Mechanistic basis of infertility of mouse intersubspecific hybrids

Abstract

According to the Dobzhansky-Muller model, hybrid sterility is a consequence of the independent evolution of related taxa resulting in incompatible genomic interactions of their hybrids. The model implies that the incompatibilities evolve randomly, unless a particular gene or nongenic sequence diverges much faster than the rest of the genome. Here we propose that asynapsis of heterospecific chromosomes in meiotic prophase provides a recurrently evolving trigger for the meiotic arrest of interspecific F1 hybrids. We observed extensive asynapsis of chromosomes and disturbance of the sex body in >95% of pachynemas of Mus m. musculus × Mus m. domesticus sterile F1 males. Asynapsis was not preceded by a failure of double-strand break induction, and the rate of meiotic crossing over was not affected in synapsed chromosomes. DNA double-strand break repair was delayed or failed in unsynapsed autosomes, and misexpression of chromosome X and chromosome Y genes was detected in single pachynemas and by genome-wide expression profiling. Oocytes of F1 hybrid females showed the same kind of synaptic problems but with the incidence reduced to half. Most of the oocytes with pachytene asynapsis were eliminated before birth. We propose the heterospecific pairing of homologous chromosomes as a preexisting condition of asynapsis in interspecific hybrids. The asynapsis may represent a universal mechanistic basis of F1 hybrid sterility manifested by pachytene arrest. It is tempting to speculate that a fast-evolving subset of the noncoding genomic sequence important for chromosome pairing and synapsis may be the culprit.

Fig. 1.

DSBs, repair, and meiotic recombination in sterile males. (A) RAD51/DMC1, MSH4, and MLH1 foci are visualized by immunolabeling on meiotic spreads from sterile hybrids and fertile controls. Synaptonemal complexes are labeled by anti-SYCP3 antibody; unsynapsed chromosomes of mid-pachynemas are labeled by HORMAD2. (B and C) The increased frequency of RAD51/DMC1 in early pachynemas of sterile hybrids reflects stalled repair of DSBs in asynapsed autosomes. The difference between reciprocal hybrids in MSH4 and MLH1 probably is controlled by an X-linked polymorphism of the meiotic recombination rate locus (39). N = number of mice, n = number of cells analyzed: RAD51, DMC1 and MSH4 (N = 3, n = 30 for zygotene, n = 60 for early pachytene; MSH4 N = 3, n = 100; MLH1, N = 5, n = 150. (D) ATR decorates only the XY pair in B6 mice; in sterile hybrids it also persists on unsynapsed autosomes (N = 4 n = 100 per genotype). (E) STAG3 cohesin labels all chromosomes irrespective of their synapsis status (N = 3, n = 50).

Fig. 2.

Asynapsis of homologous chromosomes in sterile F1 males. (A) Asynapsis of pachytene spermatocytes revealed by immunostaining of SYCP3 or HORMAD2. Early (histone H1t-negative) pachynemas show multiple asynapsed autosomes decorated by the phosphorylated form of histone H2AFX (γH2AFX). H1t-positive mid–late pachynemas show one or two pairs of asynaptic autosomes engulfed in the sex body. (B) The exceptional multivalents and ring-like chromosomes indicate partial and/or nonhomologous synapsis. (C) Asynapsis was rare in B6 mice and was absent in intraspecific hybrids (B6 × BALB/c)F1 (abbreviated B6CF1) and (PWD × PWK)F1 (abbreviated PKF1). PB6F1 meiocytes with more than four univalents disappear in mid-pachynema and diplonema. (D) Distribution of pachynemas according to the number of asynapsed autosomes estimated by counting the CREST-stained centromeres on SYCP3-labeled synaptonemal complexes. Individual pachytene stages could not be identified in this experiment. In total 111 aberrant pachynemas were counted. (E) Frequency of mid–late pachynemas with autosomal univalents within the sex body in sterile PB6F1 and semifertile reciprocal B6PF1 hybrids. (F) X–Y asynapsis correlates with male sterility; however, the sex chromosomes could not be reliably identified in the majority of early pachynemas. PAR, pseudoautosomal region.

Fig. 3.

Five autosomes were tested for asynapsis by DNA FISH and HORMAD2 immunolabeling on pachytene spreads from PB6F1 sterile males. (A and B) (Left) A single DNA cloud signals properly aligned chromosomes and HORMAD2-positive axes restricted to sex chromosomes. (Center) A cell in which asynapsis does not involve the studied chromosome. (Right) Arrows show partial asynapsis of Chr 2 and complete asynapsis of Chr 16, respectively. (C–E) Separate DNA clouds and HORMAD2-labeled univalents demonstrate asynapsis of Chrs 17 (C), 18 (D), and 19 (E). (F) The frequency of asynapsis of five examined chromosomes in sterile PB6F1and semifertile reciprocal B6PF1 hybrids. n, total number of cells scored; nd, not done.

Fig. 4.

Gene-expression profiling of 14.5-dpp male testes of sterile males and fertile controls. (A) The number of genes with suggestive differences in expression per chromosome (unadjusted P value < 0.05) between sterile PB6F1 hybrids and a pool of fertile (B6PF1, B6, PWD) controls was compared with counts expected by pure chance (Poisson model, P < 0.01). The X chromosome showed a significantly high number of misexpressed genes compared with autosomes. Three mice were analyzed per genotype. (B) Median absolute log fold-change between PB6F1 and B6PF1 average gene expression was calculated for Chr X cytogenetic bands. Maximum is attained for A7.1. (C) Log fold-changes of Chr X gene expression between PB6F1 and B6PF1.

Fig. 5.

Asynapsis at prophase I of oocytes of reciprocal hybrids and B6 controls. (A) Almost half the pachynemas of hybrid females show one or more pairs of asynaptic autosomes detected by SYCP3/SYCP1 or HORMAD2/SYCP3 immunostaining. The unsynapsed chromosomes embedded in clouds of histone γH2AFX show a tendency to clustering. (B) The high frequency of asynapsis in intersubspecific hybrids does not depend on the fertility status of their male counterparts. PB6*fF1 is the hybrid (PWD × B6.Hst1^f) carrying the C3H allele of Hst1/Prdm9 gene. The males of this phenotype are fertile (13, 31). Three females per genotype and 40 cells per mouse were analyzed. (C and D) Chromosome alignment in meiosis I by time-lapse analysis. The values represent the amount of DNA located near the equatorial plane in C3B6F1 (green, n = 6), B6PF1 (red, n = 7), and PB6F1 (orange, n = 7) oocytes at indicated time points before anaphase.