Regulation of steroid production and key genes in catfish Heteropneustes fossilis using recombinant gonadotropins.

The two gonadotropins, FSH and LH, stimulate growth and development of the gonads through gonadal biosynthesis of steroid hormones and growth factors. To date, cDNA sequences encoding gonadotropin subunits have been isolated and characterized from a large number of fish species. Recently, we successfully cloned and characterized gonadotropins (LHß, FSHß, and GPα) from the pituitary glands of the catfish, Heteropneustes fossilis. In the present study, we describe herein the production of recombinant stinging catfish, H. fossilis (hf) FSH (rhfFSH) and LH (rhfLH) using the methylotrophic yeast P. pastoris expression system. We further explored the hypothesis that the recombinant gonadotropins can modulate the hypothalamus-pituitary-ovarian (HPO) axis genes (avt, it, gnrh2, kiss2, and cyp19a1a) and regulate their transcriptional profile and steroid levels in relation to their annual developmental stage during preparatory and pre-spawning phases under in-vitro conditions. We found that the different concentrations of recombinant rhfFSH and rhfLH significantly stimulated E2 levels in the preparatory and prespawning season, and also upregulated gonadal aromatase gene expression in a dose dependent manner. Our results demonstrate that the yeast expression system produced biologically active recombinant catfish gonadotropins, enabling the study of their function in the catfish.

Air sac and gill vasotocin receptor gene expression in the air-breathing catfish Heteropneustes fossilis exposed to water and air deprivation conditions.

Heteropneustes fossilis is a facultative air-breathing freshwater catfish and inhabits ponds, ditches, swamps, marshes and rivers that dry up in summers. It possesses a pair of unique tubular accessory respiratory organ (air sac), which is a modification of the gill chamber and enables it to live in water-air transition zones. In the catfish, three vasotocin (Vt) receptor gene paralogs viz., v1a1, v1a2 and v2a were identified for Vt actions. In the present study, the receptor gene transcripts were localized in the gill and air sac by in situ hybridization, and their expression levels in relation to water and air deprivation conditions were investigated by quantitative RT-PCR. The catfish were exposed to 1 h and 2 h in gonad inactive (resting) and gonad active (prespawning) phases. The gene paralogs showed overlapping distribution in the respiratory epithelium of primary and secondary lamellae of gills and reduced lamellae of the air sacs. In water deprivation (forced aerial mode of respiration) experiment, v2a expression showed a high fold increase in the air sac, which was unchanged or inhibited in the gill. Both v1a1 and v1a2 expression was significantly upregulated in the air sac but showed varied responses in the gill. The gill v1a1 expression was unchanged in the resting phase and modestly upregulated in the prespawning phase. The gill v1a2 expression was modestly upregulated at 1 h in both phases but unchanged at 2 h. In the air deprivation experiment (forced aquatic respiration), the v2a expression in the air sac was inhibited except for a mild stimulation at 1 h in the prespawning phase. In the gill, the v2a expression was stimulated with a steep upregulation at 2 h in the prespawning phase. Both v1a1 and v1a2 expression was significantly high in the gill but only modestly increased or unchanged in the air sac. The expression patterns point to a functional distinction; the V2 type receptor expression was higher in the air sac during forced aerial respiration, and the V1 type receptor expression was highly prominent in the gill during forced aquatic respiration. Water and air deprivation treatments caused a significant increase in plasma cortisol level, and the stimulation was higher in the water deprivation fish in the resting phase but equally prominent in the water and air deprivation groups in the prespawning phase. The results indicate that the changes in the expression patterns of Vt receptor genes may be a sequel to stress (hypoxic, metabolic and osmotic), and both Vt and cortisol may interact to counter the stress responses. This study shows that Vt has a new role in the control of air sac functions.

Cloning and characterization of estrogen hydroxylase (cyp1a1 and cyp1b1) genes in the stinging catfish Heteropneustes fossilis and induction of mRNA expression during final oocyte maturation.

Estrogen hydroxylases (EHs) are cytochrome P450 Family 1 (Cyp1, Clan 2) proteins involved in estrogen hydroxylations at 2-, 4- or 16- carbon positions to form catecholestrogens. EHs are encoded by CYP1A1, CYP1A2 and CYP1B1 in mammals. In the catfish Heteropneustes fossilis, cyp1a1 and cyp1b1 cDNAs were cloned and characterized from liver and ovary. The cyp1a1 cDNA is 2071 bp long and codes for a 518 amino acids (aa) long protein. The cloned cyp1b1 cDNA is 1927 bp long and codes for a 509 residue protein. The deduced proteins clustered distinctly into teleost Cyp1a1 and Cyp1b1 clades, distinct from the tetrapod clusters and featured common function domains and homology with other teleost proteins. In the qPCR assay, the transcripts were the most abundant in the liver, followed by brain and ovary, and moderate in gill, kidney and muscle. Evidence was presented to show the involvement of the genes in reproduction. Expression of brain and ovarian transcripts showed significant seasonal variations with the highest abundance in the spawning phase. In situ hybridization showed the transcripts in the follicular layer (theca and granulosa) of the ovarian follicles. Periovulatory changes in the expression cyp1a1 and cyp1b1 were obtained during final oocyte maturation (FOM) and ovulation induced by human chorionic gonadotropin (hCG), both in vivo and in vitro, and by 2-hydroxyestradiol-17ß (catecholestrogen) in vitro. In the brain, the transcript levels increased with time but in the ovary, the increase was maximal at 16 h and decreased at 24 h. The periovulatory activation of the cyp1 genes was reported in this study and discussed on the basis of complex regulation of FOM and ovulation.

