Oocytes Exhibit Lipid Accumulation, Endoplasmic Reticulum Stress, Mitochondrial Dysfunction, and Apoptosis in Response to High Fat Diet.

Female obesity is associated with decreased fecundity with increased time-to-pregnancy and decreased chance of pregnancy. In obesity, accumulation of lipid in non-adipose tissues, or lipotoxicity, is associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction and ultimately apoptosis. We have previously shown that obese women have increased triglycerides in follicular fluid; thus, the present study examined whether diet-induced obesity causes lipotoxicity in granulosa cells and the cumulus oocyte complex (COC). COCs and granulosa cells were collected from ovaries of adult mice fed high fat or control diet for 4 weeks. COCs and oocytes were assessed for 1) lipid content by neutral lipid stain BODIPY 493/503; 2) ER homeostasis by measuring expression of ER stress marker genes; 3) Mitochondrial function by measuring membrane potential; and 4) apoptosis by DNA laddering and TUNEL assays. Quantification of lipid levels showed that in immature COCs lipid content was significantly higher in both cumulus cells and oocytes of mice fed high fat diet compared to control. Expressed as total lipid, oocytes from mice fed high fat diet had markedly increased lipid content compared to oocytes of mice fed control diet both before and following ovulation. The presence of ER stress was determined by measuring the expression of ER stress marker genes (ATF4, GRP78, CHOP10 and Hsp70) in COCs isolated from preovulatory follicles of eCG-treated mice. There were significantly higher levels of ATF4 and GRP78 expression in COCs from high fat diet fed mice relative to control. Mitochondrial damage initially manifests as a decrease in the mitochondrial membrane potential (MMP), determined by the ratio of red to green fluorescence using JC-1 probe. In mice fed high fat diet, the mean ratio of red/green fluorescence was significantly reduced in both immature, preovulatory, and ovulated oocytes compared to oocytes from control diet mice, demonstrating that oocytes from high fat diet fed mice have reduced MMP, indicative of mitochondrial damage. The incidence of apoptosis in granulosa cells and COCs of preovulatory follicles was investigated. DNA laddering assay showed that granulosa cells from high fat diet fed mice have greater 180bp and 360bp band intensities compared to cells from control diet fed mice, indicating increased DNA fragmentation. TUNEL assay on ovarian sections confirmed this result by demonstrating more TUNEL-positive cells within the ovarian follicles of mice fed high fat diet compared to control diet. The apoptotic index reached 44.03% in COCs and 31.83% in granulosa cells from high fat diet fed mice compared to 7.6% and 7.65%, respectively, in control diet fed mice. Indicators of lipotoxicity were analyzed in ovarian cells of women of varying BMI in order to determine whether lipotoxicity pathways may be activated in the human ovary in response to obesity. ATF4 expression was significantly increased in granulosa cells from obese women compared to granulosa cells of moderate or overweight women, suggesting that ER stress pathways are activated in granulosa cells of obese women. Thus, lipid accumulation, ER stress, mitochondrial dysfunction and apoptosis are markedly increased in ovarian cells of mice fed HFD. ER stress is also increased in granulosa cells from obese women. These results indicate that lipotoxicity may contribute to the reduced pregnancy rates observed in response to obesity.

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