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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. · 2.27 Impact Factor
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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. · 1.53 Impact Factor
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I Swapna,
M Rajasekhar,
A Supriya,
K Raghuveer,
G Sreenivasulu,
M K Rasheeda, K C Majumdar,
H Kagawa,
H Tanaka,
A Dutta-Gupta,
B Senthilkumaran
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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. · 2.23 Impact Factor
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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. · 1.53 Impact Factor
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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. · 1.53 Impact Factor
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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. · 1.53 Impact Factor
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ABSTRACT: A simple assay for identifying DNA-binding proteins is described that involves loading of protein fractions onto nitrocellulose membrane using a slot-blot apparatus, incubating with 32P-labeled DNA probe in buffer in the presence of excess of nonspecific E. coli DNA at room temperature, and washing with increasing concentration of NaCl (from 50 to 500 mM) to obtain optimum signal. A simple and rapid scheme of purification of a sex and tissue-specific DNA-binding protein, which binds specifically to the GATA repeats of Bkm (banded krait minor satellite DNA), designated as Bkm-binding protein (BBP), is also described. This requires only a DNA affinity column after the initial ammonium sulfate precipitation. The insert (545 bp) of the Drosophila clone 2(8) containing 66 copies of GATA repeats was used to prepare the sequence-specific DNA-Sepharose affinity column. The slot-blot-binding assay and the simple scheme of purification described here may be used for routine screening and purification of sequence-specific DNA-binding proteins in general.
Analytical Biochemistry 11/1996; 241(1):23-9. · 3.00 Impact Factor
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ABSTRACT: Genetic fingerprint profiles have been successfully used for establishing biological relationships, in linkage analysis, and in studies of population structure but have not so far been used for ascertaining phylogenetic relationships among related groups of species and genera. This is largely because these profiles are thought to evolve too rapidly to be informative over large time intervals. However, we show here that among the Crocodilia, whose phylogeny is a debated issue, these profiles can provide phylogenetically useful information. By using the probe Bkm-2(8), DNA fingerprints with distinct bands distributed in the size range 0.5-23.0 kb were obtained for individuals of 18 species belonging to seven of the eight genera of crocodilians. These genetic profiles showed individual-, species-, and restriction enzyme-specific patterns. In addition, striking differences were observed in the copy number of Bkm-related sequences in genomes of different crocodilian species. The qualitative data from DNA fingerprint profiles, and quantitative data on copy number variation in Bkm-related sequences, suggest that these genera belong to two distinct groups, one of which includes Alligator, Paleosuchus, and Caiman; the other includes Crocodylus, Osteolaemus, Tomistoma, and Gavialis. A close relationship between Tomistoma and Gavialis is also suggested by these results.
Proceedings of the National Academy of Sciences 11/1994; 91(22):10601-5. · 9.68 Impact Factor
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ABSTRACT: The mouse Y chromosome remains highly condensed in all somatic tissues but decondenses extensively in testis. We have isolated a mouse Y chromosome-specific repeat M34 (11.5 kb) and shown that this is distributed along the Y chromosome except the sex-determining region (the Y short arm) in which GATA repeats are predominantly concentrated. It has 32 copies of GATA repeats in a 2.7 kb fragment. About 200-300 copies of M34 on the Y chromosome are interspersed among other sequences. A 1.2 kb fragment (p3) of M34, containing GATA repeats, also has scaffold attachment region (SAR) motifs which bind to nuclear matrices. A strong affinity of histone H1 to SAR motifs is implicated in maintaining the condensed state of the Y chromosome in somatic tissues. The probable significance of molecular organization of the Y chromosome is discussed.
Nucleic Acids Research 07/1994; 22(12):2289-95. · 8.03 Impact Factor
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ABSTRACT: Individual-specific DNA fingerprints of crocodilians were obtained by the use of Bkm-2(8) probe. Pedigree analyses of Crocodylus palustris, C. porosus and Caiman crocodilus revealed that the multiple bands (22-23 bands with Aludigest) thus obtained were inherited stably in a Mendelian fashion. Unique fingerprints permitted us to identify individuals, assign parentage, and reconstruct the DNA profile of a missing parent. Average band sharing between unrelated crocodiles was found to be 0.37. Band sharing between animals of known pedigrees increased predictably with relatedness and provided a basis for distinguishing relatives from non-relatives. Similar results obtained in other species/genera, using the same probe, suggest that this approach may be applicable to all species of crocodilians, and could facilitate genetic studies of wild and captive populations.
MGG - Molecular and General Genetics 05/1993; 238(1-2):49-58.
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ABSTRACT: In the rat ascitic histiocytoma, AK-5, chromosome numbers vary between 33 and 41 with a peak at 40 chromosomes. None of the metaphase spreads showed double minute chromosomes. The karyotype of this tumor was characterized by hypodiploid chromosome constitution. Giemsa banding analysis revealed 12 clonal marker chromosomes (M1-M12). Tentative identification of these markers were: M1 = ins(1q); M2 = t(5;?); M3 = t(8;10); M4 = t(8;?); M5 = t(10;X); M6 - t(15;?); M7 = t(5;6); M8 = del(13p); M9-M12 = unidentified. M3 being a large near metacentric chromosome serves as a characteristic marker for this tumor. All marker chromosomes except M2 and M4 were present in single copy per cell. In some metaphases M2 was present in 2 copies while M4 was present in 2 or 3 copies per cell. The total cell cycle duration of AK-5 cells was 15.5 h and the different phases, G1, S, G2 + M were estimated as 1.2, 12.2, and 2 h, respectively.
Anticancer research 15(2):289-94. · 1.73 Impact Factor
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ABSTRACT: Semen samples from 11 Indian leopards (Pantherapardus) from 3 different zoos in India were collected by electroejaculation. A computer-aided semen analyzer (CASA) was used for assessing the quality of the semen vis-à-vis sperm motility. The volume of the ejaculate, sperm density, and the number of motile and morphologically normal spermatozoa were found to be 1.57 +/- 1.26 mL, 55.78 million +/- 38.67 million per mL, 57.05% +/- 16.96% and 71.92% +/- 15.32%, respectively. Although the spermatology varied between individuals in the study, Box-Whisker-plot analysis suggested that the distribution was normal (P > .05). The ejaculated sperm were cryopreserved after diluting in test-yolk buffer. The post-thaw motility was 32.14% and did not differ at 30 or 60 days after cryopreservation. CASA indicated that the progressive velocity (VSL) of cryopreserved spermatozoa was decreased and, as a consequence, they moved more slowly than the neat (VSL 76.3 microm/sec in neat vs 53.8 microm/sec in cryopreserved spermatozoa) and the trajectories were less planar. However, both cryopreserved and neat spermatozoa penetrated the zona-free hamster oocyte with equal efficiency (79% neat vs 80% cryopreserved). The study also reports application of CASA for feline spermatozoa and provides information for the first time on the spermatology of the Indian leopard. This baseline data could be used in captive breeding programs. The results are compared and discussed with the available information on other felines.
Journal of Andrology 22(1):25-33. · 2.97 Impact Factor
