[show abstract][hide abstract] ABSTRACT: In the present study the expression of 13 genes known to be involved in sex differentiation and steroidogenesis in catfish was analyzed during gonadal ontogeny by quantitative real-time RT-PCR. Dmrt1 and sox9a showed exclusive expression in male gonads while ovarian aromatase (cyp19a1) and foxl2 were abundant in differentiating female gonads. Most of the genes related to steroidogenesis were expressed only after gonadal differentiation. However, genes coding for 3β-hydroxysteroid dehydrogenase (3β-hsd), 17α-hydroxylase/C17-20 lyase type 1 (cyp17) and steroidogenic acute regulatory protein (star) were barely detectable during gonadal differentiation. Ovarian aromatase, cyp19a1, which is responsible for estradiol-17β biosynthesis in females, was expressed very early in the undifferentiated gonads of catfish, around 30-40 days post hatch (dph). The steroidogenic enzyme, 11β-hydroxylase (cyp11b1) required for the production of 11-ketotestosterone (11-KT) was expressed only after differentiation of testis. These results suggest that estradiol-17β has a critical role in ovarian differentiation, while the role of 11-KT in testicular differentiation is doubtful. In conclusion, dimorphic expression of dmrt1 and sox9a in gonads during early development is required for testicular differentiation, and sex-specific expression of cyp19a1 and foxl2 in females plays a critical role in ovarian development. Our study reveals that the critical period of gonadal differentiation in catfish starts around 30-40 dph when sex-specific genes showed differential expression.
Sexual Development 06/2011; 5(4):213-23. · 2.22 Impact Factor
[show abstract][hide abstract] ABSTRACT: The growth hormone (GH) gene along with its regulatory sequences has been isolated from the blood and pituitary gland of Labeo rohita. This GH gene is approximately 2.8 kb long and consists of five exons and four introns of varying sizes with AG/TA in its exon-intron junctions. The promoter has a single cyclic AMP response unit (CRE) element, TATA, CAT and several Pit 1 binding sequences. The 1169-bp gene transcript starts 54 bp upstream of the ATG initiation codon and has two polyadenylation signals, ATTAAA, after the TAG stop codon. The mature mRNA has the poly (A) tail inserted 16 bp downstream of the second polyadenylation signal. Four chimeric 'autotransgenes' were constructed having either histone 3 or beta-actin promoter and cDNA or the total GH gene. The functionality of the individual components of the autotransgene was determined in the Chinese hamster ovary (CHO) cells by transfection experiments. Based on the results, the transcription of the GH gene is initiated at the transcription start signal of the respective promoters and terminates at the 3' regulatory sequence of the GH gene. Expression of GH in CHO cells shows that the fish promoters are active, the splicing signal is recognized, and the mRNA produced is stable and translated. The GH protein produced is effectively translocated and secreted into the medium. These results indicate the usefulness of CHO cells in determining the property of individual components of autotransgenes constructed from L. rohita and overall functional commonality between fish and mammal.
Fish Physiology and Biochemistry 01/2009; 34(4):413-36. · 1.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: DNA fingerprints were obtained in three species of commercially important freshwater fishes, Labeo rohita (Hamilton). Catla catla (Hamilton) and Oreachromis mossambicus (Peters), using Bkm 2(8) and M13 multilocus probes. Bkm 2(8) gave a higher number of bands when compared with M13. However, the number of bands obtained by each probe in O. mossambicus was similar. The higher band-sharing coefficient observed in this species may be attributed to inbreeding as it arose from a small founder population. In L rohita and C. catla, the Bkm 2(8) detected similar DNA fingerprints when two enzymes Hinfi and Taqi were used. The M13 probe also gave similar fingerprints with three restriction enzymes (Hinfi, Taqi, Alui). Comparison of the DNA fingerprints obtained by Bkm 2(8) and Ml 3 showed that these two probes detected different alleles. The overall similarity of the DNA fingerprint patterns in L. rohita and C. catla may be due to their genetic closeness as indicated by their same chromosome number, C-value and their ability to produce fertile hybrids. A similar argument also holds true for the Oreochromis species where interspecies hybridization results in fertile offspring.
