Altered expression of testis-specific genes, piRNAs, and transposons in the silkworm ovary masculinized by a W chromosome mutation

Abstract

Background: In the silkworm, Bombyx mori, femaleness is strongly controlled by the female-specific W chromosome. Originally, it was presumed that the W chromosome encodes female-determining gene(s), accordingly called Fem. However, to date, neither Fem nor any protein-coding gene has been identified from the W chromosome. Instead, the W chromosome is occupied with numerous transposon-related sequences. Interestingly, the silkworm W chromosome is a source of female-enriched PIWI-interacting RNAs (piRNAs). piRNAs are small RNAs of 23-30 nucleotides in length, which are required for controlling transposon activity in animal gonads. A recent study has identified a novel mutant silkworm line called KG, whose mutation in the W chromosome causes severe female masculinization. However, the molecular nature of KG line has not been well characterized yet.

Results: Here we molecularly characterize the KG line. Genomic PCR analyses using currently available W chromosome-specific PCR markers indicated that no large deletion existed in the KG W chromosome. Genetic analyses demonstrated that sib-crosses within the KG line suppressed masculinization. Masculinization reactivated when crossing KG females with wild type males. Importantly, the KG ovaries exhibited a significantly abnormal transcriptome. First, the KG ovaries misexpressed testis-specific genes. Second, a set of female-enriched piRNAs was downregulated in the KG ovaries. Third, several transposons were overexpressed in the KG ovaries.

Conclusions: Collectively, the mutation in the KG W chromosome causes broadly altered expression of testis-specific genes, piRNAs, and transposons. To our knowledge, this is the first study that describes a W chromosome mutant with such an intriguing phenotype.

Figure 1

Genetic analysis of the KG line. (A) Mating scheme. KG♀×KG♂ and KG♀×WT♂, but not WT♀×KG♂ generated masculinized females. Sib-crosses weakend masculinized phenotypes, but KG♀×WT♂ reactivated masculinization. (KG♀×WT♂)♀×WT♂ individuals were used in this study. See also Table 1. (B) The molecular structure of KG W chromosome. Genomic PCR analyses were performed by using eight W chromosome-specific RAPD markers [17]. Arrowheads indicate W chromosome-specific amplicons.

Figure 2

Misexpression of testis-specific genes in the KG ovaries. (A) Abnormal splicing of Bmdsx mRNAs in the KG ovaries. The primers that can discriminate female- and male-type Bmdsx were used. (B) Misexpression of testis-specific genes in the KG ovaries. The amounts of indicated mRNAs were measured by qPCR, normalized to rp49, and described as a box plot. Expressions of testis-specific genes in the WT and KG ovaries were enlarged. n = 3 for the WT ovaries, WT testes, and KG testes. n = 8 for the KG ovaries. * p < 0.05, Student's t-test. NS, non significant.

Figure 3

Profiling of piRNAs in the KG ovaries. Heat map showing relative abundances of sense and antisense transposon piRNAs in the KG ovaries of three individuals (WT ovarian piRNAs = 1). Blue indicates relative expression (RE) meets 0.6 ≤ RE ≤ 1.6. Light blue corresponds to RE < 0.6 and red indicates RE > 1.6.

Figure 4

Profiling of piRNAs deriving from the sex-determining region in the KG ovaries. Relative abundances of total transposon piRNAs enriched in the sex-determining region [8]. Error bar indicates ± standard deviation (n = 3).

Figure 5

Overexpression of transposons in the KG ovaries. The amounts of indicated mRNAs were measured by qPCR, normalized to rp49, and described as a box plot. n = 3 for the WT testes and KG testes. n = 6 for the WT ovaries. n = 8 for the KG ovaries. * p < 0.05, one-way ANOVA, Tukey's multiple comparison test. NS, non significant.