Sterility and Gene Expression in Hybrid Males of Xenopus laevis and X. muelleri

BackgroundReproductive 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 FindingsPhenotypic 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/SignificanceOur 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.

研究背景
生殖隔离（reproductive isolation）是界定独特生物物种的核心特征之一，但目前我们对生殖隔离的基因表达基础仍所知甚少。强大分子生物学工具的问世，使得我们能够鉴定参与生殖隔离过程的基因，并将研究焦点聚焦于区分不同生物物种的分子机制。本研究量化了非洲爪蟾属（Xenopus）杂交雄性的不育表型，针对睾丸组织的基因表达模式开展微阵列分析（microarray analysis），以鉴定相较于其两个亲本物种——非洲爪蟾（Xenopus laevis）与穆氏爪蟾（X. muelleri）——杂交雄性中异常表达的基因。

研究方法与主要结果
我们采用新型精子检测方法，对不育雄性杂交种（X. laevis × X. muelleri）的精子发生（spermatogenesis）表型特征进行分析，可量化活精子、死精子及未分化精子的数量，活动精子与非活动精子的比例，以及精子形态。结果显示，相较于亲本物种，杂交种的成熟精子丰度显著降低。杂交精子的体积更大，且伴随大量未分化的精子细胞。我们将睾丸组织基因表达的微阵列分析结果，与基于基因组杂交得到的序列分化校正相结合，以筛选与不育表型相关的候选基因。转录组（transcriptome）分析揭示了一种显著的不对称异常表达模式：相较于X. laevis，杂交种中仅有约140个基因出现异常表达；而相较于X. muelleri，杂交种中异常表达的基因数量接近4000个。

结论与意义
本研究结果为不育杂交雄性的精子表型特征与基因表达之间建立了重要关联。广泛的基因异常表达模式提示了可能存在的特殊机制，可解释杂交种中X. laevis基因组的显性调控模式。这些发现为杂交种中等位基因显性（allelic dominance）的相关研究提供了有力支撑，同时为从转录组层面解析物种分化机制提供了新的研究视角与启示。