Absolute mRNA levels and transcriptional regulation of the mouse testis-specific thioredoxins

Abstract

Thioredoxins function as general protein disulphide reductases. Mammalian male germ cells are equipped with a set of three testis-specific thioredoxins named Sptrx-1, -2 and -3, respectively. Sptrx proteins are expressed either in different structures within the sperm cell or at different stages of sperm development, suggesting a refined regulation in the timing and levels of expression for each Sptrx gene. Previous studies based on qualitative northern-blot and in situ hybridization analyses restricted the presence of Sptrx mRNAs to adult testis, but nothing is known about their transcriptional regulation or relative expression levels in this tissue.In this report we investigate the transcriptional profiles of the mouse Sptrx genes in terms of the germ cell-specific regulation by promoter analysis in GC-2spd(ts) cells. Besides, we perform a comprehensive quantification of the Sptrx mRNA molecules by real-time PCR in wholeanimal experiments. By these means, we show that transcription is differentially regulated for each Sptrx gene and identify the 5'-flanking regions anticipated to contain the cis-regulatory elements responsible, at least in part, for the transcriptional silencing and/or activation of the Sptrx genes. In addition we show remarkable age-associated variations between the Sptrx mRNA expression patterns. Finally we corroborate the testis-specific expression pattern of Sptrx-2 and Sptrx-3, but surprisingly we find a wider expression pattern for Sptrx-1

Alternative splicing of c-fos pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species

“…Here we used a quantitatively rigorous approach based on RT and real-time PCR amplification [ 14 - 17 ] to provide novel information on the actual steady-state copy numbers of the two c- fos transcripts. Quantitations were carried out in adult mouse organs, whole mouse embryos at different developmental stages, and murine cultured cells.…”

“…The work described herein aimed to gain further insights into the functional significance of the alternative splicing of c- fos . To this end, we first undertook a systematic and meticulous quantitation of the real copy numbers of c- fos transcript variants by means of a real-time PCR methodology [ 14 - 17 ]. We tested how the organ context, gestational age, synthesis and turnover rates, and serum stimulation of quiescent cells modulate the absolute expression profiles of the investigated c- fos transcripts.…”

Identification of the Major Proteins Produced by Cultured Germline Stem Cells in Chicken

“…Thioredoxins function as general protein disulfide reductases. Mammalian male germ cells are equipped with a set of 3 testis‐specific thioredoxins (Sptrx‐1, −2, and −3) that are expressed either in different structures within the sperm cell or at different stages of sperm development (Jimenez et al, 2005). Three of the spots were identified as keratins, which might have been introduced during the handling of the 2‐DE gels.…”

New Methodologies for Assessing the Presence and Ecological Effects of Pesticides in Doñana National Park (SW Spain)

“…Absolute quantification (real quantification) gives the molecule copy number of each transcript in each of the samples under study using a calibration curve. Although relative quantification is much easier to perform because a calibration curve is not necessary, relative data are much less informative than absolute data (Jurado et al, 2003;Prieto-Álamo et al, 2003;Jiménez et al, 2005;RuizLaguna et al, 2005RuizLaguna et al, , 2006. The commercialization of real-time PCR equipments allows for absolute expression data with precision levels unattainable for those generated by conventional end-time PCR.…”