Expression In Gonadal Tissues Research Articles

Suitable reference gene for quantitative real-time PCR analysis of gene expression in gonadal tissues of minnow Puntius sophore under high-temperature stress

BackgroundHigh ambient temperature is known to affect fish gonadal development and physiology in a variety of ways depending on the severity and duration of exposure; however, the underlying molecular mechanisms are poorly understood. Gonadal gene expression influence the gonadal development, physiology and the quality of egg/sperm produced in teleosts and the mechanistic understanding of spatio-temporal changes in the gonadal gene expression could be instrumental in controlling the fate of egg/sperm and the quality of seed produced. Real time-quantititative polymerase chain reaction (RT-qCR), is a high throughput, sensitive and reproducible methodology used for understanding gene expression patterns by measuring the relative abundance of mRNA transcripts. However, its accuracy relies upon a suitable reference gene whose expression levels remain stable across various experimental conditions. In the present study, we evaluated the suitability of ten potential reference genes to be used as internal controls in RT-qPCR analysis in gonadal tissues (ovary and testis) of minnow Puntius sophore exposed to high temperature stress for different time periods (7 days, 60 days). Expression analysis of ten different constitutively expressed genes viz. 18S ribosomal RNA (18S rRNA), beta actin (βactin), β-2 microglobulin (b2mg), eukaryotic elongation factor-1 (eef1), glyceraldehyde-3phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), ribosomal binding protein L13 (rpl13), tubulin (tub), tata box binding protein (tbp), ubiquitin (ubi) was carried out by using RT-qPCR and the stability in their expressions were evaluated by using four different algorithms; namely, delta Ct, BestKeeper, geNorm and NormFinder.ResultsIn ovary, eef1 was found to be the most suitable reference gene in all the algorithms used. In testis, b2mg was found to be the most suitable reference gene in delta Ct, BestKeeper, NormFinder analysis while tbp and eef1 were found to be the most suitable reference genes in geNorm analysis.ConclusionsIn conclusion, eef1 and b2mg were found to be the most suitable reference genes in ovary and testis, respectively, of Puntius sophore exposed to high temperature stress, and could be used as internal controls for gene expression analysis in gonadal tissues of Puntius sophore.

Hypoxia alters gene expression in the gonads of zebrafish ( Danio rerio)

The objectives of this study were to characterize gene expression responses to hypoxia in gonads of mature zebrafish ( Danio rerio), and to start characterizing modes of action by which hypoxia could potentially alter reproduction. Adult male and female zebrafish were maintained under normoxia (7 mg O 2/L), moderate hypoxia (3 mg O 2/L), and severe hypoxia (1 mg O 2/L) for 4 and 14 days and changes in gene expression in gonadal tissues ( n = 5 per sex per treatment) were evaluated using a commercial 21,000 gene zebrafish oligonucleotide microarray. Differentially expressed genes were determined using ANOVA ( p < 0.05), and enriched gene ontology (GO) categories ( p < 0.01) identified using GeneSpring GX software. Short-term (4 d) exposure to hypoxia affected expression of genes associated with the initial adaptive responses such as: metabolism of carbohydrates and proteins, nucleotide metabolism, haemoglobin synthesis, reactive oxygen species metabolism, and locomotion. Prolonged (14 d) hypoxia affected a suite of genes belonging to different GO categories: lipid metabolism, reproduction (e.g., steroid hormone synthesis), and immune responses. Results of the present study demonstrate that reproduction likely would be affected by hypoxia via multiple modes of action. These include previously hypothesized mechanisms such as modulation of expression of steroidogenic genes, and downregulation of serotonergic pathway. In addition, we propose that there are multiple other points of disruption of reproductive system function linked, for example, to reorganization of lipid transport and other mechanisms involved in responding to hypoxia (e.g., hydroxysteroid dehydrogenase alterations, downregulation of contractile elements, etc.).

Cloning of the Bovine β-Carotene-15,15’-Oxygenase and Expression in Gonadal Tissues

Beta-carotene-15,15'-oxygenase (betaCO), found mainly in intestinal mucosa and liver, is the enzyme responsible for cleaving beta-carotene into retinal, which can be used or stored at these sites or carried by the bloodstream to different target cells within the body. We isolated the cDNA for bovine betaCO and demonstrated its expression in gonadal tissues. A cDNA of 2130 base pairs (bp) was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), using degenerate oligonucleotides; the deduced protein shared an identity of 75% with its homologues from other mammalian species. In order to evaluate the expression of this enzyme, we performed RT-PCR and in situ hybridizations in the ovary and testis of bovines. RT-PCR showed the expression of betaCO in testis, ovary, and cultured granulosa cells. In situ hybridization of complete ovary and testis revealed expression in granulosa cells and the corpus luteum in the ovary and in germinal and interstitial cells in the testis. These results suggest that beta-carotene could act as a local source of retinoids, which have been shown to be important during proliferation, differentiation, and maturation of both female and male germinal cells.

