Fatty Acid Binding Protein 3 And Transzonal Projections Are Involved In Lipid Accumulation During In Vitro Maturation Of Bovine Oocytes

Abstract

Oocytes that undergo in vitro maturation (IVM) are metabolically abnormal and accumulate excess lipid content. However, the mechanism of lipid accumulation and the role of cumulus cells in this process are unclear. Recently, it was shown that fatty acid binding proteins (FABPs) performed intra- and extracellular fatty acid transport. We postulated that FABP3 might be responsible for fatty acid transport from cumulus cells to the oocytes via transzonal projections (TZPs) during IVM. Transcript and protein levels of FABP3 were analyzed in both in vivo- and in vitro-matured cumulus-oocyte-complexes and were increased in IVM samples. Further analysis showed increased lipid content in oocytes and cumulus cells in IVM samples compared to in vivo-derived. We therefore speculated that altered traffic of fatty acids via FABP3 during IVM was the mechanism leading to the excess of lipids accumulated within IVM oocytes. Furthermore, we demonstrated an increase in FABP3 levels and lipid content during the first 9 h of IVM, further strengthening the possibility of fatty acid transport via FABP3 and TZPs. Additionally, disruptions of TZPs during IVM decreased lipid accumulation in oocytes. Our results shed light on a possible mechanism involving FABP3 and TZPs that causes excess lipid accumulation in oocytes during IVM.

Figure 1

Quantification of lipid content in cumulus cells and oocytes derived from immature, in vivo-matured and in vitro-matured cumulus-oocyte complexes (COCs). (A) Lipid content in cumulus cells was determined by western blot analysis of perilipin 2 (PLIN2) using histone 3 (H3) as a normalizer. (B) Lipid quantification in oocytes was performed by fluorescence confocal microscopy of lipid droplets. The values for lipid content represent the ratio of area of lipid droplets to total oocyte area. Representative immature, in vivo-matured and in vitro-matured oocytes stained with BODIPY 493/503 are shown in Supplementary Fig. S1. (C) Relative amounts of fatty acid binding protein 3 (FABP3) transcripts in immature, in vivo-, and in vitro- matured cumulus cells and oocytes. (D) FABP3 protein levels in cumulus cells normalized by x-Tubulin (TUBA). Lowercase letters above bars in the same graph indicate significant differences (P  < 0.05). Values are presented as mean ± standard error of the mean. The full western blot images in (A) and (D) are shown in Supplementary Fig. S2a and S2b, respectively. *The order of the groups is different between the graph and the representative immunoblot images.

Figure 2

Confocal microscopic analysis demonstrating the presence of fatty acid binding protein 3 (FABP3) within the transzonal projections (TZPs) of immature cumulus-oocyte-complexes (COCs). FABP3 was immunostained using 488 Alexa Fluor (green); TZPs (actin) were labeled with Alexa Fluor 647 phalloidin (red) and nuclei were stained with DAPI (blue). (A) Photomicrographs of immature COCs were obtained in 63 x objective and (B) in 63 x objective zoomed 3.5 x. (C) Digital zoom showing FABP3 (arrow) within TZPs (asterisk) in zona pellucida (OO = oocyte; CC = cumulus cells). A total of 12 immature COCs were analysed and showed similar pattern; 6 additional COCs are shown in Supplementary Fig. S3.

Figure 3

Confocal photomicrographs of fatty acid binding protein 3 (FABP3) immunodetection in transzonal projections (TZPs) after 9 h of in vitro maturation (IVM). FABP3 is immunostained using 488 Alexa Fluor (green); TZPs (actin) are stained with Alexa Fluor 647 phalloidin (red); nuclei are stained with DAPI (blue). (A) Photomicrographs of COCs after 9 h of IVM were obtained in 63x objective and (B) in 63x objective zoomed 3.5x. (C) Digital zoom showing FABP3 (arrow) along TZPs (asterisk) in the zona pellucida (OO = oocyte; CC = cumulus cells). A total of 12 COCs in vitro-matured for 9 hours were analysed and showed similar pattern; 6 additional COCs are shown in Supplementary Fig. S4.

Figure 4

Confocal microscopic analysis of fatty acid binding protein 3 (FABP3) in tranzonal projections (TZPs) of denuded and partially denuded oocytes cultured in the maturation media for 9 h. (A) Images acquired from denuded oocytes show no FABP3 and TZPs within the zona pellucida. (B) The image of a portion of the denuded oocytes demonstrating the complete absence of TZPs and FABP3 within the zona pellucida. (C) Images acquired from a partially denuded oocyte demonstrating the presence of FABP3 at the same location as TZPs within the zona pellucida. (D) The portion of the partially denuded COCs demonstrating the complete absence of TZPs and FABP3 within the zona pellucida in the denuded area, and the presence of FABP3 and TZPs in the cumulus-enclosed area. FABP3 (arrow) is immunostained using 488 Alexa Fluor (green); TZPs (actin; asterisk) are stained with Alexa Fluor 647 phalloidin (red); nuclei are stained with DAPI (blue). Photomicrographs were obtained in 63x objective (A and C), and merged with z-stack captures with digital zoom (B and D) to illustrate the absence (B) or presence (D) of FABP3 along the TZPs. (OO = oocyte; CC = cumulus cells). ** indicates the lack of cumulus cells and TZPs. A total of 10 denuded oocytes and 11 partially denuded oocytes in vitro-matured for 9 hours were analysed and showed similar pattern; 6 additional denuded oocytes and 6 additional partially denuded oocytes are shown in Supplementary Figs S5 and S6, respectively.

