Proteomic changes in oocytes after in vitro maturation in lipotoxic conditions are different from those in cumulus cells

Abstract

Maternal lipolytic metabolic disorders result in a lipotoxic microenvironment in the ovarian follicular fluid (FF) which deteriorates oocyte quality. Although cellular stress response mechanisms are well defined in somatic cells, they remain largely unexplored in oocytes, which have distinct organelle structure and nuclear transcription patterns. Here we used shotgun proteomic analyses to study cellular responses of bovine oocytes and cumulus cells (CCs) after in vitro maturation under lipotoxic conditions; in the presence of pathophysiological palmitic acid (PA) concentration as a model. Differentially regulated proteins (DRPs) were mainly localized in the endoplasmic reticulum, mitochondria and nuclei of CCs and oocytes, however the DRPs and their direction of change were cell-type specific. Proteomic changes in PA-exposed CCs were predominantly pro-apoptotic unfolded protein responses (UPRs), mitochondrial and metabolic dysfunctions, and apoptotic pathways. This was also functionally confirmed. Interestingly, although the oocytes were enclosed by CCs during PA exposure, elevated cellular stress levels were also evident. However, pro-survival UPRs, redox regulatory and compensatory metabolic mechanisms were prominent despite evidence of mitochondrial dysfunction, oxidative stress, and reduced subsequent embryo development. The data provides a unique insight that enriches the understanding of the cellular stress responses in metabolically-compromised oocytes and forms a fundamental base to identify new targets for fertility treatments as discussed within.

Non-esterified fatty acids in the ovary: friends or foes?

“…A growing number of publications report that SFAs have detrimental effects on oocyte quality. A recent analysis in bovine oocytes revealed that blastocysts derived after PA treatment (150 μM) were inferior in quality and had a high proportion of apoptotic cells in the inner cell mass [75]. This observation is in line with an earlier report showing that exposing bovine oocytes to high concentrations of SA resulted in a significant reduction of the number of oocytes that reach the blastocyst stage with high expression of the maternally imprinted gene IGF2R [64] (Fig.…”

Differential effects of high fat diet-induced obesity on oocyte mitochondrial functions in inbred and outbred mice

“…Perhaps, these responses are not fully functional in mature oocytes at the transcriptional level. We have previously shown that exposure of bovine COCs to lipotoxic concentrations during IVM resulted in several UPR-related proteomic changes 24 , which were persistent until the blastocyst stage 36 . Interestingly, we observed that BiP and ATF4, as well as SOD2 and CAT were significantly higher in B6 control oocytes compared to the Swiss controls, and were equally high in the B6 HFD group.…”

“…In contrast, high MMP can also be detrimental, and was linked with increased embryonic arrest at the 2-cell stage in mice and with high fragmentation rate in 8-cell embryos in humans 35 . Interestingly, the increase in MMP at the final stage of IVM in bovine oocytes due to lipotoxicity and oxidative stress has been shown to be temporary, and was followed by a subsequent reduction in MMP (uncoupling) during early cleavage stages 24,36 . Uncoupling occurs particularly in mitochondria that suffer most from high ROS accumulation, which may result in a heterogeneous population of hyperactive and inactive mitochondria within the same oocyte.…”

“…Mitochondrial inner membrane potential (MMP) and ROS level in oocytes were estimated using a combined JC-1 (Invitrogen) and CellROX Deep Red (ThermoFisher) staining, respectively, followed by confocal microscopy 24 (see Appendix A.2 for the detailed staining method).…”

“…In addition, mitochondria also act as biological sensors of cellular stress and regulate unfolded protein responses (UPR) and signaling cascades to promote cell survival or initiate apoptosis, similar to those regulated by the endoplasmic reticulum (ER) 23 . Recent data suggest that mitochondrial dysfunction may affect UPR signaling in oocytes and embryos following short term in vitro exposure to lipotoxic conditions 24 . However, there is no data available about the activation of these mechanisms in oocytes in obese individuals following long-term exposure to oxidative stress or lipotoxicity.…”