A new paradigm for profiling testicular gene expression during normal and disturbed human spermatogenesis

Abstract

The aim of this study was to identify gene expression patterns of the testis that correlate with the appearance of distinct stages of male germ cells. We avoided the pitfalls of mixed pathological phenotypes of the testis and circumvented the inapplicability of using the first spermatogenic wave as done previously on rodents. This was accomplished by using 28 samples showing defined and highly homogeneous pathologies selected from 578 testicular biopsies obtained from 289 men with azoospermia (two biopsies each). The molecular signature of the different developmental stages correlated with the morphological preclassification of the testicular biopsies, as shown by resampling-based hierarchical clustering using different measures of variability. By using analysis of variance (ANOVA) and extensive permutation analysis, we filtered 1181 genes that exhibit exceptional statistical significance in testicular expression and grouped subsets with transcriptional changes within the pre-meiotic (348 genes), post-meiotic (81 genes) and terminal differentiation (38 genes) phase. Several distinct molecular classes, metabolic pathways and transcription factor binding sites are involved, depending on the transcriptional profile of the gene clusters that were built using a novel clustering procedure based on not only similarity but also statistical significance.

Expression of SNURF–SNRPN upstream transcripts and epigenetic regulatory genes during human spermatogenesis

“…10 For validation of the gene-expression data described here, we used a second, independent dataset of human spermatogenesis. 11 Except for EZH2 and MBD1, concordant expression profiles were found. The apparent discrepancy regarding the latter two genes may, at least in the case of MBD1, be because of different probes on the two array platforms and the presence of different splice forms (see below).…”

“…To corroborate these findings, genes displayed in the heatmap of Figure 3 were interrogated on a dataset from a completely different, single-oligonucleotide microarray platform 11 by using the intersection obtained from HUGO annotations. This cross-platform comparison validates the expression changes described here and obviates the need to validate single genes, as has been shown in a study in which 93% of genes common to two microarray platforms could be confirmed in their differential expression by quantitative real-time PCR 12 .…”

“…11 This was done by building an intersection of the two platforms using HUGO gene abbreviations. Hierarchical cluster analysis was carried out with the mean expression values from the different stages of spermatogenic impairment and clustered with the TIGR MeV software v4.3 (The TIGR Consortium, http://www.tm4.org/mev.html) using Manhattan distance and average linkage.…”

Altered gene expression signature of early stages of the germ line supports the pre‐meiotic origin of human spermatogenic failure

“…Some recent microarray studies have assessed global gene expression analysis in testicular biopsies from infertile men to identify the genes critical for spermatogenesis (Fox et al, 2003;Rockett et al, 2004;Ellis et al, 2007;Feig et al, 2007;von Kopylow et al, 2010;Chalmel et al, 2012). In these studies specific germ cell transcription patterns are inferred from infertile testicular phenotypes in men and a pattern of significantly decreased regulated genes has been attributed to the degree of spermatogenic failure and the loss of specific stages of germ cells.…”

Structural and functional analysis of the promoters of the human glycerol kinase gene family

“…Prior to their utilisation for in situ promoter studies, the cells were genetically characterised and were found to express Pgk-2 and protamine transcripts (data not shown). Pgk-2 transcription is known to occur only in post-meiotic germ cells (Feig et al, 2007), while protamine is only expressed in round spermatids stage (Hecht, 1986). To understand testis-specific gene regulation during spermatogenesis, it is essential to analyse in detail the cis-elements in pro-moters that are expressed at similar stages of spermatogenesis.…”