Aquaculture Research 06/2008; 28(2):129 - 138. · 1.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: The growth hormone (GH) gene isolated and cloned from various Labeo species (L. rohita, L. calbasu, L. fimbriatus, L. gonius, L. bata, and L. kontius) is shown to contain a single copy in the haploid genome, with an overall size of ∼2.5 kb. The GH gene in all the Labeo species studied has five exons and four introns of various sizes with the exon/intron boundary sequence of GT/AG. The length
variation of the GH gene between the species is found to be due to length variation in the form of several deletions in the
third intron. The length of individual exons is the same in all the species with an open reading frame (ORF) of 630 bp (210
amino acids) except in L. rohita, which has a 9 bp deletion in the fourth exon, resulting in a shorter GH of 621 bp (207 amino acids). The similarity in the
nucleotide and amino acid sequences between the different Labeo species is greater than 97%, in spite of eight amino acids being altered in the GH protein of Labeo that reside outside the conserved domain sequence required for its function. Nucleotide substitutions are seen in the form
of 20 transitions and three transversions in the ORF of the GH gene. Both types of transitions (A–G; T–C) and only one type
of transversion (A–C) are detected in the GH gene. Codon preference in GH gene shows a strong preference for G and C in the
wobble position of the codons. Genetic interrelationships determined between Labeo and other species of fishes using nucleotide sequence of GH cDNA supports the overall teleost classification of Nelson (Fishes
of the World. Wiley, New York, 1984) with separate clades for Ostariophysi, Protacanthopterygii, and Acanthopterygii. Besides, the unweighted pair group method
with arithmetic means (UPGMA) analysis clearly distinguishes between the species having five exons and four introns in the
GH gene from the species having six exons and five introns in the same gene. The Labeo species analyzed in the present study could be clustered into two groups using the maximum-parsimony method on the intron
sequences data of the GH gene.
Fish Physiology and Biochemistry 01/2007; 33(4):311-333. · 1.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: We used thiourea-induced thyroid hormone depletion as a strategy to understand the influence of thyroid hormones on testicular recrudescence of the air-breathing catfish, Clarias gariepinus. Treatment with 0.03% thiourea via immersion for 21 days induced hypothyroidism (thyroid hormone depletion) as evidenced by significantly reduced serum T(3) levels. Thiourea-treated males had narrowed seminiferous lobules with fewer spermatozoa in testis, very little or no secretory fluid, reduced protein and sialic acid levels in seminal vesicles when compared to controls. The histological changes were accompanied by reduction in serum and tissue levels of testosterone (T) and 11-ketotestosterone (11-KT), a potent male specific androgen in fish. Qualitative changes in the localization of catfish gonadotropin-releasing hormone (cfGnRH) and luteinizing hormone (LH, heterologous system) revealed a reduction in the distribution of immunoreactive neuronal cells and fibers in thyroid depleted fish. Interestingly, thiourea-withdrawal group showed physiological and histological signs of recovery after 21 days such as reappearance of spermatozoa and partial restoration of 11-KT and T levels. These data demonstrate that thyroid hormones play a significant role in testicular function of catfish. The mechanism of action includes modulating sex steroids either directly or through the hypothalamo (GnRH)-hypophyseal (LH) axis.
Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology 06/2006; 144(1):1-10. · 2.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: A computer-aided semen analysis system was used to assess the % motile cells following storage of carp semen in 11 different buffers at 2, 5 or 22° C. BWW and TLP were the most suitable storage buffers because carp semen stored at 5° C in these buffers following activation showed no significant decrease in % motile spermatozoa up to 24 h. But, in most of the other buffers (Fish Ringer, Cytomix, Cortland, FRT, Mannitol, FPS, NAS and TSM) the motility potential was lost by 2 h. Storage was best at pH 6–9 and at 5° C. Carp spermatozoa exhibit three distinct motility patterns, namely ‘linear’, ‘circular’ and ‘haphazard’, the proportion of spermatozoa with a particular motility pattern depending on storage buffer and time. All spermatozoa with a linear trajectory had high VSL, STR and LIN; those moving in circles had low VSL, STR, LIN and BCF and those with a haphazard trajectory were distinct in that they had the highest ALH and their VSL, STR, LIN and BCF were higher than the circular moving spermatozoa and lower than the spermatozoa exhibiting linear trajectory. The study also demonstrates a pronounced time-dependent decrease in VCL, VAP, VSL and ALH of carp spermatozoa following activation with water or low osmolality solutions. This study provides for the first time data related to seven motility parameters of carp spermatozoa and demonstrates how these parameter values could be used to evaluate quality of carp milt following storage in different buffers. It confirms that carp spermatozoa exhibit linear or circular trajectories and provides evidence for a third type of trajectory described as haphazard. All three motility patterns could be discriminated objectively on the seven motility parameters.
