Distinct prophase arrest mechanisms in human male meiosis

Abstract

To prevent chromosomal aberrations being transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. Here, we unravel two clearly distinct meiotic arrest mechanisms that occur during prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y chromosome-encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63, possibly causing spermatocyte apoptosis. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to silence the X chromosome-encoded gene ZFX Discovery and understanding of these meiotic arrest mechanisms increases our knowledge of how genomic stability is guarded during human germ cell development.

Keywords: Human spermatogenesis; Infertility; Meiosis; Meiotic arrest; Meiotic silencing; p63.

Fig. 1.

Histological evaluation of patient testis sections. Immunohistochemical localization of γH2AX in paraffin-embedded testis sections of fertile men (f) and patients with meiotic maturation arrest reveal two types of meiotic prophase arrest patients: type I (I) and type II (II) meiotic arrest. Type I meiotic arrest patients display meiotic prophase arrest and disturbed γH2AX distribution and no XY-body formation, type II meiotic arrest patients display meiotic prophase arrest but normal γH2AX distribution and XY-body formation. Left-hand panels show a global overview of the γH2AX staining in different germ cell populations within the testis and the right-hand panels show higher magnification images of γH2AX staining in spermatocytes. Depicted are: Sertoli cells (Ser), elongated spermatids (ES), A_pale and A_dark spermatogonia, spermatocytes (SP) and apoptotic spermatocytes (arrows). Scale bars: 3 μm (left); 10 μm (right).

Fig. 2.

Immunofluorescence staining of meiotic spread preparations of fertile men (f) and type I (I) and type II (II) meiotic arrest patients. (A,B) γH2AX (A), MLH1 (labeling meiotic crossovers; B) and SYCP3 (A,B) immunostaining. Scale bar: 5 μm. Insets on the right are a magnification of the neighboring panels showing MLH1 staining in relation to SYCP3. (C) Average number of MHL1 foci (±s.d.) in pachytene spermatocytes of fertile men (n=10) and type I (n=7) and type II (n=10) arrested human spermatocytes. *P≤0.00001 (one-way ANOVA).

Fig. 3.

Transcriptomic analysis of fertile (f), type I and II spermatocytes. (A,B) Multidimensional scaling of leptotene/zygotene spermatocytes (L/Z, green) and pachytene spermatocytes (f, orange) from fertile men (data taken from Jan et al., 2017) alongside type I (turquoise), type II (dark blue) and meiotic metaphase (metaphase, red) arrested spermatocytes (A) and differential gene expression analysis (adjusted P-value<0.05; B) reveal distinct transcriptomic profiles for type I and II arrested spermatocytes. Depicted in B are comparisons between leptotene/zygotene (L/Z) and pachytene spermatocytes from fertile men; pachytene spermatocytes from fertile men and type I and II arrested spermatocytes; leptotene/zygotene (L/Z) and type I and II arrested spermatocytes; and, finally, between type I and type II arrested spermatocytes. The total number of DEGs for every comparison are shown as well as the number of genes upregulated (red arrows) and the number of genes downregulated (green arrows). (C) Analysis of genes upregulated during the leptotene/zygotene to pachytene transition in normal spermatogenesis reveals a leptotene/zygotene-like expression pattern in type I and II arrested spermatocytes.

Fig. 4.

Clustering of upregulated or downregulated genes in type I, type II (or in both) arrested spermatocytes. K-means cluster analysis of DEGs between fertile (f), type I and type II spermatocytes reveals genes that are aberrantly expressed in type I arrested spermatocytes (clusters 1, 2), type II arrested spermatocytes (clusters 3, 4, 5) and in both type I and II arrested spermatocytes (clusters 6, 7, 8). Blue line represents the mean expression profile of genes in a cluster; profiles of individual genes are depicted by gray lines.

Fig. 5.

Patient type-specific expression of genes reflecting aberrant sex chromosome silencing, DNA damage repair and cell cycle regulation. (A-C) Beeswarm plots depicting different expression levels of ZFY (adjusted P≤0.0389) and ZFX (adjusted P≤0.0899) (A), TP53 (adjusted P≤0.8030) and TP63 (adjusted P≤0.0469) (B) and CCNA1 (adjusted P≤0.0051), CCNA2 (adjusted P≤0.0626), CCNE1 (adjusted P≤0.0001) and CDC25A (adjusted P≤0.0031) (C) in fertile (f; orange), type I (turquoise) and type II (dark blue) spermatocytes.

Fig. 6.

Aberrant sex chromosome silencing in type I and II arrested spermatocytes. Boxplots showing the amount of genes expressed from the X and Y chromosomes relative to the total amount of genes expressed in the same sample for leptotene/zygotene (L/Z), fertile control pachytene (f), type I arrested and type II arrested spermatocytes. A significant difference was detected between fertile and arrested spermatocytes (two-way ANOVA, Tukey HSD) (*adjusted P≤0.0001).