Figure 5

Confocal microscopic analysis of cumulus-oocytes complexes (COCs) after 18 h of IVM. (A) Confocal photomicrographs of immunodetection of fatty acid binding protein 3 (FABP3) in tranzonal projections (TZPs) in 18-h matured COCs. (B) Photomicrograph showing a detailed view of the COC using a 63x objective and zoomed 3.5x. (C) The digital zoom showing the details of the terminal portions of the TZPs disconnected from the ooplasm; FABP3 are immunolocalized at the terminal portion of the TZPs. FABP3 is immunostained using 488 Alexa Fluor (green); TZPs (actin) are stained with Alexa Fluor 647 phalloidin (red); nuclei are stained with DAPI (blue). Photomicrographs were obtained in 63 x objective (A) and in 63 x objective zoomed 3.5 x (B). Digital zoom (C) showing a few FABP3 molecules (arrow) in the terminal portion of TZPs (asterisk) and the perivetellin space (PVS) between oocyte (OO) and zona pellucida. (CC = cumulus cells; PB = polar body). A total of 15 COCs in vitro-matured for 18 hours were analysed and showed similar pattern; 6 additional COCs are shown in Supplementary Fig. S7.

Figure 6

Confocal microscopic analysis of cumulus-oocytes complexes (COCs) at different stages of oocyte maturation (merge of z-stack images) in 63 x objective with digital zoom. (A) Photomicrograph showing the localization of fatty acid binding protein 3 (FABP3) and tranzonal projections (TZPs) in immature COCs. (B) Photomicrograph showing the localization of FABP3 and TZPs in COCs after 9 h of maturation. (C) Photomicrograph showing the localization of FABP3 and TZPs in COCs after 18 h of maturation. Arrows indicate FABP3 immunolocalized in TZPs. FABP3 is immunostained using 488 Alexa Fluor (green); TZPs (actin) are stained with Alexa Fluor 647 phalloidin (red); nuclei are stained with DAPI (blue).

Figure 7

Fatty acid binding protein 3 (FABP3) protein levels and lipid content in oocytes from immature cumulus-oocytes complexes (COCs) and COCs in vitro-matured for 9 and 18 h. (A) FABP3 protein quantification in oocytes was performed by western blot analysis using x-Tubulin (TUBA) as the normalizer. The full western blot image is shown in Supplementary Fig. S2c. (B) Lipid quantification in oocytes. The values for lipid content represent the ratio of the area of lipid droplets to the total oocyte area. Letters above bars in the same graph indicate significant difference (P < 0.05). Values are represented as mean ± standard error of the mean.

Figure 8

Confocal microscopic analysis of fatty acid binding protein 3 (FABP3) in cumulus-oocytes complexes (COCs) after in vitro maturation (IVM) in the absence (control) and presence of cytochalasin B. (A) and (C) The photomicrographs showing the presence of tranzonal projections (TZPs) and FABP3 in the control group. (B) and (D) Photomicrographs showing the lack of most parts of TZPs and FABP3 within the zona pellucida. FABP3 was immunostained using 488 Alexa Fluor (green); TZPs (actin) are stained with Alexa Fluor 647 phalloidin (red); nuclei are stained with DAPI (blue). Photomicrographs were obtained in 63x objective (A and C); and in 63 x objective zoomed 3.5 x (B and D). CC = cumulus cells; ZP = zona pellucida; OO = oocyte. **Indicate the disassembly and blocking of TZP formation by cytochalasin B. A total of 10 control COCs and 10 cytochalasin treated COCs in vitro-matured for 9 hours were analysed and showed similar pattern; 6 additional control COCs and 6 additional cytochalasin B treated COCs are shown in Supplementary Fig. S10. (E) Lipid content in oocytes from COCs after IVM for 9 h in the absence (control) or presence of cytochalasin B. Lipid quantification was performed by fluorescence confocal microscopy to detect lipid droplets. Representative control and cytochalasin B-treated oocytes stained with BODIPY 493/503 are shown in Supplementary Fig. S11. The values for lipid content represent the ratio of the area of lipid droplets to the total oocyte area. Letters above bars indicate significant differences (P  < 0.05). Values are represented as mean ± standard error of the mean.