Identification of kisspeptin2 cDNA in the catfish Heteropneustes fossilis: Expression profile, in situ localization and steroid modulation.

Kisspeptin (Kiss) is considered an upstream regulator of gonadotropin-releasing hormone in mammals but its role in non-mammalian vertebrates is not unequivocally established. In the catfish Heteropneustes fossilis, a 605 bp long cDNA was identified from the brain by cloning as well as by retrieving from the catfish transcriptome database. The open reading frame (ORF, 93-405 bp) codes for a 113 amino acids long precursor protein. Homology and phylogenetic analyses showed that the predicted protein belongs to the vertebrate Kiss2 type with a high degree of conservation in the Kiss2-10 region (FNFNPFGLRF). The kiss2 transcripts were expressed highly in the brain and gonads in a dimorphic manner with a female bias. In the brain, kiss2 transcripts showed regional differences with higher expression in the medulla oblongata and forebrain regions. The kiss2 transcripts showed significant seasonal variations with the highest expression in the brain in spawning phase and in the gonads in prespawning phase. The kiss2 transcripts were localized in the brain (nucleus preopticus, habenular nucleus, nucleus recessus posterioris, nucleus recessus lateralis) and stratum periventriculare (radial glial cells) of optic tectum, pituitary and ovary (follicular layer and germinal vesicle). Ovariectomy (1, 2, 3 and 4 weeks) decreased brain kiss2 mRNA levels and a single injection of estradiol-17ß (E2; 0.5 µg/g body weight) in 3- week ovariectomized (OVX) and sham operated fish resulted in an increase in the transcript levels after 24 h. The E2 receptor antagonist Tamoxifen (TMX) produced biphasic effects on the kiss2 expression in the dose- response study. TMX inhibited the expression in the OVX fish, but elicited a stimulatory effect in the OVX + E2-treated fish. Testosterone (T) decreased, and progesterone (P4) inhibited (resting phase) or stimulated (prespawning phase) the transcript level in 3-week OVX fish. In the 3-week sham groups, E2 increased, and TMX, T and P4 inhibited the kiss2 transcript levels. The results suggest that Kiss2 is an important regulator of the brain- pituitary- gonadal- endocrine axis, and in habenular and optic tectum functions.

Expression profile of kisspeptin2 and gonadotropin-releasing hormone2 mRNA during photo-thermal and melatonin treatments in the female air-breathing catfish Heteropneustes fossilis.

In seasonally breeding vertebrates, extrinsic factors like photoperiod and temperature are major determinants, controlling the annual reproductive cycle. In teleosts, kisspeptin, which occurs in two molecular forms: kisspeptin1 (Kiss1) and kisspetin2 (Kiss2), has been reported to alter gonadotropin (Lh and Fsh) secretion but its effect on gonadotropin-releasing hormone (Gnrh) secretion is not unequivocally proved. In the catfish Heteropneustes fossilis, we isolated and characterized kiss2 and gnrh2 cDNAs and the present work reports effects of altered photo-thermal conditions and melatonin (MT, a pineal hormone) on their expressions in the brain. The exposure of the catfish to long photoperiod (LP, 16 h light) at normal temperature (NT) or high temperature (HT, 28 °C) at normal photoperiod (NP) for 14 or 28 days stimulated both kiss2 and gnrh2 expression in both gonad resting and preparatory phases with the combination of LP + HT eliciting maximal effects. Short photoperiod (SP, 8 h light) under NT or HT altered the gene expression according to the reproductive phase and temperature. MT that mediates photo-thermal signals to the brain inhibited brain kiss2 and gnrh2 gene expression in the NP + HT, LP + NT, and SP + NT groups. The altered photo-thermal conditions elicited changes in steroidogenic pathway as evident from changes in plasma E2, progesterone, and testosterone levels. The results show that brain kiss2-gnrh2 signaling is involved in photo-thermal-mediated mechanisms controlling reproduction.

Stress hormones modulate lipopolysaccharide stimulation of head kidney interleukin-6 production in the catfish Heteropneustes fossilis: In vivo and in vitro studies.

