212 Mare aging affects metabolomic profile of oocytes and follicular cells at different maturation stages

Oocyte quality and female fertility decline with advanced maternal age. The oocyte develops and matures in the ovarian follicular environment and relies on follicular cells, specifically cumulus and granulosa cells, to acquire developmental competence. Herein, we utilised a nontargeted metabolomic approach to identify and quantify metabolic disturbances promoted by mare advanced maternal age on the oocyte and somatic follicular cells during three maturation stages. Follicular samples (including oocytes, cumulus cells, and granulosa cells) were collected by transvaginal, ultrasound-guided aspiration of dominant preovulatory follicles (≥35 mm, uterine oedema) from young mares (10 ± 0.6 years, n = 8) and old mares (23 ± 0.8 years, n = 12), housed in dry lots and fed grass/alfalfa hay, at three stages of follicle/oocyte development: 0 h (no induction of follicular maturation, oocyte presumptively at germinal vesicle stage), 24 h (~24 h after follicle maturation induction with hCG and deslorelin acetate, anticipated metaphase I stage of oocyte development), and 42 h (follicle collection at 24 h from follicle maturation induction plus 18 h of culture, with the oocyte confirmed at metaphase II). Single samples were collected from most mares, with a few old mares providing two samples. Relative abundance of metabolites in the samples were determined by gas chromatography- and liquid chromatography-mass spectrometry and compared between the age groups for each cell type at each time point by Student’s t-tests with Bonferroni adjustment factor. From a total of 453 identified metabolites, 194 were annotated. In oocytes, 21 annotated metabolites were affected by mare age at 0 h (young and old, respectively, n = 8 and 10), 10 at 24 h (n = 8 and 14), and 14 at 42 h (n = 8 and 9). In cumulus cells, 10 annotated metabolites differed between age groups at 0 h (n = 8 and 8), 22 annotated metabolites at 24 h (n = 8 and 14), and 8 at 42 h (n = 8 and 10). In granulosa cells, 23 annotated metabolites were affected by mare age at 0 h (n = 8 and 11), 56 at 24 h (n = 8 and 15), and 11 at 42 h (n = 6 and 9). Among the main metabolites affected by advanced mare age, glutamic acid, putrescine, and triacylglycerols were consistently higher, whereas ceramides and alanine were mostly lower in relative abundance in oocytes, cumulus cells, and granulosa cells from old than young mares. Monosaccharides were more abundant in cumulus cells and less abundant in oocytes from old compared with young mares at 24 h, and a similar trend was observed for free fatty acids at 42 h. Overall, many of our age-related findings point toward impaired mitochondrial metabolic function and the associated oxidative stress in oocytes and follicular cells from old mares. Some of our findings also suggest limited anaerobic energy metabolism and potential impaired transfer of carbohydrate and free fatty acid substrates from cumulus cells to the oocyte in old mares. The identification of these metabolic differences and possible related pathways may contribute to development of therapeutic interventions targeting age-induced follicular cell dysfunction.

The study was funded by the Cecil and Irene Hylton Foundation and Abney Scholarship.