Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage

Abstract

BackgroundSpermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis.ResultsWe have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation.ConclusionsThis work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2618-1) contains supplementary material, which is available to authorized users.

In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites

“…PRDM9 is recruited to promoters in a Spo11-dependent manner Class 2A sites are mainly promoters that are mostly active at the beginning of meiotic prophase (99% are expressed in spermatocytes at leptotene/zygotene) (da Cruz et al 2016). Their Spo11 dependency could imply that DSB formation or progression throughout meiotic prophase is required for PRDM9 interaction with these sites.…”

Spermatogenesis and the Evolution of Mammalian Sex Chromosomes

“…Newly arisen male-biased genes are often X-linked, but over time the movement of these genes from the X to the autosomes has led to the de-enrichment of X-linked male meiotic genes [46]. Meiotic silencing may also select for X-linked genes that are critical in the later stages of meiosis to be highly expressed earlier in development (prior to meiosis or in early meiosis), so that transcripts persist through MSCI [48,49]. If true, then the constraints of MSCI may partially explain the enrichment for X-linked genes that are expressed earlier in spermatogenesis.…”

“…DNA becomes highly condensed and the morphological features of the mature spermatozoa take shape through the process of spermiogenesis (Figure 1A). Spermiogenesis is a highly specialized developmental process and many genes expressed during this phase are specific to postmeiotic cells [48,49,71,72]. Similar to MSCI, sex-linked genes are also partially silenced during postmeiotic development through a process known as postmeiotic sex chromosome repression (PSCR) .…”

“…The resulting haploid products undergo spermiogenesis to produce highly specialized spermatozoa. Spermatogenic genes on the autosomes (blue) are on average expressed throughout spermatogenesis, although there are groups of genes that are more highly expressed early (prior to MSCI) or late in spermatogenesis [31,48,71,102,103]. Genes on the X and Y chromosomes (orange) are silenced during meiosis (MSCI) and remain transcriptionally repressed in postmeiotic development (PSCR).…”

Mutations in Genes Coding for Synaptonemal Complex Proteins and Their Impact on Human Fertility

“…Although most available data were obtained in rodents and humans, recent studies have shown a single evolutionary origin of the metazoan SC [Fraune et al, 2012b[Fraune et al, , 2013[Fraune et al, , 2014. All 7 mammalian SC components are meiosis-specific [da Cruz et al, 2016] and contain α-helical domains that are predicted to form coiled coils, which are capable of homotypic and heterotypic protein interactions. Moreover, most SC components have been shown to be able to assemble higher order structures [Yuan et al, 1998;Öllinger et al, 2005;Davies et al, 2012;Lu et al, 2014].…”