Interleukin-6 (IL-6) is a pleiotropic cytokine secreted by immune tissues such as monocytes/macrophages and have pro-inflammatory/anti-inflammatory and neuroendocrine actions. In this study, we report the modulatory effects of stress hormones, the cortisol agonist dexamethasone and catecholamines on lipopolysaccharide (LPS) - induced stimulation of head kidney IL-6 in the catfish Heteropneustes fossilis. In the in vivo study, the intraperitoneal administration of LPS stimulated, and dexamethasone time-dependently inhibited IL-6 level. In the in vitro study, the incubation of macrophage cultures with LPS stimulated IL-6 level significantly in all incubation times. Dexamethasone did not alter the basal IL-6 level but inhibited time-dependently the LPS-induced stimulation. Likewise, catecholamines did not alter the basal level of IL-6. Both epinephrine and norepinephrine inhibited the LPS-induced stimulation of IL-6. Dopamine, on the other hand, was ineffective. The results indicate that IL-6 is a useful marker of head kidney macrophage activity for studying endocrine-immune interactions in the catfish.

Reproductive hormones modulate differentially brain and ovarian vasotocin receptor gene expression in early and late recrudescent catfish, Heteropneustes fossilis.

Investigations on the role of the reproductive hormones on VT receptor gene expression are lacking in teleosts. Previously we reported that gonadotropin and steroid hormones modulate the secretion and gene expression of brain and ovarian vasotocin (VT) in the catfish Heteropneustes fossilis. In continuation, in the present study we investigated the role of estradiol-17ß (E2), the maturation-inducing steroid (MIS) 17α, 20ß-dihydroxy-4-pregnen-3-one (17, 20ß-DP), and human chorionic gonadotropin (hCG) on the expression of VT receptor genes (v1a1, v1a2 and v2a) in the brain and ovary of the catfish in early (previtellogenic, preparatory) and late (post vitellogenic, prespawning) phases of the ovarian cycle. The steroid treatments (in vivo and in vitro) modulated only the v1a1 and v1a2 expression in both tissues, but not the v2a expression. The E2-induced modulation of the v1a1 and v1a2 gene expression varied with the reproductive phase. In the preparatory phase, E2 up regulated the expression of brain and ovarian v1a1 and v1a2 gene expression, the response varied with the dose and duration. In the prespawning phase, E2 inhibited the expression in a dose- and duration-dependent manner. On the other hand, 17, 20ß-DP up regulated the expression of brain and ovarian v1a1 and v1a2 in both phases, and the response was higher in the prespawning phase and varied with dose and duration. In contrast to the steroid effects, the hCG treatment modulated the expression of all the VT receptor genes only in the prespawning phase and the response varied with dose and duration. The results indicate differential modulatory roles of steroid hormones and hCG on the VT receptor gene expression, to mediate VT's reproductive or osmoregulatory functions. While the hCG effect on v1a type receptor expression may be steroid- dependent, that of v2a expression seems to be steroid-independent.

Molecular cloning and characterization of a gonadotropin-releasing hormone 2 precursor cDNA in the catfish Heteropneustes fossilis: Expression profile and regulation by ovarian steroids.

Gonadotropin-releasing hormone 2 (Gnrh2) is one of the three classes of Gnrh distributed in vertebrates and is highly conserved. In the present study, the cDNA encoding Gnrh2 was isolated and characterized in the ostariophysan catfish Heteropneustes fossilis (hf). The cDNA is 611 bp long with an open reading frame (ORF) of 261 bp that encodes a highly conserved protein of 86 amino acids. The deduced Gnrh2 precursor protein clustered with the vertebrate Gnrh2 type. The sequence identity of hfgnrh2 is 94% with African catfish (Clarias gariepinus) gnrh2 mRNA (accession no. X78047). The hfgnrh2 transcripts were expressed only in the brain and gonads with a higher expression in the female brain and ovary in both resting and prespawning phases. The expression was higher in the prespawning phase than the resting phase. The gnrh2 expression in the brain and ovary showed significant seasonal variations but with opposite patterns. In the brain, the expression was the highest in the preparatory phase, decreased progressively to low levels in the postspawning and resting phases. In the ovary, the transcript level was low in the resting and preparatory phases, increased sharply in the prespawning phase reaching the peak level in the spawning phase and declined sharply in the postspawning phase. The gnrh2 mRNA showed the highest expression in the hind brain-medulla oblongata and moderate to low expression in forebrain regions and pituitary. Ovariectomy resulted in a duration-dependent inhibition of hfgnrh2 mRNA levels in the resting and prespawning phases. Steroid (E2, testosterone and progesterone) replacement treatments (0.5 µg/g body weight) in the 3- week ovariectomized fish restored the inhibition due to ovariectomy, elevated the expression over and above the sham level in the resting phase (E2 group), and raised the levels almost to that of the sham group (testosterone and progesterone groups) in the prespawning phase. In the sham control groups, the steroid replacement resulted in a significant reduction in the mRNA levels. The expression of the gnrh2 mRNA in the brain-pituitary-gonadal axis and its regulation by gonadal steroids suggest that Gnrh2 may have a reproductive role in the catfish.

