Prespawning Phase Research Articles

Triclosan, an antimicrobial drug, induced reproductive impairment in the freshwater fish, Anabas testudineus (Bloch, 1792).

Triclosan (TCS), an antimicrobial drug, is known to occupy different compartments in aquatic ecosystems. The present study focused to evaluate the reproductive toxicity of triclosan, at environmentally relevant (0.009 and 9μg L-1) and sublethal (176.7μg L-1) concentrations for 90days in the pre-spawning phase of the fish, Anabas testudineus. The reproductive biomarkers, namely, gonadal steroidogenic enzymes, expression of aromatic genes, levels of serum gonadotropins, sex hormones, and histology of gonads were analyzed. The weight of the animal, brain weights along with gonadosomatic index decreased while mucus deposition increased significantly at all concentrations of triclosan as the primary defensive mechanism to prevent the entry of toxicants. Triclosan disrupted gonadal steroidogenesis as evidenced by a reduction in the activities of gonadal steroidogenic enzymes. The expressions of cyp19a1a and cyp19a1b genes were up-regulated in the brain of both sexes and testis, while down-regulated in the ovary indicating estrogenic effects of the compound. The endocrine-disrupting effects of triclosan were confirmed. The current results suggest that chronic exposure to triclosan altered reproductive endpoints thereby impairing normal reproductive functions in fish.

Structural modeling and gene expression analysis of phosvitinless vitellogenin (vgc) in the Indian freshwater murrel, Channa punctatus (Bloch, 1793)

Vitellogenin (Vg) is a female-specific egg-yolk precursor protein, synthesized in the liver of fish in response to estrogens. In the present study, complete gene of phosvitinless vitellogenin (vgc) was sequenced, its 3D structure was predicted and validated by web-based softwares. The complete nucleotide sequence of vgc was 4126 bp which encodes for 1272 amino acids and showed the presence of three conserved domains viz. LPD_N, DUF1943 and DUF1944. The retrieved amino acid sequence of VgC protein was subjected to in silico analysis for understanding the structural and functional properties of protein. mRNA levels of multiple vg genes have also been quantified during annual reproductive cycle employing qPCR. A correlation has been observed between seasonal changes in gonadosomatic index with estradiol levels and hepatic expression of three types of vg genes (vga, vgb, vgc) during ovarian cycle of murrel. During preparatory phase, when photoperiod and temperature are low; low titre of E2 in blood induces expression of vgc gene. A rapid increase in the levels of E2 favours induction of vgb and vga genes in liver of murrel during early pre-spawning phase when photoperiod is long and temperature is high in nature. These results suggest that among three vitellogenin proteins, VgC is synthesized earlier than VgA and VgB during oogenesis.

Endemic fish calling: Acoustics and reproductive behaviour of the Neretva dwarf goby Orsinigobius croaticus.

The Neretva dwarf goby Orsinigobius croaticus (Gobiiformes, Gobionellidae) is an endemic fish native to the freshwaters of the Adriatic Basin in Croatia and Bosnia and Herzegovina, a Mediterranean Biodiversity Hotspot. Due to its limited distribution range, specific karst habitat and endangered status, laboratory studies on reproductive biology are scarce but crucial. Herein, we investigated the sound production and acoustic behaviour of the endangered O. croaticus during reproductive intersexual laboratory encounters, utilising an interdisciplinary approach. We also performed dissections and micro-computed tomography (μCT) scanning of the pectoral girdle to explore its potential involvement in sound production. Finally, comparative acoustic analysis was conducted on sounds produced by previously recorded soniferous sand gobies to investigate whether acoustic features are species-specific. The endemic O. croaticus is a soniferous species. Males of this species emit pulsatile sounds composed of a variable number of short (~15 ms) consecutive pulses when interacting with females, usually during the pre-spawning phase in the nest, but also during courtship outside the nest. Pulsatile sounds were low-frequency and short pulse trains (~140 Hz, <1000 ms). Male visual behaviour rate was higher when co-occurring with sounds and females entered the male's nest significantly more frequently when sounds were present. Characteristic body movements accompanied male sound production, such as head thrust and fin spreading. Furthermore, μCT scans and dissections suggest that O. croaticus shares certain anatomical similarities of the pectoral girdle (i.e. osseous elements and arrangement of levator pectoralis muscles) to previously studied sand gobies that could be involved in sound production. Multivariate comparisons, using sounds produced by eight soniferous European sand gobies, effectively distinguished soniferous (and sympatric) species based on their acoustic properties. However, the discrimination success decreased when temperature-dependent features (sound duration and pulse repetition rate) were excluded from the analysis. Therefore, we suggest both spectral and temporal features are important for the acoustic differentiation of sand gobies.

