Lipid homeostasis is essential for oogenesis and embryogenesis in the silkworm, Bombyx mori

Abstract

Reproduction, a fundamental feature of all known life, closely correlates with energy homeostasis. The control of synthesizing and mobilizing lipids are dynamic and well-organized processes to distribute lipid resources across tissues or generations. However, how lipid homeostasis is precisely coordinated during insect reproductive development is poorly understood. Here we describe the relations between energy metabolism and reproduction in the silkworm, Bombyx mori, a lepidopteran model insect, by using CRISPR/Cas9-mediated mutation analysis and comprehensively functional investigation on two major lipid lipases of Brummer (BmBmm) and hormone-sensitive lipase (BmHsl), and the sterol regulatory element binding protein (BmSrebp). BmBmm is a crucial regulator of lipolysis to maintain female fecundity by regulating the triglyceride (TG) storage among the midgut, the fat body, and the ovary. Lipidomics analysis reveals that defective lipolysis of females influences the composition of TG and other membrane lipids in the BmBmm mutant embryos. In contrast, BmHsl mediates embryonic development by controlling sterol metabolism rather than TG metabolism. Transcriptome analysis unveils that BmBmm deficiency significantly improves the expression of lipid synthesis-related genes including BmSrebp in the fat body. Subsequently, we identify BmSrebp as a key regulator of lipid accumulation in oocytes, which promotes oogenesis and cooperates with BmBmm to support the metabolic requirements of oocyte production. In summary, lipid homeostasis plays a vital role in supporting female reproductive success in silkworms.

Keywords: Bombyx mori; Fecundity; Lipid metabolism; Lipidomics.

Fig. 1

Excessive accumulation of lipid droplets in the midgut of BmBmm mutants. A Relative content of TG in the midgut and fat body from WT females (fifth instar, day 3) were determined in five biological replicates (each replicate contains tissue from three animals). B Relative TG levels in the midgut of female and male silkworms from WT and ΔBmBmm (fifth instar, day 3) were determined in five biological replicates (each replicate contains tissue from three animals). Data are normalized to WT. C The H&E staining of midgut, and tissues were taken from WT and ΔBmBmm females (fifth instar, day 3). Scale bar represents 50 μm. D TEM images of enterocytes in the midgut. Tissues were taken from WT and ΔBmBmm females on the 3rd day of the 5th instar. Scale bar represents 5 μm. E Oil red O-stained images of midgut. Samples were obtained from WT and ΔBmBmm females (fifth instar, day 1) and after 72 h of starvation. Scale bar represents 100 μm. Error bars represent means ± SDs. **p < 0.01; ***p < 0.001

Fig. 2

The absence of BmBmm affects lipid droplet size in the fat body and energy mobilization. A Body weight analysis of female and male 5th instar larvae from WT (n = 94) and ΔBmBmm (n = 100) per day. B Relative TG levels in the fat body of female and male silkworms from WT and ΔBmBmm (fifth instar, day 3) were determined in five biological replicates (each replicate contains tissue from three animals). Data are normalized to WT. C The lipid droplets of WT and ΔBmBmm females visualized by BODIPY staining of the fifth-instar larval fat body. Scale bar represents 20 μm. D Quantification of lipid droplet area in adipocytes in panel C. E TEM images of fat body taken from WT and ΔBmBmm females (fifth instar, day 3). Arrows indicate giant lipid droplets. Scale bar represents 5 μm. Quantitative analyses of lipid droplet areas are summarized in F. G Relative mRNA levels of lipid droplet size-regulating genes in the fat body from WT and BmBmm^−/− females were determined by qRT-PCR in three biological repeats (each replicate contains three silkworms). Data are normalized to WT. H Relative expression levels of BmBmm in the midgut and fat body before and after starvation treatment were determined in three biological replicates (each replicate contains three silkworms). I Survival statistics of larvae under starvation stress conditions. Females and males of WT and ΔBmBmm were starved on the 1st day of the fifth instar. Error bars represent means ± SDs. *p < 0.05; **p < 0.01; ***p < 0.001; ns non-significant

