BmdsxM knockout in Masc-R females fully restores ovary development
Our previous study using the Masc-R strain suggested a potential role of Masc and Bmdsx in the sexual differentiation of the gonads and germ cells [33]. However, it remains unclear whether Masc directly induces spermatogenesis or promotes male differentiation in germ cells indirectly by inducing the expression of BmdsxM; the latter is supported by the fact that the ectopic expression of the Masc mRNA in Masc-R females results in the expression of both BmdsxM and BmdsxF. Therefore, to eliminate the influence of BmdsxM expression, we generated Masc-R females that do not express BmdsxM by crossing the Masc-R strain with a BmdsxM deletion mutant strain established in this study (Additional files 5 and 6). The BmdsxM mutant strain had a 7-bp deletion in the Bmdsx exon 5 that encodes for the male-specific open reading frame (ORF), producing a truncated version of the BmDSXM protein (Additional file 8). qRT-PCR analysis demonstrated that the homozygous mutation in BmdsxM abolished BmdsxM expression in the Masc-R females, while it increased BmdsxF expression by as much as seven-fold compared with Masc-R females heterozygous for the BmdsxM mutation (Fig. 1A and 1B). Morphological analysis of the internal genitalia confirmed that Masc-R females heterozygous for the BmdsxM mutation (genetically the same as the Masc-R/+ females) formed degenerated ovaries (Fig. 1E), consistent with what has been previously reported for Masc-R females [33]. Testis-like tissues were observed at the apical end of ovarioles (Fig. 1H).
In comparison, Masc-R females homozygous for the BmdsxM mutation (thus only expressing BmdsxF even in the presence of the Masc-R gene) had fully developed ovaries as observed in normal females (Fig. 1F and 1G). Additionally, the morphological features of the apical end of ovarioles were similar to those observed in normal females (Fig. 1I and 1J). No testis-like tissues were observed in Masc-R/+, BmdsxMΔ7/ MΔ7 females. These results demonstrated that the Masc-R transgene indirectly promotes the development of male gonads and the germ cells by inducing expression of BmdsxM.
BmdsxM knockout in Masc-R females restores the ability of egg production
We next investigated the fertility of Masc-R females homozygous for the BmdsxM mutation. As reported previously, Masc-R expression in females caused a significant decrease in the number of mature eggs (Fig. 2A, Masc-R/+, BmdsxMΔ7/+) [33]. Interestingly, the homozygous mutation in BmdsxM restored the number of mature eggs produced by Masc-R females (Fig. 2A, Masc-R/+, BmdsxMΔ7/ MΔ7). The hatchability of eggs laid by the Masc-R females homozygous for the BmdsxM mutation was also similar to that of normal females (Fig. 2B). These results demonstrated that BmdsxM knockout fully restored the ability of egg production in Masc-R females.
BmdsxM knockout in Masc-R females induces the expression of genes essential for oogenesis
To get more insight into the gene expression profile in the gonads of Masc-R/+, BmdsxMΔ7/ MΔ7 females, we performed qRT-PCR to quantify expression levels of Bmovo-1 and Bm-nosO, both of which are important for oogenesis in the silkworm [34, 35]. While Masc-R expression in females suppressed the expression of these two genes (Fig. 1C and 3D), the expression levels of Bmovo-1 and Bm-nosO were restored in Masc-R females homozygous for the BmdsxM mutation. These results further supported our previous findings that the egg production ability was restored in Masc-R females homozygous for the BmdsxM mutation (Fig. 2A and 2B).
Combined, these results demonstrated that BmdsxM depletion in Masc-R females restored the ability of egg production by inducing the expression of genes important for oogenesis and egg formation.
BmdsxF depletion in Masc-R females promotes female to male sex reversal in internal and external genitalia
We performed similar analyses using females with forced Masc-R expression and lack of BmdsxF expression, which were generated by crossing the Masc-R strain with a BmdsxF deletion mutant strain that was established in this study (Additional files 5 and 9). This mutant strain had an 85-bp deletion in the female-specific Bmdsx exon (exon 3) and its adjacent intron sequence, resulting in no BmDSX protein production (Additional files 5 and 8). qRT-PCR analysis demonstrated that the homozygous mutation in BmdsxF abolished BmdsxF expression in the Masc-R females. Although Masc-R females expressed BmdsxM despite being female, BmdsxM expression levels were lower compared with Masc-R females heterozygous for the BmdsxF mutation (Fig. 3A and 3B). Importantly, Masc-R females homozygous for the BmdsxF mutation developed internal genitalia that consisted of male-specific accessory glands, seminal vesicles, vas deferens, and ejaculatory duct and whose shape resembled normal male genitalia (Fig. 3F and 3G). In addition, testes similar in morphology with testes from normal males were observed at the apical end of the vas deferens in Masc-R/+, Bmdsx FΔ85/ FΔ85 females (Fig. 3I and 3J). However, unlike normal males, the apical end of the vas deferens in these females was divided into several tubes (Fig. 3G and Additional file 11).
In comparison, Masc-R females heterozygous for the BmdsxF mutation (genetically the same as the Masc-R/+ females) formed degenerated ovaries (Fig. 3E) that were consistent with those previously reported in Masc-R females [33]. Moreover, testis-like tissues were observed at the apical end of ovarioles (Fig. 3H). These results indicated that BmdsxF depletion in Masc-R females drives female to male sex reversal in the internal genitalia.