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 (LHβ, FSHβ, 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.

Variation in immuno-reproductive milieu of testis in Clarias magur from pre-spawning to spawning phase: An indication towards non-canonical role of immune elements in testes

Immune mechanisms are major players in ensuring the normal functioning of testicular functions. However, apart from their role in active defence against pathogens, prior studies have also suggested a possibility for reproduction-related (non-immune) functions of certain immune elements. This study employs a comparative transcriptomics approach followed by network analysis for tracking the variation in the immuno-reproductive milieu of Clarias magur testis in spawning versus pre-spawning phase. The results show a significant modulation of both reproduction and immune-relevant genes in spawning versus pre-spawning phase. The functional enrichment of the upregulated reproduction-relevant gene network also shows immune-related biological processes which indicates a probability of involvement of these candidates in spermatogenesis-related events for switching from pre-spawning to spawning phase. The upregulated immune network is highly dense with 40 hubs, 10 cluster sub-networks and 142 functionally enriched pathways in comparison to its downregulated counterpart with only 5 hubs, 1 cluster and 1 enriched pathway. These findings indicate that the synchronisation in modulation of both reproductive and immune-related factors is critical for progression of testicular events guiding the switch from pre-spawning to spawning phase. The reproductive phase-dependent variation in plasma sex steroid levels and the selected genes for quantitative PCR also corroborated this hypothesis. The study also serves as a preliminary screening step for probable immune candidates that may be involved in reproductive functions of testis in addition to defence.

Sex steroids in diploid and triploid gibel carp (Carassius gibelio) of both sexes in different phases of the reproductive cycle

The changes in concentrations of 17ß-estradiol (E2), testosterone (T), 11-ketotestosterone (11-KT) in the gonads and plasma of diploid and triploid gibel carp (Carassius gibelio) from the Siemianowka Reservoir, the Vistula River drainage were quantified and compared using an enzyme immunoassay. The phase of gonad maturity was based on histological analysis and the gonadosomatic index. All fish had properly developed gonads in the pre-spawning, spawning and late spawning phases of the reproductive cycle in the fish collected in April, June and October, respectively. Diploid and triploid females and males did not differ in mean GSI in all reproductive phases. In October, ovaries of most females contained vitellogenic oocytes, testes had large numbers of spermatozoa. The hormone concentrations in both tissues depended mainly on the reproductive phase, but not on ploidy. The patterns of changes in E2 concentrations was similar in females and males. In the pre-spawning phase, diploid and triploid females showed differences in the concentrations of 11-KT in gonads and plasma, and T in plasma. Diploid males differed in pattern of T concentrations in gonads and plasma, while all males showed a similar pattern of 11-KT plasma concentrations. Spermatozoa observed in triploid males suggest that they participate in reproduction. Gibel carps, regardless of ploidy, had an extended period of reproduction, which makes this invasive species a potentially greater threat to native ichthyofauna. The sex androgen concentrations that differed between 2 n and 3 n females could be physiological factors potentially contributing to the coexistence of gynogenetic C. gibelio females and sexual diploids.

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 1h and 2h 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 1h in both phases but unchanged at 2h. In the air deprivation experiment (forced aquatic respiration), the v2a expression in the air sac was inhibited except for a mild stimulation at 1h in the prespawning phase. In the gill, the v2a expression was stimulated with a steep upregulation at 2h 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.

