A Global Expression Switch Marks Pachytene Initiation during Mouse Male Meiosis

Abstract

Male spermatogenesis is an essential and complex process necessary to gain totipotency and allow a whole new organism to develop upon fertilization. While single-gene based studies have provided insights into the mechanisms underlying spermatogenesis, detailed global profiling of all the key meiotic stages is required to fully define these processes. Here, by isolating highly enriched mouse meiotic cell populations, we have generated a comprehensive gene expression atlas of mammalian meiosis. Our data define unique signatures for the specific stages of meiosis, including global chromosome X inactivation and reactivation. The data also reveal profound switches in global gene expression at the initiation of pachynema that are reminiscent of the commitment to meiosis observed in budding yeast. Overall, this meiotic atlas provides an exhaustive blueprint and resource for mammalian gametogenesis and meiosis.

Figure 1

Architecture of the mammalian testis and purification of meiotic fractions. (a) Schematic drawing of a section of a seminiferous tubule with mitotic spermatogonia, spermatocytes, and spermatids surrounded by two Sertoli nurse cells; (b) Representative pictograms of FACS-purified meiosis prophase I meiotic stages labeled for phosphorylated histone H2AX and synaptonemal complex protein 3 (immunofluorescence analyses), as well as the split sexual chromosome in round spermatids (fluorescent in situ hybridization); (c) A representative Hoechst FACS profile is shown, with the various meiotic cell populations used in this study indicated: spermatogonia (Sp), pre-leptotene (pL), leptotene-zygotene (L/Z), early-pachytene (eP), middle-pachytene (mP), late-pachytene (lP), diplotene (D), and round spermatids (RS).

Figure 2

Dynamics of meiotic gene expression. The heatmap shows probe sets having meiotic fraction-specific genes, which are indicated. Each horizontal line corresponds to a probe set with yellow and blue indicating normalized high and low expression, respectively. Genes are divided into 8 K-means clusters. Complete gene lists and GO terms tables are available in the Supporting Information (Supplementary Dataset S1 and Dataset S2).

Figure 3

Meiotic sex chromosome inactivation and reactivation. The average expression of 874 X-linked present probe sets is shown. Complete inactivation is clearly visible at the late pachynema stage. The three phases of chr X meiotic developmental stage are indicated below the heatmap. Probe sets were serially ordered relative to their chromosome location from centromere to telomere but are not to scale. A schematic of chr X is shown on the left for orientation purposes.

Figure 4

Meiotic fraction-specific genes and commitment. (a) The top 50 probe sets having the most restricted expression patterns are shown for each of the eight meiotic fractions studied. A high degree of correlative gene expression is observed in cell populations before and after the early-pachytene switch; (b) Heatmaps of centrin and tektin probe sets are displayed. An expression switch is observed suggesting a totally new regulation of these fundamental regulators of centrioles; (c) Centrosome reduction during spermiogenesis. The male germ cells possess intact centrosomes containing centrioles and centrosomal proteins until the round spermatid stage. The microtubules of the distal centriole extend as axoneme of the spermatid tail. The centrioles are degenerated to various extents in spermatozoa of different species. Rodent spermatozoa lose both centrioles completely (shown here). The schematics are not drawn to scale (adapted from Manandhar et al. [24]). Nu: nucleus.