Reproductive stage- and sex-dependant effects of neurohypophyseal nonapeptides on gonadotropin subunit mRNA expression in the catfish Heteropneustes fossilis: An in vitro study.

In the present study, in vitro effects of synthetic vasotocin (VT), isotocin (4Ser, 8Ile- oxytocin; ITb) and the recently cloned IT gene paralog product (8Val-Isotocin, ITa) were studied on the expression of pituitary gonadotropin (GtH) subunit mRNA levels. In male pituitaries of early (preparatory phase) and late (prespawning phase) recrudescing catfish, Heteropneustes fossilis, VT (10 nM, 100 nM and 1000 nM) stimulated fshß expression dose-dependently. But in females, the dose-dependent effect was found only in the preparatory phase. In males, VT stimulated lhß expression only at higher doses. In females, VT produced a significant dose-dependent increase of the lhß expression only in the prespawning phase. VT stimulated the expression of gpα, dose-dependently in the preparatory phase in males and in the prespawning phase in females. The incubation of the pituitaries with ITb did not alter the fshß expression in either sex in both preparatory and prespawning phases. In males, ITb stimulated the expression of lhß and gpα only at the highest concentration (1000 nM) in both phases. In females, ITb stimulated both lhß and gpα expression only at 1000 nM in the preparatory phase and dose-dependently in the prespawning phase. The incubation of the pituitaries with ITa produced effects similar to ITb on the expression of fshß, lhß, and gpα. The results show that the basic peptide VT modulates both fshß and lhß expressions, which are influenced by the sex and reproductive stage. The neutral peptide ITA/ITb exerts an insignificant effect on the fshß expression regardless of sex or season. Both VT and ITa/ITb elicit a significant effect on the lhß expression in late recrudescent phase especially in females.

Ovaprim, a commercial spawning inducer, stimulates gonadotropin subunit gene transcriptional activity: A study correlated with plasma steroid profile, ovulation and fertilization in the catfish Heteropneustes fossilis.

The commercial fish spawning inducer Ovaprim (OVP) containing a salmon gonadotropin-releasing hormone analogue and domperidone (a dopamine receptor-2 antagonist) has been widely used as an effective spawning inducer in artificial breeding of fishes. It induces a preovulatory LH surge resulting in final oocyte maturation (FOM) and ovulation through a mechanism involving a steroidogenic shift to secrete a maturation-inducing steroid (MIS). In the present study, a 0.5µL/g body weight dose of OVP each injected at 0h and 24h intraperitoneally into gravid female catfish, Heteropneustes fossilis resulted in periovulatory changes in gonadotropin (GtH) subunit gene expression and steroid hormone levels. The OVP injections induced ovulation time-dependently from 6h onwards with 100% ovulation recorded from 24h to 48h. The fertilization rate was high from 6h to 18h and declined from 24h onwards. The OVP treatment up regulated the expression of GtH subunit genes differentially. The expression of glycoprotein-α (GPα) and luteinizing hormone (LHß) peaked at 6h and 12h, and declined at 18h and 24h after the first injection. The second OVP injection at 24h elicited only a transient increase in the GPα expression at 6h and a sustained increase in the LHß expression from 6h to 18h after the second injection, but both transcripts decreased subsequently. The follicle-stimulating hormone (FSHß) expression responded to the OVP treatment from 12h onwards and maintained a constant level from 18h to 36h after the first injection; the second dose had little effect. Plasma steroids were differentially altered: the levels of estradiol-17ß decreased while that of the MIS 17,20ß-dihydroxy-4-pregnen-3-one; 17,20ß-DP increased, causing the steroidogenic shift preceding FOM and ovulation. The present results indicate that LHß expression coincides with the ovulation response and the late induction and maintenance of the FSH expression may be related to post-ovulatory events in the ovary.

Effects of the fish spawning inducer ovaprim on vasotocin receptor gene expression in brain and ovary of the catfish Heteropneustes fossilis with a note on differential transcript expression in ovarian follicles.