Carotenoid profiling of Mytilus coruscus with unialgal cultures: Insights into biosynthetic pathways in tissues

The carotenoid profiling of Mytilus coruscus generally depends on the dietary microalgae, and the ingested carotenoids are modified and processed depending on the properties of different tissues. Although much research effort has focused on analyzing carotenoid profiling between microalgae and shellfish, the detailed metabolic pathways of dietary carotenoids in shellfish are poorly understood. In the present study, we carried out feeding experiments with different unialgal cultures (Chaetoceros mulleri and Dicrateria inornata) during the growth stages and examined the carotenoid profiles between the dietary microalgae and the corresponding tissues in order to study the resorption and metabolic transformations of dietary carotenoids in M. coruscus. The results showed that 1) carotenoids mainly occurred in the pre-spawning phase of M. coruscus; the contents of most carotenoids showed the same trends as in dietary microalgae. 2) An association analysis of the total content of carotenoids and relevant gene expression demonstrated that the main tissue for carotenoid synthesis in M. coruscus was the hepatopancreas. The synthetic reactions of the allenic to acetylenic carotenoids pathway and the 4-keto oxidative pathway in M. coruscus coexisted. 3) Quantitative analysis revealed that the carotenoids mytiloxanthin, alloxanthin, and pectenolone containing acetylene groups were selectively assimilated in gonad (69.3–74.1%), mantle (83.2–102%), and muscle (86.9–100%), forming a large percentage of the carotenoids present in those tissues. These results provide new insights into the biosynthetic and metabolic transformation pathways of carotenoids in M. coruscus.

Activin receptor type IIB in rohu (Labeo rohita): molecular characterization, tissue distribution and immunohistochemical localization during different stages of gonadal maturation.

Activin receptor type IIB (ActRIIB) is a transmembrane serine/threonine kinase receptor which plays a pivotal role in regulating the reproduction in vertebrates including teleost. Earlier studies have documented its importance in governing gonadal maturation in higher vertebrates. However, reports on the regulation of fish reproductive system by ActRIIB gene are still limited. Here, we report the identification and characterization of ActRIIB cDNA of Labeo rohita, a commercially important fish species of the Indian subcontinent. The full-length gene encoding rohu ActRIIB was cloned and found to be of 1674bp in length. Functional similarities were evident from evolutionary analysis across vertebrates. Real-time PCR to measure the expression of ActRIIB transcript in rohu revealed significant mRNA levels in gonads followed by non-reproductive tissues, including the brain, pituitary and muscle. With respect to different gonadal maturation stages, predominant expression of ActRIIB mRNA was observed during the pre-spawning phase of both sexes. To further delineate its role in rohu reproduction, a recombinant protein of the extracellular domain of ActRIIB (rECD-ActRIIB) was produced, and polyclonal antibody is raised against the protein for its immuno-localization studies during different gonadal maturation stages. Strong immunoreactivity was noticed in the pre-vitellogenic oocytes which decreased dramatically in the fully mature oocytes. Similarly, the strong and intense immunoreactivity was found in the spermatids and spermatocytes of the immature testis, and eventually the intensity reduced with the progression of the maturation stage. These results provide the first evidence of the presence of ActRIIB in rohu gonadal tissues. Taken together, our observations lay the groundwork for further understanding and investigating on the potential role of ActRIIB in fish reproduction system in the event of gonadal maturation.

Age, Growth and Ovarian Histology of Sardinella aurita (Valenciennes, 1847) in the South of Atlantic Moroccan Coast

Otoliths reading and length frequency distribution were used for age determination and growth estimation of Sardinella aurita (round sardinella) stock of Southern Atlantic coast of Morocco. Both otoliths' method for age determination and Bhattacharya’s method for length frequency analysis showed five age groups. The growth performance index revealed that there is significant accordance among method of length frequency distribution and otoliths reading for stock assessment studies of Sardinella aurita stock in the south of Morocco. The microscopic observation of female gonads using histology method was investigated for the first time in our study area and confirms the presence of five principal stages of ovary of sardinella aurita: immature, maturing virgin and recovering spent, mature (or pre-spawning phase), spawning, post-spawning or spent. In addition, our results of the fecundity showed that the mean relative fecundity obtained is estimated at 193 ± 98 oocytes/g which is lower compared to those obtained in other areas in the Atlantic coast.

