Characterization of genes expressed early in mouse spermatogenesis, isolated from a subtractive cDNA library

Abstract

Spermatogenesis provides an excellent model for the study of gene expression during differentiation of defined cell lineages. In vivo studies have revealed a complex set of regulatory interactions between different cell types during mammalian spermatogenesis (Fritz 1994). These interactions result in a multiple cascade of gene expression. Different genes participating in the regulation of these processes have been reported when testis-specific promoters or testis-specific splicing of transcripts were used (Wolgemuth and Watrin, 1991; Barouki 1992). However. we know very little about the genetic systems involved in the differentiation processes at early spermatogenesis stages. Expressed sequence tags, ESTs, corresponding to transcripts detected in both fetal female and adult male gonads have been partially characterized from a fetal ovary cDNA library (L6pezAlafi6n and del Mazo 1995). Interest in these types of ESTs has been recently reported in physical and genetic mapping and in studies of markers for gene expression. An especially interesting result was the finding of ESTs with a developmentally regulated, gonadal or germ-cell-specific pattern of expression. In testis, 7117 ESTs, human sequences and tentative human sequences have been reported (Adams et al. 1995). The construction of cDNA subtractive libraries allows the isolation of clones either differentially expressed during a process or that show tissue-specific expression (Hedrick et al. 1984). Paramagnetic technology was used to develop an mRNA subtraction strategy, based on the approach described by L6pez-Fernfindez and del Mazo (1993), with the aim of constructing a cDNA library enriched in clones corresponding to genes activated during early spermatogenesis and probably inactive either in somatic tissues or in testis at previous stages of differentiation (for example, at birth). For selection of clones specifically or preferentially expressed at such stages, the use of a subtractive library was linked to a double differential screening (Benton and Davis 1977; Luo et al. 1994; L6pez-Alafidn and del Mazo, 1995). For characterization of new testis-specific genes with putative roles in mouse early spermatogenesis, 17 selected cDNAs were sequenced, and the pattern of expression throughout testis development was analyzed. The subtractive cDNA library was constructed from poly[A]+RNA corresponding to genes transcribed in testis at day 10 of postnatal life (p.n.), coincident with the beginning of cytological meiotic differentiation. The subtraction method was focused on the enrichment of transcripts expressed in neither newborn testis nor somatic tissues. This cDNA library contained 8 x 104 primary plaques and was amplified to a titer of 1.5 x 109 pfu ml -l. A rate of 43% recombinants was obtained. The testis-specific cDNA clones were selected by hybridizing duplicated filters containing approximately 10,000 clones with a mixture of sscDNA prepared from somatic tissues or from adult testis RNAs. From this screening, 159 clones that were selected hybridized positively with testis and negatively with somatic