Ovaprim (OVP), a commercial formulation of a salmon GnRH analogue and the dopamine receptor-2 blocker domperidone, is a successful spawning inducer for fish breeding. It induces a preovulatory surge in LH, which stimulates the synthesis of a maturation-inducing steroid (MIS, 17,20ß-dihydroxy-4-pregnen-3-one) that initiates germinal vesicle breakdown (GVBD) and ovulation. Coincidently, the OVP treatment also stimulates vasotocin (VT) secretion in the brain and ovary of the catfish Heteropneustes fossilis that also stimulates the synthesis of the MIS. VT mediates its effect through V1- and V2-type receptors. In the present study in the catfish, we report that OVP stimulates the expression of VT receptor genes v1a1, v1a2 and v2a in the brain and ovary. A single intraperitoneal administration of OVP (0.5µL/g body weight) or incubation of post-vitellogenic ovarian follicles with 5µL/mL OVP, for 0, 4, 8, 12, 16, and 24h stimulated ovulation and GVBD, respectively, in a time-dependent manner. The OVP treatment in vivo stimulated brain VT receptor transcript levels 4h onwards. The peak expression was noticed at 12h (v1a1), 8 and 12h (v1a2), and 8, 12 and 16h (v2a), coinciding with FOM and ovulation. The VT receptor genes are expressed in the ovarian follicles compartmentally; both v1a1 and v1a2 are expressed in the isolated follicular layer (theca and granulosa) but absent in denuded oocytes. V2a is expressed in the denuded oocytes and not in the follicular layer. The OVP injection stimulated the v1a1 and v1a2 expression from 4h onwards in both intact follicle and isolated follicular layer, the peak expression was observed at 16h. The v2a expression was up-regulated in both intact follicles and denuded oocytes at 4h (denuded oocytes) or 8h (intact follicle) onwards with the peak expression at 12h and 16h (denuded oocytes) or at 16h (intact follicles). Under in vitro conditions, the OVP incubations elicited similar pattern of changes with the peak stimulation at 16h for all the genes. In conclusion, the VT receptor genes are differentially expressed in the ovarian follicles and OVP induced periovulatory stimulation of the VT receptor genes, coinciding with FOM and ovulation.

Structural and functional diversity of nonapeptide hormones from an evolutionary perspective: A review.

The article presents an overview of the comparative distribution, structure and functions of the nonapeptide hormones in chordates and non chordates. The review begins with a historical preview of the advent of the concept of neurosecretion and birth of neuroendocrine science, pioneered by the works of E. Scharrer and W. Bargmann. The sections which follow discuss different vertebrate nonapeptides, their distribution, comparison, precursor gene structures and processing, highlighting the major differences in these aspects amidst the conserved features across vertebrates. The vast literature on the anatomical characteristics of the nonapeptide secreting nuclei in the brain and their projections was briefly reviewed in a comparative framework. Recent knowledge on the nonapeptide hormone receptors and their intracellular signaling pathways is discussed and few grey areas which require deeper studies are identified. The sections on the functions and regulation of nonapeptides summarize the huge and ever increasing literature that is available in these areas. The nonapeptides emerge as key homeostatic molecules with complex regulation and several synergistic partners. Lastly, an update of the nonapeptides in non chordates with respect to distribution, site of synthesis, functions and receptors, dealt separately for each phylum, is presented. The non chordate nonapeptides share many similarities with their counterparts in vertebrates, pointing the system to have an ancient origin and to be an important substrate for changes during adaptive evolution. The article concludes projecting the nonapeptides as one of the very first common molecules of the primitive nervous and endocrine systems, which have been retained to maintain homeostatic functions in metazoans; some of which are conserved across the animal kingdom and some are specialized in a group/lineage-specific manner.

Identification and characterization of a catechol-o-methyltransferase cDNA in the catfish Heteropneustes fossilis: Tissue, sex and seasonal variations, and effects of gonadotropin and 2-hydroxyestradiol-17ß on mRNA expression.

Catechol-O-methyltransferase (COMT) is involved in the methylation and inactivation of endogenous and xenobiotic catechol compounds, and serves as a common biochemical link in the catecholamine and catecholestrogen metabolism. Studies on cloning, sequencing and function characterization comt gene in lower vertebrates like fish are fewer. In the present study, a full-length comt cDNA of 1442bp with an open-reading frame (ORF) of 792bp, and start codon (ATG) at nucleotide 162 and stop codon (TAG) at nucleotide 953 was isolated and characterized in the stinging catfish Heteropneustes fossilis (accession No. KT597925). The ORF codes for a protein of 263 amino acid residues, which is also validated by the catfish transcriptome data analysis. The catfish Comt shared conserved putative structural regions important for S-adenosyl methionine (AdoMet)- and catechol-binding, transmembrane regions, two glycosylation sites (N-65 and N-91) at the N-terminus and two phosphorylation sites (Ser-235 and Thr-240) at the C-terminus. The gene was expressed in all tissues examined and the expression showed significant sex dimorphic distribution with high levels in females. The transcript was abundant in the liver, brain and gonads and low in muscles. The transcripts showed significant seasonal variations in the brain and ovary, increased progressively to the peak levels in spawning phase and then declined. The brain and ovarian comt mRNA levels showed periovulatory changes after in vivo and in vitro human chorionic gonadotropin (hCG) treatments with high fold increases at 16 and 24h in the brain and at 16h in the ovary. The catecholestrogen 2-hydroxyE2 up regulated ovarian comt expression in vitro with the highest fold increase at 16h. The mRNA and protein was localized in the follicular layer of the vitellogenic follicles and in the cytoplasm of primary follicles. The data were discussed in relation to catecholamine and catecholestrogen-mediated functions in the brain and ovary of the stinging catfish.