Preclinical studies of gene transfer for the treatment of desmoid disease in familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) arises following mutation or loss of the adenomatous polyposis coli (APC) gene. Desmoid tumours are proliferations of fibroblasts and occur as an extracolonic manifestation of FAP. They are a leading cause of death after colectomy. The aim of this study was to assess the potential for APC gene transfer into fibroblasts in vitro and in vivo as a basis for consideration of gene therapy in the prevention or treatment of desmoid tumours. The APC gene was transferred by lipofection into fibroblasts in tissue culture and into peritoneum and small bowel mesentery in vivo. Reverse transcriptase-polymerase chain reaction was used to determine whether or not transfection was successful. Transgene expression was recorded in vitro to 7 days after transfection. High levels of transgene expression were also seen in samples of peritoneum (all eight mice), small bowel mesentery (seven of eight), liver (seven of eight) and intestinal tissues (five to six of eight) following intraperitoneal treatment. Interestingly, transgene expression in gonadal tissues was occasionally noted. Liposomal transfection of APC gave prolonged high-level expression of the transgene, an important basis for gene therapy. No adverse effects were recorded. Further work is needed in animal models of desmoid disease to assess the clinical effects of gene therapy.

Variations in DNA (cytosine-5)-methyltransferase-1 expression during oogenesis and early development of the zebrafish.

We report the determination of zebrafish DNA (cytosine-5-) methyltransferase ( dnmt-1) temporal and spatial patterns of expression in gonadal tissues and during early development. Only one dnmt-1 message of around 5 kb was observed in all tissues examined and its levels were highest in gonadal tissues. During the course of oogenesis, early oocytes contain significant amounts of dnmt-1 transcript while message abundance declines as oocytes mature. During early embryogenesis message levels remain low until the blastula stage. Methyltransferase enzyme assays reveal that the maternal dnmt-1 message accumulated during oogenesis is translated into protein presumably providing necessary dnmt-1 stockpiles to support early embryonic development prior to zygotic gene activation. Such spatial and temporal regulation of dnmt-1 expression suggests specific functions for the enzyme during oogenesis and early development of zebrafish.

Characterization of Ke 6, a New 17β-Hydroxysteroid Dehydrogenase, and Its Expression in Gonadal Tissues

The abnormal regulation of the Ke 6 gene has been linked to the development of recessive polycystic kidney disease in the mouse. In this report, we have shown that Ke 6 is a 17beta-hydroxysteroid dehydrogenase and can regulate the concentration of biologically active estrogens and androgens. The Ke 6 enzyme is preferentially an oxidative enzyme and inactivates estradiol, testosterone, and dihydrotestosterone. However, the enzyme has some reductive activity and can synthesize estradiol from estrone. We find that the Ke 6 gene is expressed within the ovaries and testes. The presence of Ke 6 protein within the cumulus cells surrounding the oocyte places it in a strategic location to control the level of steroids to which the egg is exposed. Previously, it had been shown that glucocorticoids can induce renal cysts in the neonatal rodent, only when given at a narrow time window of postnatal kidney development. We propose that the reduction in the level of Ke 6 enzyme, which occurs in the cpk, jck, and pcy mice, may lead to abnormal elevations in local level of sex steroids, which either directly or indirectly via abnormal glucocorticoid metabolism result in recessive renal cystic disease, a developmental disorder of the kidney.

Differential Expression of the Proto-oncogenes c- abl and c- mos in Developing Mouse Germ Cells

C- abl and c- mos, two proto-oncogenes with unique patterns of expression in gonadal tissues, were examined by in situ hybridization with respect to their expression in the germ line. C- abl transcripts were observed to be most abundant in late round spermatids and elongating spermatids. C- mos transcripts were also expressed in round spermatids, but at much reduced levels compared to the levels of c- abl. C- abl mRNAs were also detected in mouse oocytes which have entered the growth phase. This temporal specificity of accumulation of c- abl transcripts was similar to that observed for c- mos, which also accumulates in growing and fully grown oocytes (follicle stages 4-6). Quantitative evaluation suggested that c- abl mRNA levels in oocytes are at least an order of magnitude lower than those of c- mos transcripts.

The regulation of neurohypophyseal peptide gene expression in gonadal tissues.

The regulation of neurohypophyseal peptide gene expression in gonadal tissues.