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

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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) that are expressed either in different structures within the sperm cell or at different stages of sperm development. 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 whole-animal 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.

Section snippets

Materials and methods

Luciferase reporter constructs. BAC clones RP23-291E22, RP23-332H6, and RP23-332H16 containing mouse Sptrx-1, Sptrx-2, and Sptrx-3 genomic regions, respectively, were obtained from BACPAC Resources Center (http://bacpac.chori.org/). All the Sptrx 5′-flanking regions were amplified by PCR using combinations of the mutagenic primers listed in Supplemental data Table 1. All the PCR products were first cloned into pGEM-Teasy (Promega), verified by sequencing, and inserted into the promoterless

Sptrx mRNA and protein expression levels in GC-spd(ts) cell line

The identification of the cis-acting and trans-acting elements directing the spermatogenic-specific transcription of Sptrx genes is essential to shed more light on their role in sperm physiology. However, a major limitation to this approach is the availability of adequate cell lines that can recapitulate different aspects of spermatogenesis. To our knowledge, GC-spd(ts) is the only spermatogenic mouse cell line available, which is able to undergo meiosis in vitro and develop a primordial tail

Discussion

Regulation of gene expression during spermatogenesis is exerted according to a complex and coordinated scheme where different cis-acting and trans-acting elements converge to either activate, modify or silence the expression of a particular group of specific genes responsible for the formation of a specialized cell as the spermatozoon. Among those gene products participating in the spermatogenic process, we have recently identified three sperm-specific thioredoxins [5]. Since control of redox

Acknowledgments

This work was supported by the Swedish Medical Research Council (Projects 03P-14096, 03X-14041, and 13X-10370), the Åke Wibergs Stiftelse, the Karolinska Institutet, and the Spanish Ministerio de Ciencia y Tecnología (Grant BMC2002-00179). A. Jiménez was supported by a postdoctoral fellowship (EX2003-0390) from the Spanish Ministerio de Educación, Cultura y Deporte. M.-J. Prieto-Álamo and J. Jurado were recipients of postdoctoral contracts (Programa Ramón y Cajal) from the Spanish Ministerio de

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    These authors contributed equally to this work.

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