Altered expression of testis-specific genes, piRNAs, and transposons in the silkworm ovary masculinized by a W chromosome mutation
© Hara et al; licensee BioMed Central Ltd. 2012
Received: 10 January 2012
Accepted: 28 March 2012
Published: 28 March 2012
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.
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.
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.
In the silkworm, Bombyx mori, females are heterogametic (ZW) whereas males are homogametic (ZZ) [1, 2]. Genetic studies have shown that at least one copy of the W chromosome is sufficient for determining femaleness, irrespective of Z chromosome copy number, suggesting that the W chromosome is a strong female-determinant in the silkworm [3, 4]. Therefore, it was presumed that the W chromosome likely encodes a female-determining gene(s) so-called Fem. However, not even a single protein-coding gene has been identified from the W chromosome. Instead, it was found that the silkworm W chromosome contains numerous transposable elements, their remnants, and simple repeats [1, 5–7]. These make it difficult to obtain a long contig sequence of the W chromosome, and thus the complete sequence of the W chromosome remains to be resolved.
Recently, we uncovered an intriguing facet of the silkworm W chromosome-the W chromosome is a source of female-enriched PIWI-interacting RNAs (piRNAs) . PIWI proteins and PIWI-interacting RNAs are at the heart of transposon silencing system in animal gonads [9–11]. piRNAs are 23-30 nucleotide-long small RNAs that can act as sequence-specific guides for PIWI proteins. Mutations in piRNA pathway proteins lead to de-silencing of transposons and result in severe developmental defects in germ line cells [9–11]. With the aid of piRNA deep-sequencing and careful analyses, we identified a number of transposons and associated piRNAs originating from the sex-determining region of the W chromosome . The role of female-enriched piRNAs remains enigmatic.
Doublesex (dsx) gene is an evolutionarily conserved transcription factor that mediates the somatic sex determination pathway [12, 13]. B. mori dsx (Bmdsx) is alternatively spliced in a sex-dependent manner, yielding male-specific BmDSX or female-specific BmDSX (BmDSXM and BmDSXF, respectively). BmDSXF is a functional switch at the bottom of sex determination pathway in the silkworm [12, 13]. B. mori P-element somatic inhibitor (BmPSI) and IGF-II mRNA binding protein (BmIMP) contribute to sex-specific splicing of Bmdsx mRNAs [14, 15].
The KG line is a mutant line that shows various degrees of female masculinization features, such as formation of chitin-like structures in the external genitalia . It has been genetically shown that masculinization is caused by a mutation in the KG W chromosome. One of the most prominent phenotypes in masculinized females is the aberrant splicing of Bmdsx mRNAs. Masculinized female tissues, including ovarian tissues, express both female- and male-type Bmdsx mRNAs, indicating that the mutation in the KG W chromosome interferes with the somatic sex determination pathway. Although this remarkable phenotype is unique to the silkworm KG line the molecular nature of KG W chromosome is largely unknown. Here, we investigated expression profiles of testis-specific genes, piRNAs, and transposons in the KG ovaries. Our results revealed that the mutation in the KG W chromosome caused misexpression of testis-specific genes, reduction of a set of piRNAs, and overexpression of transposons in the KG ovaries.
Results and discussion
Genetic analyses of the KG line
Genetic analysis of the KG line
G0♀ × G0♂
G1♀ × G1♂
G2♀ × G2♂
G3♀ × G3♂
G3♀ × WT♂
WT♀ × (G3♀ × WT♂)♂
Our results suggested that the mutation in the KG W chromosome became suppressed during sib-crosses. We envision that masculinized phenotypes depend on interaction between the mutated W chromosome and normal autosomes/Z chromosomes. For example, a masculinizing factor is located on autosomes/Z chromosomes while the W chromosome harbors an anti-masculinization factor, which might be mutated in the KG W chromosome. Thus, the results of sib-cross experiments can be explained by incompatibility between a masculinizing and anti-masculinization factors of the WT and the original KG line, respectively. Natural selection against the severe KG phenotypes may attribute to the diminishing masculinization as well.
Several W chromosome-specific RAPD makers are available for the silkworm W chromosome [17–19]. We utilized these markers to understand the structure of KG W chromosome. As shown in Figure 1B, when compared to the WT W chromosome, the KG W chromosome retained 8 RAPD markers surveyed, indicating that the KG W chromosome does not have a large deletion, unlike is observed in the W chromosome of sex-limited yellow-cocoon strain [8, 17].
KG ovaries misexpressed a number of testis-specific genes
To understand the molecular nature of the masculinized phenotypes observed in the KG line, we focussed on the masculinized ovaries. To obtain females showing severe masculinization, we used individuals generated by (KG × WT) female × WT male. For simplicity, these are described as the KG females/males or the KG line in this manuscript (Figure 1A).