Effects of altered photoperiod and temperature on expression levels of gonadotrophin subunit mRNAs in the female stinging catfish Heteropneustes fossilis.

Differential effects of photoperiod and temperature on the temporal modulation of gonadotrophin subunit genes (glycoprotein α, gpα), follicle-stimulating hormone ß (fshß) and luteinizing hormone ß (lhß) expression were investigated in the stinging catfish Heteropneustes fossilis. Female H. fossilis were exposed to varying photoperiod and temperature conditions for 14 and 28 days in the early preparatory phase of the annual reproductive cycle. Gonadotrophin subunit gene expression, gonado-somatic index (IG ), ovarian histology and plasma steroid hormone levels were evaluated. The exposure of H. fossilis to long photoperiod (LP) of 16 h light or high temperature (HT) at 28 ± 2° C (mean ± s.e.), alone or in combination, resulted in significant increases in gpα, fshß and lhß messenger (m)RNA levels, IG , plasma oestradiol-17ß (E2 ), testosterone (T) and progesterone (P4 ) levels. The ovaries were filled with advanced yolky oocytes. On the other hand, the short photoperiod (SP) of 8 h light exposure decreased the transcript levels with higher inhibition in the normal temperature (NT) group at 18 ± 2° C (mean ± s.e.) than the HT group at 28 ± 2° C. Furthermore, the inhibition reached the highest level in total darkness (TD) of 24 h light deprivation under NT conditions at 18 ± 2° C. Consequently, the SP and TD treatments inhibited the IG , plasma E2 and T levels and ovarian development. The exposure to high temperature at 28 ± 2° C also modified the short photoperiod effect by elevating plasma E2 level. The plasma T level changed only mildly while the plasma P4 level showed the greatest fluctuations; the level reached the nadir in the SP + HT group but increased in the SP + NT group on day 28. A two-way ANOVA of the data showed differential effects of photoperiod and temperature; photoperiod produced a highly significant effect on fshß expression while temperature had a highly significant effect both on lhß and gpα levels. Thus, the differential expression of the gpα by the environmental variables ensures temporal synchronization of ovarian development and spawning.

Morpho-histology of head kidney of female catfish Heteropneustes fossilis: seasonal variations in melano-macrophage centers, melanin contents and effects of lipopolysaccharide and dexamethasone on melanins.

In the catfish Heteropneustes fossilis, the anterior kidney is a hemopoietic tissue which surrounds the adrenal homologues, interrenal (IR) and chromaffin tissues corresponding to the adrenal cortical and adrenal medulla of higher mammals. The IR tissue is arranged in cell cords around the posterior cardinal vein (PCV) and its tributaries and secretes corticosteroids. The chromaffin tissue is scattered singly or in nests of one or more cells around the epithelial lining of the PCV or blood capillaries within the IR tissue. They are ferric ferricyanide-positive. Leukemia-inhibitory factor (LIF)-like reactivity was noticed in the lining of the epithelium of the IR cell cords and around the wall of the PCV and blood capillaries. No staining was observed in the hemopoietic cells. IL-1ß- and TNF-α-like immunoreactivity was seen in certain cells in the hemopoietic tissue but not in the IR region. Macrophages were identified with mammalian macrophage-specific MAC387 antibodies and are present in the hemopoietic mass but not in the IR tissue. Pigments accumulate in the hemopoietic mass as melano-macrophage centers (MMCs) and are PAS-, Schmorl's- and Perls'-positive. The pigments contain melanin (black), hemosiderin (blue) and lipofuscin/ceroid (oxidized lipid, yellowish tan), as evident from the Perls' reaction. The MMCs were TUNEL-positive as evident from FITC fluorescence, indicating their apoptotic nature. The MMCs showed significant seasonal variation with their density increasing to the peak in the postspawning phase. Melanins were characterized spectrophotometrically for the first time in fish anterior kidney. The predominant form is pheomelanin (PM), followed by eumelanin (EM) and alkali-soluble melanin (ASM). Melanins showed significant seasonal variations with the level low in the resting phase and increasing to the peak in the postspawning phase. Under in vitro conditions, lipopolysaccharide (10 µg/mL) treatment increased significantly the levels of PM and EM levels both at 16 and at 32 h and the ASM level at 32 h. On the other hand, the synthetic glucocorticoid dexamethasone (100 nM) decreased significantly the levels of EM, PM and ASM time-dependently. The results indicate that the anterior kidney is an important site of immune-endocrine interaction.