To extend our findings to other sexually dimorphic traits, we performed a morphological analysis of the external genitalia. Unlike similar previous studies of lepidopteran insects, we prepared cuticle specimens of the external genitalia, which enabled more accurate determination of the morphological changes in cuticle structures. The external genitalia of Masc-R/+ females heterozygous for the BmdsxF mutation had morphological characteristics similar to those of normal females (Fig. 4A–6D). In comparison, the external genitalia of Masc-R females homozygous for the BmdsxF mutation were malformed, with partial development of several male-specific genital organs, such as the uncus, clasper, penis, and 9th tergite, which is unique to males (Fig. 4E and 4F). The shape of the ventral plate was also similar to that of normal males. These results strongly support our previous findings that BmdsxF depletion in Masc-R females promotes maleness.
BmdsxF knockout in Masc-R females enhances spermatogenesis
We next investigated whether the testis-like tissues and the testes found in Masc-R females heterozygous or homozygous for BmdsxF mutation have the ability to produce spermatozoa. In the silkworm, males produce two types of sperm bundles, one of which consists of eupyrene sperm and the other of which is composed of apyrene sperm [36, 37]. The testis-like tissues observed in Masc-R females heterozygous for the BmdsxF mutation contained sperm bundles that resembled apyrene sperm bundles (Fig. 5A and 5B). Similarly, the testes of Masc-R females homozygous for BmdsxF mutation contained apyrene sperm bundles (Fig. 5C). Although the testis-like tissues also contained sperm bundles that represented eupyrene sperm bundles, their size was smaller than that produced by normal male animals, and their shape was abnormal (Fig. 5D, 5E, and 5G). The testes of Masc-R females homozygous for BmdsxF mutation contained sperm bundles that resembled eupyrene sperm bundles, the size and shape of which were similar to those observed in male animals (Fig. 5D, 5F, and 5G). These results demonstrated that BmdsxF depletion, and thus the expression of BmdsxM alone, promoted spermatogenesis, although the genetic sex of germ cells were all ZW females.
BmdsxF knockout in Masc-R females induces the expression of genes involved in spermatogenesis
To evaluate the spermatogenesis observed in Masc-R/+, BmdsxF∆85/F∆85 females, we performed qRT-PCR to quantify the expression of Bombyx orthologs of the Maelstrom (Mael) and always early (aly) genes (designated BmMael and BmAly, respectively), which are important for spermatogenesis and meiotic progression and spermatid differentiation in the silkworm [38, 39]. Although the expression of these two genes in Masc-R females heterozygous for the BmdsxF mutation (genetically the same as Masc-R/+ females) was higher than that in normal females, the levels were still lower than those in normal males (Fig. 3C and 3D). The expression of BmMaelstrom and BmAly in Masc-R females homozygous for the BmdsxF mutation was significantly higher than in Masc-R females heterozygous for the BmdsxF mutation and were comparable to those in normal males (Fig. 3C and 3D). These results support the above findings that BmdsxF depletion in Masc-R females promotes spermatogenesis by increasing the expression of genes important for spermatogenesis (Fig. 4).
Combined, these results demonstrate that BmdsxF depletion in Masc-R females restored the ability to produce eggs by inducing the expression of genes important for spermatogenesis, meiotic progression, and spermatid differentiation.
MASC protein interacts with the lncRNA from the Bmdsx-AS1 gene
Our results revealed that Masc-R promotes the development of male characteristics in genitalia, including the gonads and the germ cells, by inducing the expression of BmdsxM. Masc is required for the male-specific splicing of Bmdsx transcripts, giving rise to BmdsxM [8, 24]. Therefore, we hypothesized that Masc might directly mediate male-specific Bmdsx splicing. To assess the potential interaction between Masc and Bmdsx, we performed RNA immunoprecipitation (RIP) in testis samples using a polyclonal antibody against MASC protein. Western blotting using whole protein extract from testes revealed that our anti-MASC antibody specifically recognized a protein with a molecular weight that was consistent with the putative molecular weight of the MASC protein (64.8 kDa, Fig. 6A, left panel). The same Western blotting with an anti-DSX-DBD antibody detected a single protein band with the putative molecular weight of the BmDSXM (30.0 kDa) and BmDSXF (29.5 kDa) proteins (Fig. 6A, right panel). Immunostaining using the anti-MASC antibody indicated that MASC protein was predominantly localized in cells at the testicular basement membrane (Fig. 6B, 6E and 6I). Similarly, immunostaining analysis using the anti-DSX-DBD antibody revealed that the BmDSX protein was also expressed in cells of the testicular basement membrane (Fig. 6F and 6J) and that it co-localized with MASC (Fig. 6G). These results were confirmed by in situ hybridization (ISH) using Masc or Bmdsx-specific riboprobes (Fig. 6C). Moreover, immunostaining demonstrated the co-localization of MASC and BmDSX in the cell nucleus (Fig. 6K, arrow heads).
The co-localization of MASC with BmDSX in the nucleus of cells found in the testicular basement membrane further supported the possibility that MASC may directly promote male-specific splicing in Bmdsx. To investigate whether MASC interacts with Bmdsx pre-mRNA, we performed RIP using the anti-MASC antibody, followed by qRT-PCR. Contrary to our expectation, we found no significant enrichment of MASC on Bmdsx pre-mRNA (Fig. 6D). Instead, specific binding of MASC on lncRNA from the Bmdsx-AS1 gene, which is a testis-specific factor involved in the male-specific splicing of Bmdsx, was observed with the same analysis (Fig. 6D) [27]. Significant enrichment was not observed in any of the other RNAs examined, some of which were genes reportedly implicated in male-specific Bmdsx splicing. These results suggested that MASC promotes male differentiation in gonads and germ cells by enhancing the expression of BmdsxM and through physical interaction with Bmdsx-AS1 lncRNA.