to the sexually inactive group. These results suggest that FSH may participate in spermatogenesis, whereas LH is more involved in spermiogenesis. The GH mRNA level increased slightly during the maturation process but no significant differences were found between the groups studied.