Fig. 3

BmBmm deficiency leads to decreased fecundity in females. A Photographs of eggs laid by WT females mated with WT and ΔBmBmm males respectively and ΔBmBmm females mated with WT males. The eggs laid by ΔBmBmm females did not hatch on the 10th day. Scale bar represents 10 mm. B Photographs of ovarioles from WT and ΔBmBmm. Scale bar represents 10 mm. C The length of ovarioles. D The number of eggs laid by females within 24 h was counted (n = 10). Female is represented by F. Male is represented by M. E The hatching rates of eggs were analyzed after 10 days since the female moths laid eggs (n = 10). Female is represented by F. Male is represented by M. F Relative TG levels of silkworm eggs laid by female moths of WT and ΔBmBmm were determined in five biological replicates (each replicate contains tissue from three animals). Data are normalized to WT. G Relative content of TG of testis from WT and ΔBmBmm males on the 7th day of pupal stage were determined in five biological replicates (each replicate contains tissue from three animals). Error bars represent means ± SDs. *p < 0.05; **p < 0.01; ***p < 0.001; ns non-significant

Fig. 4

Comparison of the relative contents of glycerides in WT and ΔBmBmm^m embryos. A The relative content of triglyceride species with an even number of carbon atoms in silkworm eggs was significantly lower in ΔBmBmm^m than in WT (n = 5, Student’s t test, p < 0.05). B The relative content of triglyceride species with an odd number of carbon atoms in silkworm eggs was significantly lower in ΔBmBmm^m than in WT (n = 5, Student’s t test, p < 0.05). C Statistics of triglyceride species whose relative content in ΔBmBmm^m was significantly higher than that in WT (n = 5, Student’s t test, p < 0.05)

Fig. 5

Maternal BmHsl affects embryonic cholesterol homeostasis. A Photographs of eggs laid by WT and BmHsl^−/− females mated with BmHsl^−/− and WT males respectively. A portion of eggs laid by BmHsl^−/− females did not hatch. Scale bar represents 10 mm. B The number of eggs laid by females within 24 h was counted (n = 10). Female is represented by F. Male is represented by M. C The hatching rates of eggs were analyzed after 10 days since the female moths laid eggs (n = 10). Female is represented by F. Male is represented by M. D and E Relative TG levels of silkworm embryos laid by WT and ΔBmHsl females (ΔBmHsl^m) at 4–6 h (D) and 108–120 h (E) post-oviposition (hpo) were determined in five biological replicates (each replicate contains tissue from three animals). Data are normalized to WT. F Relative levels of free cholesterol, cholesteryl esters and total cholesterol of silkworm embryos laid by WT and ΔBmHsl^m females on 5th and 10th day post-oviposition (dpo) were determined in four biological replicates (each replicate contains tissue from three animals). Data are normalized to WT. Error bars represent means ± SDs. *p < 0.05; **p < 0.01; ***p < 0.001; ns non-significant

Fig. 6

The lipogenesis signaling is stimulated in the fat body of ΔBmBmm females. A Volcano plot of differential genes from fat body of WT and ΔBmBmm females. B Top 30 of GO enrichment terms of up-regulated differential genes in fat body of ΔBmBmm females. C List of metabolic genes whose transcript levels were significantly changed in fat body of ΔBmBmm females in RNA-Seq. D Relative mRNA levels of lipid metabolism-related genes in the fat body from WT and BmBmm^−/− females were determined by qRT-PCR in three biological repeats (each replicate contains three silkworms). Data are normalized to WT. Error bars represent means ± SDs. *p < 0.05; **p < 0.01; ns non-significant

Fig. 7

BmSrebp may support female fecundity by regulating lipid synthesis. A Photographs of eggs laid by WT and ΔBmSrebp females mated with WT males respectively. The eggs laid by ΔBmSrebp females can normally hatch. Scale bar represents 10 mm. B The number of eggs laid by females within 24 h was counted (n = 10). Female is represented by F. Male is represented by M. C The hatching rates of embryos were analyzed after 10 days since the female moths laid eggs (n = 10). Female is represented by F. Male is represented by M. D Photographs of ovarioles from WT and ΔBmSrebp. Scale bar represents 10 mm. E Immunofluorescent staining of oocytes in ovariole from WT and ΔBmSrebp females at the wandering stage. Cell nuclei were stained with Hoechst, shown in blue; lipid droplets were stained with BODIPY, shown in green. Scale bar represents 57.9 μm. F Relative mRNA levels of lipid metabolism-related genes in the fat body from WT and ΔBmSrebp females were determined by qRT-PCR in three biological repeats (each replicate contains three silkworms). Data are normalized to WT. G Putative model for the role of BmSrebp and BmBmm in lipid homeostasis and oogenesis. Error bars represent means ± SDs. *p < 0.05; **p < 0.01; ns non-significant