To determine if the mutated KG W chromosome affects expression of testis-specific genes, we examined the abundance of several testis-specific mRNAs via quantitative PCR (qPCR). For this analysis, we focused on previously reported testis-specific genes . Our results demonstrated that, when compared to the WT ovaries, several testis-specific genes were overexpressed in the KG ovaries (Figure 2B). The expression levels of these genes in the KG ovaries were still less abundant than those in the KG testes. The expression levels were quite similar between WT and KG testes. These data indicated that the mutation in the KG W chromosome disrupted sex-specific transcriptome in the KG ovaries. At this point, we cannot determine if misexpressions of these testis-specific genes are simply dependent on heterogeneous Bmdsx expression in the KG ovaries.
Reduction of female-enriched piRNAs in the KG ovaries
We previously reported that the silkworm W chromosome is a source of female-enriched piRNAs . With the aid of our guidelines, we can deduce genomic origins of transposons based on piRNA expression data-piRNAs derived from the W chromosome are expressed more abundantly in the ovary than in the testis. Thus, piRNA expression data from the KG ovaries are likely to be useful to understand the molecular nature of KG W chromosome. To this end, we constructed, sequenced, and analyzed piRNA libraries from KG ovaries of the three individuals generated by (KG × WT) female × WT male. Cloned reads were mapped to the silkworm genome to infer a sequencing depth for normalization. We focused on piRNAs mapped to 121 well-annotated transposons.
1U and antisense bias of piRNAs deriving from the KG ovaries
1U bias (%)
Overexpression of transposons in the KG ovaries
Here we thoroughly analyzed an intriguing silkworm mutant called the KG line, whose mutated W chromosome causes severe female masculinization. Our genetic analyses showed that masculinization became suppressed during the course of sib-crosses over generations, but became reactivated when crossing a KG female with a WT male. These data indicated an interaction between the W chromosome and autosomes/Z chromosomes regarding masculinized phenotypes. Interestingly, the KG female ovaries misexpressed testis-specific genes. Expression levels of these testis-specific genes in the KG ovaries were higher than in the WT ovaries but less than in the WT testes. Comprehensive piRNA profiling revealed that a set of female-enriched piRNAs including those deriving from the sex-determining regions was reduced in the KG ovaries. Accordingly, we detected transposon de-silencing in the KG ovaries. Our current study suggests a possible link between the sex differentiation pathway and the piRNA pathway in the silkworm.
Bombyx mori strains
Wild type (WT) strain p50T (WT) and the KG line were reared on fresh mulberry leaves in an insect-rearing chamber under short-day conditions (12 L: 12D). Every batch was divided into two groups: one was for collecting ovaries and testes at the pupal stage and another was for confirming masculinized phenotypes at the adult stage.
Genomic PCR with several W chromosome-specific RAPD markers was performed as described previously .
piRNA library construction
Total RNA was prepared using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer's protocol. The total RNA (10 μg) was loaded onto a 15% denaturing polyacrylamide gel containing 8 M urea, electrophoresed, and then stained with SYBRGold (Invitrogen). Signals were visualized using LAS-1000 film (Fujifilm, Tokyo, Japan). As silkworm piRNAs are visible as a distinct band by SYBRGold staining, we could easily gel-purify piRNA-containing fraction. Small RNA libraries were constructed using a small RNA cloning kit (Takara, Kyoto, Japan). DNA sequencing was performed using the Solexa genetic analysis system (Illumina, CA, USA). One nanogram of the prepared cDNA was used for the sequencing reactions with the Illumina GA. 10,000-15,000 clusters were generated per "tile", and 36 cycles of the sequencing reactions were performed. The protocols of the cluster generation and sequence reactions were according to the manufacturer's instructions.
Solexa sequencing generated reads of up to 36 nucleotides in length. The 3' adaptor sequences were identified and removed, allowing for up to two mismatches. Reads without adaptor sequences were discarded. Reads shorter than 23 nucleotides or longer than 30 nucleotides were excluded, resulting in reads of 23-30 nucleotides. Alignment to the B. mori genome , 121 annotated transposons, and 1668 ReAS clones  were performed with SOAP2 (ver. 2.20) allowing no mismatch . The total number of perfect genome-mapping reads reflects the sequencing depth. To compare the reads among different data sets, reads were expressed in reads per million (RPM) by normalizing to the total number of perfect genome-mapping in each library. Raw excel data for transposon piRNA profiles in the KG ovaries will be provided upon request.
Quantitative PCR (qPCR)
Primers used in this study
Seq. (5' to 3')
Male specific sperm protein-RT-Fw
Male specific sperm protein-RT-Re
piRNAs sequenced in this study are deposited as DRA000275 in the DNA database of Japan (DDBJ).
randomly amplified polymorphic DNA.
We thank P. B. Kwak, A. Tsutsumi, and Y. Tomari for their critical comments on the manuscript, and M. Kawamoto for the technical assistance. Sh.K. is a recipient of fellowship from the Japan Society for the Promotion of Science. This work was supported in part by the Grants-in-Aid for Scientific Research (No. 22115502 to Su.K., and No. 17018007 to T.S.), the National Bio-Resource Project "Silkworm", and the Professional Program for Agricultural Bioinformatics from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
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