Journal of Fish Biology 04/2005; 50(6):1309 - 1328. · 1.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Thyroid hormone is known to have profound effect on the efficient functioning of the reproductive system. The GnRH-gondaotropin (GTH) axis is the crucial axis involved in regulation of the various aspects of reproduction like spermatogenesis, synthesis of sex steroids and regulation of courting and spawning behavior. Earlier study from our laboratory has shown that thyroid hormone depletion causes a decrease in GTH and GnRH levels in pituitary and preoptic area, respectively. GnRH secretion in pre-optic area is largely regulated by the monoaminergic system mainly dopamine (DA), epinephrine (E), norepinephrine (NE) and serotonin (5-HT). The expression pattern of catfish GnRH transcripts tends to corroborate our earlier findings. Hence, in the current study we aimed to investigate the levels of monoamines in the preoptic area-hypothalamus (POA-H), so as to determine whether thyroid hormone depletion modulates the monoaminergic neurotransmission thereby affecting GnRH secretion. The levels of NE and L-dopa decreased significantly while that of DA was unaltered following thyroid hormone depletion. DA has an inhibitory effect on GnRH secretion. Findings from the present study suggest that the inhibitory tone is unaltered while the stimulatory tone influencing GnRH neurons in POA-H is decreased during thiourea induced thyroid hormone depletion.
Fish Physiology and Biochemistry 04/2005; 31(2-3):189-92. · 1.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the present study, thiourea-induced thyroid hormone depletion and thyroxine (T(4)) 'overdose' were used as a strategy to understand the influence of thyroid hormones on ovarian recrudescence of juvenile (3-months-old), immature (8-months-old) and adult (1-year-old) air-breathing catfish, Clarias gariepinus. Thiourea-induced thyroid hormone depletion in juvenile catfish impaired ovarian development, but no significant effect was observed in immature catfish and during late stage of ovarian recrudescence of mature catfish. T(4) treatment in females undergoing late stages of ovarian recrudescence induced rapid oocyte growth by promoting its early entry into maturational phase as evident from the presence of more number of vitellogenic and post-vitellogenic follicles, decrease in aromatse immunoreactivity and reduced estradiol-17beta levels. Hence, thyroid hormones have an important role to play during early stages of ovarian development and vitellogenesis of catfish and also indicating that thyroid has a stage dependent effect on ovary.
Fish Physiology and Biochemistry 04/2005; 31(2-3):267-70. · 1.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Transgenic Labeo rohita founder population was analyzed for the presence of autotransgene having histone 3 promoter and growth hormone (GH) cDNA (LRH3-GHcDNA) or total GH gene (LRH3-GH2.8) by PCR with transgene specific primers. Transgene specific amplification was seen with LRH3-GHcDNA in five out of seven individuals and all three fishes with LRH3-GH2.8, indicating their transgenic nature. Transgene integration was also studied by Southern hybridization of DNA isolated from blood of the transgenic fishes with two different probes (histone 3 promoter and cDNA of L. rohita). Autotransgene integration was confirmed in all PCR positive transgenic individuals. The site of integration of the transgene in the genome of the four transgenic fish could be determined by inverse PCR. Two individuals showed integration at the same site whereas in the remaining two individuals the integration sites were different.
Fish Physiology and Biochemistry 04/2005; 31(2-3):281-7. · 1.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Changes in the phosphorus-containing metabolites were monitored by 31P nuclear magnetic resonance in the developing embryos of Clarias batrachus. Phosphomonoester, yolk phosphoprotein, phosphocreatine, ATP, and inorganic phosphate (Pi) were consistently observed in all
the developmental stages of C. batrachus. None of these phosphometabolites exhibited any significant change in their concentration up to the blastula stage, whereas
distinct decrease in all except inorganic phosphate was observed in the fry stage. Concomitantly an increase in the concentration
of inorganic phosphate was observed. Further, from the resonance positions of α, β, and γ phosphate groups of ATP, it was
evident that the ATP molecules in vivo were liganded either to Ca2+ or Mg2+. This study also revealed that the intracellular pH of the developing embryos was approximately 7.05 up to the gastrula stage,
after which it decreased in the fry stage to 6.98 units.