Consequences of sodium dodecyl sulfate exposure on the antioxidant status and steroidogenesis in fish gonad

A native Indian catfish (Heteropneustes fossilis) and an Indian major carp (Cirrhinus cirrhosus) were treated with sodium dodecyl sulfate (SDS) to elucidate its consequences on antioxidant status and sex steroid synthesis. Fishes were exposed to 1/10th of LC50 of SDS for 30 days. Different enzymatic and non-enzymatic antioxidants, major sex steroids, steroidogenic enzymes, gonadosomatic index (GSI), and serum cortisol level were measured in both male and female fishes. Levels of enzymatic and non-enzymatic antioxidants altered significantly in both the fish species. Steroid hormones in both male and female fishes decreased significantly in the SDS-treated group. Profiles of steroidogenic enzymes (hydroxysteroid dehydrogenase; 3β-HSD and 17β-HSD) show significant decrease in the SDS treatment group in both carp and catfish than that of the control group irrespective of sexes. GSI also decreases significantly in the SDS-treated group when compared with the control group in spawning phase of carp and catfish irrespective of sexes. Serum cortisol level was increased significantly after SDS exposure. Higher level of cortisol was noted in the spawning phase than that of pre-spawning phase. Current study proves that SDS could interrupt the normal steroidogenesis process in fish, leading to impaired sex hormone production. Detrimental effect of SDS was noted to be more prominent on female steroidogenesis than that of male fish. A species or season-specific variation in steroidogenesis was also observed which indicates that SDS-induced toxicity depends on the sexual status of the specific fish species.

δ

The European eel, a catadromous endangered species, displays plasticity in phenotypic traits and in trophic behaviour, features that can eventually affect growth. The aim of this study was to compare variation in carbon (δ13C) and nitrogen (δ15N) stable isotopes in muscle tissue of the adult eel life stages (i.e. yellow and silver eel), as function of growth. Eel samples were obtained from nine sites across the Mediterranean region, that included seven coastal lagoons of different size and ecological structure, and two freshwater habitats corresponding to one river and one lake. A large variability was found in both δ13C and δ15N values, suggesting that eels fed over a broad range of potential food sources. Eels from each location showed specific range values of δ13C, in most sites irrespective of stage, reflecting lagoon- or freshwater-specific carbon sources. A positive correlation was found between individual δ15N values and annual growth rates, suggesting that eels generally seem to feed on prey of higher δ15N values among the trophic resources available in each foraging habitat. This allows individuals to grow faster and to attain earlier the pre-spawning phase (i.e. the silver stage). A better understanding of eel trophic ecology across different life stages and different habitats is one of the prerequisites for the effective management of local stocks.

Advancement in spawning period of Labeo dyocheilus (McClelland, 1839) in the mid Himalayan regions by hormonal manipulation using Ovatide

An attempt was made to advance the spawning period of Labeo dyocheilus (McClelland, 1839) by pretreatment with the synthetic gonadotropin releasing hormone analogue, Ovatide. Total 12 females and 24 males (1: 2 ratio) were used for breeding operations conducted during 30th May to 30th June 2016 comprising six sets. Female fishes of set I to IV were pretreated with intramuscular injection of the ovatide at the rate 0.3 ml kg-1 body weight twice at an interval of two weeks while males were injected @ 0.1 ml kg-1 body weight once before 15 days of induced breeding trial for achieving advance maturity and spawning during pre-spawning phase. Fishes of set V and VI were not subjected to any pretreatment with ovatide. On 30th of June all the six breeding sets were were treated with ovatide for induced spawning. Female and males were injected at 0.7 ml kg-1 and 0.3 ml kg-1 of body weight, respectively and left overnight in FRP tanks for spawning. Results indicated that in sets I to IV, fishes successfully spawned and fertilised eggs were collected in morning hours whereas fishes of set V and VI failed to spawn. Fertilisation and survival rates recorded were 79-82% and 67-71%, respectively. Results indicated that treatment of ovatide during prespawning phase is beneficial for advancing the gonadal maturity, successful induced spawning and prolonging the breeding period which may play a key role in production of substantial quantity of seed of L. dyocheilus.

