A primary effect of palmitic acid on mouse oocytes is the disruption of the structure of the endoplasmic reticulum

Abstract

Exposure of mouse oocytes to saturated fatty acids (FAs) such as palmitic acid (PA) has been shown to increase lipid content and cause an endoplasmic reticulum (ER) stress response and changes in the mitochondrial redox state. PA can also disrupt Ca2+ stores in other cell types. The links between these intracellular changes, or whether they are prevented by mono-unsaturated FAs such as oleic acid (OA), is unclear. Here, we have investigated the effects of FAs on mouse oocytes, that are maturated in vitro, using coherent anti-Stokes Raman scattering and two-photon fluorescence microscopy. When oocytes were matured in the presence of PA, there were changes in the aggregation pattern and size of lipid droplets that were mitigated by co-incubation in OA. Maturation in PA alone also caused a distinctive disruption of the ER structure. This effect was prevented by incubation of OA with PA. In contrast, maturation of mouse oocytes in medium containing PA was not associated with any significant change in the redox state of mitochondria or the Ca2+ content of intracellular stores. These data suggest that a primary effect of saturated FAs such as PA on oocytes is to disrupt the structure of the ER and this is not due to an effect on the mitochondria or Ca2+ stores.

Figure 1

Oocyte maturation and survival during IVM in the presence of FAs. Oocytes treated with 200 μM PA (n = 269) showed a significant reduction (P < 0.05) from controls (n = 324) in their ability to mature to the MII stage. Oocytes matured in 200 μM OA (n = 223) or 200 μM PA + 200 μM OA (n = 23) showed no significant reduction or gain in maturation success from controls during IVM (P > 0.2). (A). After 15-h incubation period for IVM, maturation to MII was recognized by the presence of polar body. These cells, together with live immature oocytes (with clear GV), were classed as survived eggs with no significant difference between groups (all P > 0.2) (B). The error bars represent the s.d.

Figure 2

CARS images of LDs in live MII oocytes matured in high-FA environments. Immature GV stage oocytes were incubated overnight in standard M2 medium (A, E, I) supplemented with 200 µM PA (B, F, J), 200 µM OA (C, G, K) or a combination of 200 µM PA and 200 µM OA (D, H, L). MII oocytes were then selected for DIC (A, B, C, D) and CARS imaging (E, F, G, H). CARS images are colour-coded; colour bar shows depth colour-coding from −25 to 25 μm of 101 z-stacks (0 μm being the approximately equatorial plane of the egg or embryo). (I, J, K, L) are representative histograms of the number of LDs making up clusters for the different growth conditions above.

Figure 3

LD contents and aggregation size with FA treatments. (A) Scatter plot of the square root of the mean squared aggregate size against the total number of LDs in ensembles of MII oocytes imaged immediately after overnight incubation in standard M2 medium (red square, n = 12), or supplemented with 200 µM PA (green triangle, n = 10), 200 µM OA (blue circle, n = 8) or combination of 200 µM PA and 200 µM OA (yellow triangle, n = 8). The distribution of each variable in the corresponding ensemble is shown as the average ± s.d. (B) LD volume V in μm³ against total number of LDs. The distribution of each variable in the corresponding ensemble is shown as the average ± s.d. All data represent multiple trials, using two to three mice per trial. (C) The total lipid content of oocytes with the three different treatments calculated as the product of the volume and total number of LDs.

Figure 4

Mitochondrial metabolism of living MII oocytes matured in high-FA environments. An example of traces of mitochondrial FAD autofluorescence signal in MII oocytes subjected to induction of maximum reduction (2 mM cyanide) followed by subsequent maximum oxidation (1 μM FCCP). (B) The average FAD redox indexes of MII oocytes matured with various FA condition. The FAD redox index of each oocyte is the resting fluorescence level before adding drugs normalized by fluorescence levels of the maximum reductions state and maximum oxidation state. Data represent multiple oocytes (n = 50, 34 or 26 for control, PA or OA condition, respectively) using two or three mice trials. Both PA and OA condition showed no significant difference (P > 0.05) from controls. (C) The luminescence of individual oocyte from each condition was measured separately after IVM; luminescence levels are shown as counts per second, and the average ATP level of each condition was calculated from multiple oocytes (n = 20, 24 or 22 for control PA or OA condition, respectively), using two or three mice trials. Both PA and OA condition showed no significant difference (P > 0.5) from controls.

