Sterility and gene expression in hybrid males of Xenopus laevis and X. muelleri

Abstract

Background: Reproductive isolation is a defining characteristic of populations that represent unique biological species, yet we know very little about the gene expression basis for reproductive isolation. The advent of powerful molecular biology tools provides the ability to identify genes involved in reproductive isolation and focuses attention on the molecular mechanisms that separate biological species. Herein we quantify the sterility pattern of hybrid males in African Clawed Frogs (Xenopus) and apply microarray analysis of the expression pattern found in testes to identify genes that are misexpressed in hybrid males relative to their two parental species (Xenopus laevis and X. muelleri).

Methodology/principal findings: Phenotypic characteristics of spermatogenesis in sterile male hybrids (X. laevis x X. muelleri) were examined using a novel sperm assay that allowed quantification of live, dead, and undifferentiated sperm cells, the number of motile vs. immotile sperm, and sperm morphology. Hybrids exhibited a dramatically lower abundance of mature sperm relative to the parental species. Hybrid spermatozoa were larger in size and accompanied by numerous undifferentiated sperm cells. Microarray analysis of gene expression in testes was combined with a correction for sequence divergence derived from genomic hybridizations to identify candidate genes involved in the sterility phenotype. Analysis of the transcriptome revealed a striking asymmetric pattern of misexpression. There were only about 140 genes misexpressed in hybrids compared to X. laevis but nearly 4,000 genes misexpressed in hybrids compared to X. muelleri.

Conclusions/significance: Our results provide an important correlation between phenotypic characteristics of sperm and gene expression in sterile hybrid males. The broad pattern of gene misexpression suggests intriguing mechanisms creating the dominance pattern of the X. laevis genome in hybrids. These findings significantly contribute to growing evidence for allelic dominance in hybrids and have implications for the mechanism of species differentiation at the transcriptome level.

Figure 1. Visualization of live (green) and dead (red) sperm in sperm density assay using dual emission filter for SYBR14 and propidium iodide.

Xenopus laevis (A); hybrid (B); X. muelleri (C); and brightfield combined with fluorescent image of undifferentiated cells of hybrids (D).

Figure 2. The number of total sperm cells in Xenopus laevis (black circles) and hybrids (striped squares) in uninjected sexually mature males and hCG injected sexually mature males.

Error bars represent±1 standard error.

Figure 3. Proportion of live (green circles), dead (red squares), and undifferentiated (black triangles) sperm cells in Xenopus laevis (A) and hybrids (B) in normal/uninjected compared to hCG injected males.

Error bars represent±1 standard error.

Figure 4. Numbers of motile (green circles) and immotile (red x's) sperm in Xenopus laevis and hybrids in normal/uninjected compared to hCG injected males.

Error bars represent±1 standard error.

Figure 5. Comparison of sperm area (µm²) in Xenopus laevis, X. muelleri, and hybrids and representative brightfield images of sperm.

Error bars represent±1 standard error and * denotes significance from a Bonferroni multiple comparison test. Hybrids have larger sperm compared to X. laevis and X. muelleri but sperm area does not differ between X. laevis and X. muelleri.

Figure 6. Number of probesets remaining at various thresholds from comparing the hybridization intensity of Xenopus muelleri vs. X. laevis.

Figure 7. Volcano plots from FDR corrected t-tests of statistical significance (vertical axis) against magnitude of expression change (horizontal axis), where each point corresponds to a gene/transcript.

Expression change (fold-change) is defined as a log₂-transformed ratio of mean nonhybrid to mean hybrid expression level. (A) Xenopus laevis vs. Hybrids; (B) Xenpous muelleri vs. Hybrids. The red line denotes FDR adjusted alpha 0.05. The horizontal deviation from 0 towards the right or left reflects hybrid underexpression or overexpression, respectively.