Controlled release of sex steroids through osmotic pump alters brain GnRH1 and catecholaminergic system dimorphically in the catfish, Clarias gariepinus

The present study aimed to evaluate osmotic pump-mediated controlled release of estrogen in males and androgen in females to analyze the impact on gonadotropin-releasing hormone (GnRH1), catecholamines (CAs) and other associated genes in the catfish, Clarias gariepinus. During pre-spawning phase, catfish were separately implanted osmotic pumps loaded with 17β-estradiol (E2) in males and 17α-methyltestosterone (MT) in females at a dose of 10 μg/100 μl or saline (100 μl) controls into both sexes to release for 21 days and all fishes were maintained as per the duration. Further, GnRH1 expression levels were analysed in the discrete regions of brain after E2 and MT treatments in male and female catfish, respectively using qPCR which revealed that GnRH1 expression was significantly higher in E2 treated male as compared to the control. On the other hand, GnRH1 expression was lower in MT treated female when compared to the control in the discrete regions of brain. In addition, certain brain and monoaminergic system related genes showed a differential response. Catfish GnRH1 could be localized in preoptic area-hypothalamus (POA-HYP) that correlated with the expression profile in the discrete regions of catfish brain. Serum levels of sex steroids in the treated male fish indicated that the treatment of E2 could maintain and impart feminization effect even in the presence of endogenous androgen during gonadal recrudescence while such an effect was not seen in females with androgen treatment. Measurement of CAs, L-3,4-dihydroxyphenylalanine, dopamine and norepinephrine levels in the male and female brain after the controlled release of E2 and MT, respectively confirmed the modulation of neurotransmitters in the E2treated male than MT treated female fish. These results collectively suggest the severity of estrogenic over androgenic compounds to alter reproductive status even at a minimal dose by targeting CAs and GnRH1 at the level of brain of catfish. This study provides insights into the reproductive toxicity of sex steroid analogues at the level of brain GnRH1 and CA-ergic system in addition to serum T, 11-KT and E2 levels during gonadal recrudescence, which is a crucial period of gametogenesis preceding spawning.

Effect of short-term intermittent exposure to waterborne estradiol on the reproductive physiology of the round goby (Neogobius melanostomus)

The objective of this study was to determine how the short-term exposure to a supraphysiological concentration of waterborne 17β-estradiol (E2) influences on melatonin (Mel) and thyroxine (T4) concentrations in plasma and E2 and 11-ketotestosterone (11-KT) concentrations in plasma and gonads in both sexes of round goby (Neogobius melanostomus) during the pre-spawning, spawning, late spawning and non-spawning phases. The experimental protocol was based on short-term, repeated exposures of fish to a supraphysiological dose of waterborne E2. Mel level was unchanged on exposure to E2 during the investigated phases, and its role in determining a time frame for spawning in both sexes of round goby seems to be stable in those conditions. T4 and sex steroids (E2 and 11-KT) were sensitive to the exposure of E2, and those changes influence gonads by accelerating oocyte development, ovulation and regression and inhibiting spermatogenesis in this species. The results demonstrate that the physiological responses of fish in all investigated phases were altered over a short window of exposure, indicating that short-term exposure to a supraphysiological dose of E2 may impact fish in the wild. Furthermore, round goby can be recommended as a very suitable model for studying endocrine disruptors, which is sensitive to even short exposure to E2.

Identification of aquaporin-1a gene transcript in Cyprinus carpio (Linnaeus, 1758) and its expression during reproduction

Aquaporins (AQPs) are a family of membranebound proteins that facilitate the hydration of cells. The identification of the AQP1a gene in common carp (Cyprinus carpio) from mRNA transcript and its expression analysis during reproduction was performed to study its role in gonad cell hydration. The AQP1a gene transcript was partially amplified using gene-specific primers and the amino acid sequence was predicted. Based on the partial transcript sequence, a phylogenetic tree was prepared using the MEGA4 software which suggested that the AQP1a gene of common carp is closely related to that of Danio rerio. Differential tissue expression was analysed using real-time PCR which indicated that the expression of AQP1a was significantly higher in the eye, kidney, and liver with 32.98±1.33 folds higher in the eye when compared to the testis. In both the male and female gonads, the highest expressions were observed after 6h of synthetic hormone injection in comparison to gonads belonging to the individual without any injection. However, the gene expression declines gradually in 12h and 24h post-injection. Expression analysis of gonad during different phases of reproduction suggests that there is a significant increment of AQP1a expression from the resting phase to the pre-spawning phase and from pre-spawning to spawning phase in the case of male gonad. However, there was no significant difference in expression during the resting and pre-spawning phase in the female gonad, although expression was significantly higher during the spawning phase. Key words Aquaporin; Gonad; Reproduction: Common carp; gene expression