Figure 5

Intracellular Ca²⁺ response of living MII oocytes matured in high-FA environments. (A) After IVM, MII oocytes were injected with a mixture of OGBD and Alexa594 (in 10:1 ratio) for ratio-metric fluorescent measurements of intracellular Ca²⁺ store. The ER Ca²⁺ store was measured by the thapsigargin (10 μM)-induced Ca²⁺ response, and all other intracellular Ca²⁺ store was measured by the ionomycin (5 μM)-induced Ca²⁺ response. (B) The experiment in (A) was carried out on MII oocytes matured with various FA condition. The average fluorescence ratio of the resting state (red bar), thapsigargin response (blue bar) and Ionomycin response (yellow bar) was calculated form multiple oocytes (n = 55, 27 or 24 for control, PA or OA condition, respectively), using two or three mice trials. For the resting state, both PA and OA condition showed no significant difference (P  > 0.1) from controls. There were also no differences for the thapsigargin response (P > 0.5) and the Ionomycin response (P  > 0.1) from controls.

Figure 6

CARS, TPF and DIC images in living MII oocytes matured in different environments. For IVM, immature GV stage oocytes were incubated overnight in standard M2 medium (A, B, C, D) or supplemented with 200 µM PA (E, F, G, H), 200 µM OA (I, J, K, L) or combination of 200 µM PA and 200 µM OA (P, Q, R, S). MII oocytes were then selected and injected with neuro-DiO in a soybean oil droplet. (A, E, I and M) DIC, (C, G, K and O) false-coloured CARS images at wavenumber 2850 cm⁻¹ and (B, F, J and N) TPF xy images accompanied by (D, H, L and P) false-coloured overlays scale bars: 10 μm. The bright circles inside the oocytes are due to the oil drop that was injected to load neuro-DiO into the ER.

Figure 7

Label-free CARS 3D images in living MII oocytes matured with high PA. For IVM, immature GV stage oocytes were incubated overnight in standard M2 medium supplemented with 200 µM PA. MII oocytes were then selected for CARS imaging. (A) Single z step CARS image (xy section) of the oocyte at wavenumber 2850 cm⁻¹. (B) Depth colour-coded images of CARS z-stacks through the same oocytes. (C) The CARS z-stacks of the same oocyte are presented as xz section, CARS was acquired; 0.1 × 0.1 μm xy pixel size; 0.5 μm z-step; 0.01 ms pixel dwell time; ~14 mW (~9 mW) pump (Stokes) power at the sample. Scale bars: 10 μm. Colour bar shows depth colour-coding from −25 to 25 μm of 101 z-stacks (0 μm being the approximately equatorial plane of the oocyte or embryo); the brightness of each colour is the maximum intensity at each corresponding z-plane. Scale bars: 5 μm.

Figure 8

CARS images of living MII oocytes matured in DPA. Immature GV stage oocytes were incubated in standard M2 medium supplemented with 200 µM PA (A, B, C, D) or 200 µM DPA (E, F, G, H) for overnight. MII oocytes were then selected for DIC (A, E) (as in Fig. 3) false-coloured CARS images at wavenumber 2850 cm⁻¹ (C, G) and at wavenumber 2090 cm⁻¹ (B, F), accompanied by (D, H) false-coloured overlays. Maximum CARS intensities are shown in photoelectrons per second. Values in C and G denote the intensity under excitation conditions identical to those in B and F, respectively, that is they are corrected for the varying temporal overlap of pump and Stokes at different wavenumbers. Scale bars: 5 μm.