Vasotocin--A new player in the control of oocyte maturation and ovulation in fish.

In this article, the physiological role of ovarian vasotocin (VT) on fish final oocyte maturation (FOM) and ovulation is reviewed based on the studies mainly available in the catfish Heteropneustes fossilis. The VT system is characterized in the follicular layer of the oocytes by both immunocytochemical and in situ hybridization techniques. The distribution was confirmed in isolated follicular layer preparations by HPLC characterization and quantification. Three VT receptor subtype genes are identified: V1a1 and V1a2 subtypes are distributed in the follicular layer and V2 subtype is present along the granulosa-oocyte membrane junction. The expression of peptide, VT precursor gene and VT receptor genes shows seasonal and periovulatory changes in the ovary. VT secretion is modulated by E2 differentially in a season-specific manner, and by progestin steroids positively. VT modulates E2 in a biphasic manner in early recrudescent phase and induces a steroidogenic shift inhibiting E2 and stimulating progestin steroid (P4, 17P4 and 17,20ß-DP) pathways in the late recrudescent phase. VT stimulates prostaglandin secretion, germinal vesicle breakdown (GVBD), oocyte hydration and ovulation. VT acts through different receptors to stimulate these processes. It uses the V1 type receptor to stimulate GVBD and ovulation, and the V2 type to stimulate oocyte hydration. VT acts as an important link in the cascade of gonadotropin control of FOM and ovulation. More research is required in other species.

Effects of ovaprim, a commercial spawning inducer, on vasotocin and steroid hormone profiles in the catfish Heteropneustes fossilis: in vivo and in vitro studies.

Ovaprim (OVP) is used as an effective spawning inducer for artificial breeding of fishes and contains a salmon gonadotropin-releasing hormone analogue and a dopamine receptor-2 antagonist, domperidone. Previously, we have shown that vasotocin (VT) stimulates ovarian final oocyte maturation, hydration, and ovulation through a mechanism involving induction of a steroidogenic shift, favouring the production of a maturation-inducing hormone (MIH). In the present study, we demonstrated that OVP stimulated brain, plasma and ovarian VT levels, suggesting multiple sites of action, apart from its well established role in the induction of a preovulatory LH surge. An intraperitoneal injection of 0.5µL/g body weight of OVP for different time intervals (0, 4, 8, 12, 16 and 24h) induced ovulation as well as increased significantly brain and plasma VT levels in a time-dependent manner. Plasma steroids were differentially altered; the levels of estradiol-17ß (E2) and testosterone (T) decreased, and the MIH (17, 20ß-dihydroxy-4-pregnen-3-one; 17, 20ß-DP) level increased time-dependently. In order to demonstrate whether OVP acts at the level of the ovary directly, in vitro experiments were conducted. The incubation of ovarian slices/follicles with OVP (1, 5 and 10µL/mL) for different time points (0, 4, 8, 12, 16 and 24h) induced germinal vesicle breakdown (GVBD) in a concentration- and time-dependent manner. Ovarian VT increased significantly in a concentration- and time-dependent manner with a maximal increment at 16h. Ovarian T and E2 levels decreased concurrently with the rise in the MIH level, dose- and duration-dependently. The results show that OVP stimulates VT at the brain and ovarian level. The direct OVP-VT cascade has the potential to stimulate FOM and ovulation, sidelining the pituitary glycoprotein hormone (LH) surge.

An in vitro study on catecholamine modulation of ovarian steroidogenic activity in the catfish Heteropneustes fossilis.

In the present study, α-methylparatyrosine (α-MPT), a tyrosine hydroxylase inhibitor was used to impair ovarian catecholaminergic activity in vitro. The consequent effects on catecholamine (CA) levels were correlated with follicular steroid production. l-dihdroxyphenylalanine (l-DOPA, the precursor of CA) and human gonadotropin (hCG) were supplemented to reverse the effect of α-MPT. The experiments were conducted in two reproductive phases, namely preparatory and pre-spawning phases in female catfish Heteropneustes fossilis. The incubation with α-MPT inhibited ovarian l-DOPA, dopamine (DA), norepinephrine (NE) and epinephrine (EP) levels and the l-DOPA supplementation compensated the inhibitory effect. The level of tyramine (TR) was increased by the α-MPT treatment but inhibited by the l-DOPA supplementation. α-MPT produced stage-specific (seasonal) effects on ovarian estradiol-17ß (E2); in the preparatory phase, E2 was decreased significantly at both 12 and 24h and in the pre-spawning phase, the level was stimulated over the respective control groups. The changes were higher at 24h in both phases. l-DOPA and hCG increased the E2 level significantly in the preparatory phase and reversed the inhibitory effect of α-MPT in the co-incubation groups. In the pre-spawning phase, α-MPT-stimulated the E2 level compared to the control groups, which was reversed by l-DOPA, hCG, or by both, in co-incubations. In contrast, the α-MPT treatment decreased progesterone (P4), 17-hydroxyprogesterone and 17,20ß-dihydroxy-4-prenen-3-one (17,20ß-DP) in a duration-dependent manner while the co-incubations with l-DOPA, hCG, or by both, significantly reversed the inhibitory effect. These results suggest that ovarian CAs (DA, NE and EP) may exert differential and stage-specific effects on E2, inhibition in the preparative phase and stimulation in the pre-spawning phase. The progestin steroids appear to be stimulated by CAs. In conclusion, this study highlights a possible direct/causal functional interaction between CA activity and gonadotropin on steroidogenic activity, and that CAs may be involved in regulating temporal secretion of the hormones through causing the shift in steroidogenic pattern.