Methionine-enkephalin-treatment suppresses the pituitary-ovary axis in the cichlid fish Oreochromis mossambicus

The involvement of endogenous opioid peptides in regulation of reproductive activity is implicated in a majority of vertebrates. Methionine-enkephalin (M-ENK) is an endogenous opioid peptide neurotransmitter produced in the central nervous system. Although a role for M-ENK in antinociception (pain suppression) and mediation of stress has been suggested, its influence on fish reproduction is not clearly understood. The aim of the present investigation was to determine the role of M-ENK on the reproductive-axis of the female tilapia Oreochromis mossambicus. Treatment of stripped O. mossambicus with 300 μg M-ENK during the ovarian cycle resulted in suppression of luteinizing hormone (LH) secretory activity. This was evidenced by a significant reduction in the area and the intensity of LH immunoreactivity in the proximal pars distalis of the pituitary gland as compared to controls during the prespawning phase. At the ovarian level, the treatment of M-ENK caused a significant increase in the mean number of follicles with chromatin nucleolar (stage I), perinucleolar (stage II) and cortical alveolar stages (Stage III). Although the mean number of early vitellogenic follicles (stage IV) did not significantly differ from that of initial controls and controls, the mean number of late vitellogenic follicles (stage V, preovulatory) and the serum levels of estradiol were significantly lower in M-ENK treated fish compared to those of controls. However, there was a significant increase in the serum levels of cortisol in M-ENK treated fish compared to controls. Together, these results suggest that treatment of M-ENK inhibits the recruitment of follicles at late vitellogenic stage through the suppression of steroidogenesis in the ovary and LH secretory activity at the level of the pituitary gland in fish. Besides, the ability of M-ENK to stimulate serum cortisol levels indicates a role for this peptide in regulation of the hypothalamo-pituitary-interrenal axis in fish.

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.

Thyrotropin-releasing hormone (TRH) in the brain and pituitary of the teleost, Clarias batrachus and its role in regulation of hypophysiotropic dopamine neurons.

Thyrotropin-releasing hormone (TRH) regulates the hypothalamic-pituitary-thyroid axis in mammals and also regulates prolactin secretion, directly or indirectly via tuberoinfundibular dopamine neurons. Although TRH is abundantly expressed in teleost brain and believed to mediate neuronal communication, empirical evidence is lacking. We analyzed pro-TRH-mRNA expression, mapped TRH-immunoreactive elements in the brain and pituitary, and explored its role in regulation of hypophysiotropic dopamine (DA) neurons in the catfish, Clarias batrachus. Partial pro-TRH transcript from C. batrachus transcriptome showed six TRH progenitors repeats. Quantitative real-time polymerase chain reaction (qRT-PCR) identified pro-TRH transcript in a number of different brain regions and immunofluorescence showed TRH-immunoreactive cells/fibers in the olfactory bulb, telencephalon, preoptic area (POA), hypothalamus, midbrain, hindbrain, and spinal cord. In the pituitary, TRH-immunoreactive fibers were seen in the neurohypophysis, proximal pars distalis, and pars intermedia but not rostral pars distalis. In POA, distinct TRH-immunoreactive cells/fibers were seen in nucleus preopticus periventricularis anterior (NPPa) that demonstrated a significant increase in TRH-immunoreactivity when collected during preparatory and prespawning phases, reaching a peak in the spawning phase. Although tyrosine hydroxylase (TH)-immunoreactive neurons in NPPa are hypophysiotropic, none of the TRH-immunoreactive neurons in NPPa accumulated neuronal tracer DiI following implants into the pituitary. However, 87 ± 1.6% NPPa TH-immunoreactive neurons were surrounded by TRH-immunoreactive axons that were seen in close proximity to the somata. Superfused POA slices treated with TRH (0.5-2 μM) significantly reduced TH concentration in tissue homogenates and the percent TH-immunoreactive area in the NPPa. We suggest that TRH in the brain of C. batrachus regulates a range of physiological functions but in particular, serves as a potential regulator of hypophysiotropic DA neurons and reproduction.

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-17β (E2), the maturation-inducing steroid (MIS) 17α, 20β-dihydroxy-4-pregnen-3-one (17, 20β-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, 20β-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.