Mechanisms Underlying Mammalian Hybrid Sterility in Two Feline Interspecies Models

Abstract

The phenomenon of male sterility in interspecies hybrids has been observed for over a century, however, few genes influencing this recurrent phenotype have been identified. Genetic investigations have been primarily limited to a small number of model organisms, thus limiting our understanding of the underlying molecular basis of this well-documented "rule of speciation." We utilized two interspecies hybrid cat breeds in a genome-wide association study employing the Illumina 63 K single-nucleotide polymorphism array. Collectively, we identified eight autosomal genes/gene regions underlying associations with hybrid male sterility (HMS) involved in the function of the blood-testis barrier, gamete structural development, and transcriptional regulation. We also identified several candidate hybrid sterility regions on the X chromosome, with most residing in close proximity to complex duplicated regions. Differential gene expression analyses revealed significant chromosome-wide upregulation of X chromosome transcripts in testes of sterile hybrids, which were enriched for genes involved in chromatin regulation of gene expression. Our expression results parallel those reported in Mus hybrids, supporting the "Large X-Effect" in mammalian HMS and the potential epigenetic basis for this phenomenon. These results support the value of the interspecies feline model as a powerful tool for comparison to rodent models of HMS, demonstrating unique aspects and potential commonalities that underpin mammalian reproductive isolation.

Keywords: Haldane's rule; feline; hybrid sterility; large X-effect; speciation.

Fig. 1.

H&E-stained testis from fertile and sterile hybrids. (A) Fertile fifth-generation Savannah backcross hybrid testis with mature, normal sperm. (B) Sterile fourth generation Savannah backcross hybrid testis showing hypospermatogenesis, with a high incidence of pachytene spermatocytes and the presence of vacuoles. (C) Fertile fourth-generation Bengal backcross hybrid testis with mature, normal sperm. (D) Sterile fourth-generation Bengal backcross hybrid testis exhibiting globozoospermia.

Fig. 2.

Manhattan plots and marker details for GWAS in two hybrid feline breeds. (A) Five markers (SAV1-5) exceeding Wellcome Trust recommendations for genome-wide significance (P_uncorrected < 5 × 10⁻⁵; −log₁₀ P = 4.30, red line) (Wellcome Trust Case Control Consortium 2007) based on analysis of the savannah cohort (n = 103). The Manhattan plot shown represents the full results under the dominant model of inheritance, but for brevity, we inserted the –log10 P value of SAV1 based on the additive model, for comparison. The full Manhattan plot under the additive model is shown in supplementary figure S9, Supplementary Material online. (B) Manhattan plot under a dominant model of inheritance for the Bengal cohort (n = 101), showing three markers (the two markers for BEN3 are in LD) exceeding genome-wide significance (red line) (Wellcome Trust Case Control Consortium 2007). (C) Table of markers, P values, coordinates in the FelCat5 assembly and most proximal gene to each marker. P-P plots for each analysis are shown in supplementary figure S10, Supplementary Material online.

Fig. 3.

(A–H) Genomic regions surrounding each top ranked marker and testis expression data for the region. A 500-kb window surrounding each significant marker is indicated by vertical lines on the chromosome ideogram. –log10 P values (y axis) for both additive and dominant inheritance models are plotted along each chromosome (x axis), with significance threshold indicated by horizontal red line. Log₂ fold testis expression change in sterile hybrids when compared with domestic cat (D), fertile hybrids (F), and wild species (W) are shown in red (significant upregulation), green (significant downregulation), and gray (nonsignificant misregulation) (supplementary table S8, Supplementary Material online).

Fig.4.

X chromosome copy number, GWAS significance, and RNA-seq gene misregulation in sterile feline hybrids. (A) Number of duplicated copies detected by CNVator across the X chromosome. Blue lines on the ideogram indicate position along the X chromosome (x axis). Purple lines indicate number of detected copies (y axis). (B) –Log₁₀ P values of SNP markers (only values above 3.3 are shown, for brevity) that approached significance on the X chromosome of each breed. Orange vertical lines indicate Bengal and red indicates Savannah. (C) Gene misexpression on the X chromosome in each sterile hybrid when compared with the domestic cat. Red indicates upregulation and green downregulation. The x axis denotes the position along the X chromosome, and the y axis indicates the log₂ (fold change) in expression for each gene when compared with domestic cat. (D) Contrasting misregulation patterns on the X chromosome and autosomes in sterile hybrids when compared with domestic cat, fertile hybrids, and wild species. x axes list the integer fold-change categories (from negative 10-fold change to positive 10-fold change) for which each gene is classified based on the magnitude of misexpression. y axes show the frequency of genes falling into these categories. Each X-autosome distribution was significantly different for all six comparisons (Komolgorov–Smirnov P < 0.01).