Functional interactions between vasotocin and prostaglandins during final oocyte maturation and ovulation in the catfish Heteropneustes fossilis.

Functional interactions between vasotocin (VT) and prostaglandins (PGs) in the regulation of final oocyte maturation (FOM) and ovulation were investigated in the catfish Heteropneustes fossilis. Incubation of post-vitellogenic follicles with VT resulted in significant increases of both PGF2α and PGE2 at 8 and 16h intervals. The rise was higher at 16h except in the 1000nM VT group, in which the PG levels decreased compared to the 100nM group (biphasic effect). VT was more effective to increase the PG levels in comparison to hCG or IT. The co-incubation of the follicles with both hCG (20IU/ml) and VT (100nM) increased significantly PGF2α level at 8h, higher than that elicited by each when incubated alone. Pre-incubation of the follicles with V1 receptor antagonist, alone or in co-incubation with VT, significantly inhibited the VT-stimulated PGF2α and PGE2 levels. Under similar conditions, V2 receptor antagonist did not affect the PGE2 levels. Both VT (100nM) and PGs stimulated FOM (germinal vesicle breakdown) and ovulation in a dose- and duration dependent manner, PGF2α was more effective. Incubation of postvitellogenic follicles with indomethacin (a non selective cyclooxygenase inhibitor) per se did not affect FOM and ovulation but significantly decreased VT and PG effects upon pre-incubation. The results suggest that the VT stimulation of PGs may be mediated mainly through the V1 receptor though the involvement of V2 receptor cannot be excluded. The article also discussed the positive interplay of gonadotropin, maturation-inducing steroid, VT and PG during FOM and ovulation.

In vitro effects of 2-hydroxyestradiol-17ß on ovarian follicular steroid secretion in the catfish Heteropneustes fossilis and identification of the receptor and signaling mechanisms.

Ovarian pieces containing postvitellogenic follicles were incubated in vitro with different concentrations of the catecholestrogen 2-hydroxyestradiol-17ß (2-OHE(2)) to evaluate its effects on steroid production and germinal vesicle breakdown (GVBD) in the catfish Heteropneustes fossilis. The incubation with 2-OHE(2) induced a shift in steroidogenic pattern: the C(19) and C(18) steroids testosterone (T) and estradiol-17ß (E(2)), respectively were significantly decreased with a concomitant significant increase in the C(21) steroids progesterone (P(4)), 17-hydroxyprogesterone (17-OHP), 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-DP), 17,20α-dihydroxy-4-pregnen-3-one (17,20α-DP) and cortisol (F). Concomitantly, the catecholestrogen induced dose-dependently GVBD response, the first sign of meiosis resumption. The co- and pre-incubations of the ovarian pieces with 2-OHE(2), and adrenergic (phentolamine, α-blocker and propranolol, ß-blocker) or estrogen (tamoxifen) receptor blockers resulted in inhibition of the stimulatory effect of the catecholestrogen on C(21) steroids and reversed the inhibition of testosterone and E(2). The α-blocker was more effective than the ß-blocker. Our results suggest that 2-OHE(2) appears to employ both adrenergic (α-type) and estrogen receptor mechanisms in mediating the effects. The co- or pre-incubation of ovarian pieces with IBMX (a cAMP elevating drug), H89 (a protein kinase A inhibitor), and PD098059 (a MAP kinase kinase inhibitor) significantly inhibited the stimulatory effect of 2-OHE(2) on the C(21) steroids. The effect of chelerythrine (a protein kinase C inhibitor), on the other hand, varied with the incubation condition. In the co-incubation, the steroids showed varied effects: 17,20ß-DP, testosterone and E(2) were elevated, and P(4) and 17-OHP were decreased. In the pre-incubation set up, all the steroids were inhibited except E(2). The inhibition by the blockers was higher in the pre-incubation groups. Taken together, the data suggest the involvement cAMP-protein kinase A, protein kinase C and MAP kinase pathways in the modulation of the steroidogenic activity.