Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging

Abstract

Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm(-1)) and lipid (~2845 cm(-1)) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans.

Figure 1. The accumulation of secreted yolk lipoprotein in the pseudocoelom of N2, fat-1, fat-2, and rme-2 mutants.

(a) CARS Images of N2, fat-1, fat-2, and rme-2 mutants containing yolk lipoprotein accumulation measured at three different vibrational frequencies. Red arrows and white rectangles represent the regions of yolk lipoprotein accumulation and surrounding buffer, respectively. E stands for embryo, I stands for intestine, and V stands for vulva. Scale bar = 30 μm. (b) The average CARS intensity ratio between yolk lipoprotein accumulation (red arrows in (a)) and surrounding buffer (white rectangles in (a)) at three vibrational frequencies. Black bar for ~1665 cm⁻¹ (protein band), white bar for ~2200 cm⁻¹ (non-resonant background), and gray bar for ~2845 cm⁻¹ (lipid band) (n = 10~15). All worms are 1D-Ad worms. Error bars represent the standard error of the mean (SEM). (P-value: *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 2. Analysis of yolk lipoprotein accumulation in the pseudocoelomic cavity of one-day-adult (1D-Ad) wild-type (N2) worms.

(a) The CARS image of 1D-Ad N2 worms containing large (upper image) and small (lower image) yolk lipoprotein accumulation (red arrows) in their pseudocoelomic cavities. The white rectangles are selected for calculating the average CARS intensity of surrounding buffer. Yellow region stands for the skin-like hypodermal cells, E stands for embryo, I stands for intestine, and V stands for vulva. Scale bar = 30 μm. (b) The CARS spectra of yolk lipoprotein accumulation obtained from pseudocoelomic cavity and the lipid droplet (LD) obtained from the skin-like hypodermal cell in 1D-Ad N2 worms. (c) The average CARS intensity ratios between LDs/surrounding buffer and between (large and small) yolk lipoprotein accumulation/surrounding buffer. A threshold of 1.47–3.12 (in red color) was applied to the CARS image to enhance the boundary of yolk lipoprotein accumulation. Error bars represent the standard deviation. (P-value: *p < 0.05, **p < 0.01, ***p < 0.001) (d) After excluding the internal organs/tissues (such as intestine, oocyte/embryo, and body wall) and applying the threshold, the pixels with intensity ratio between 1.47–3.12 are converted into red color, and pixels with intensity ratio <1.47 and >3.12 are converted into white color. The boundary of yolk lipoprotein accumulation can be easily distinguished (indicated by red arrows).

Figure 3. Analysis of yolk lipoprotein accumulation in PUFA-deficient mutants.

(a) The CARS images of N2, PUFA-deficient mutants (fat-1, fat-2, fat-3, and fat-4), and fat-2 worms supplemented with PUFAs. Red arrows indicate the regions of yolk lipoprotein accumulation. E stands for embryo, I stands for intestine, and V stands for vulva. Scale bar = 30 μm. (b) The total area and (c) the average CARS intensity of yolk lipoprotein accumulations in the pseudocoelomic cavity of uterus in different worms. (n = 57 for N2, and n = 19~32 for mutants and PUFA-supplemented fat-2 worms) All worms are 1D-Ad worms. Error bars represent the SEM. (d–k) The area contribution curves of yolk lipoprotein accumulations in various strains. (P-value: *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant).

Figure 4. Quantitative analysis of lipid content in oocytes.

(a) CARS and two-photon excitation fluorescence images of the oocytes of a wild-type worm with vit-2p::vit-2::gfp transgene expression. n: nucleus. (b) The correlation between Δ integrated GFP signal and Δ integrated CARS signal of each oocyte. The red line represents the linear regression (R² > 0.99). Each data point represents the average ± SEM (n = 17). (c) The CARS images of the oocytes and (d) The normalized amount of lipid stored in the oocytes of N2, fat-1, fat-3, fat-4, fat-2, and fat-2 worms supplemented with 18:1,n9, 18:2,n6, 18:3,n6, and 18:3,n3 (n = 9~11). All are 1D-Ad worms. Error bars represent the SEM. Scale bar = 30 μm. (P-value: *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant).

Figure 5. Analyses of oocyte growth, ovulation rate, and egg number of N2 worms and PUFA-deficient mutants.

(a) The cellular size of the oocytes (n = 9~11), (b) ovulation rate (n = 20~32), and (c) egg number (n = 10~13) of N2, fat-1, fat-3, fat-4, fat-2, and fat-2 worms supplemented with 18:1,n9, 18:2,n6, 18:3,n6, and 18:3,n3. (d) The normalized rate of lipid delivery into oocytes in N2, fat-1, fat-3, fat-4, fat-2, and fat-2 worms supplemented with 18:1,n9, 18:2,n6, 18:3,n6, and 18:3,n3. The data are normalized by wild-type (N2). All worms are 1D-Ad worms. Error bars represent SEM. (P-value: *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant).

Figure 6. The correlation between various quantitative results and egg number.

The scatter plots of (a) lipid delivery rate, (b) ovulation rate, (c) lipid content, (d) oocyte size, and (e) total area of yolk lipoprotein accumulation versus egg number, together with the values of the Pearson’s product-moment correlation coefficient (r). The analyses were conducted using R.