Are there significant differences in carbohydrate metabolism trends between in vivo and in vitro grown mouse antral follicles during oocyte final maturation?
Glucose metabolism characterization during GV to MII transition revealed altered metabolic patterns mainly in cumulus cells of in vitro grown and matured mouse antral follicles.
What is known already
For some cancer patients fertility restoration is dependent on using efficient in vitro follicle culture systems. As human donor ovarian tissue available for research is limited, establishing such culture systems relies on data generated from animal models. The culture system previously developed in our laboratory supports in vitro growth of mouse preantral follicles with good oocyte maturation rates but lower developmental competence compared to in vivo grown oocytes. Tracking and comparing the metabolic changes after meiotic maturation in in vitro and in vivo follicles could serve as a screening tool for improving culture conditions and identifying metabolic quality markers.
Study design, size, duration
Mouse secondary follicle culture was performed. In vitro grown oocytes, their corresponding cumulus (CC) and granulosa cells (GC) were collected from antral follicles, at germinal vesicle stage (GV) on day 9, and at metaphase 2 (MII) on day 10, after hCG/EGF stimulation. In vivo age-matched controls were obtained after intraperitoneal injections with eCG for GV, or with eCG and hCG for MII. In vivo GC after ovulation were not included.
Participants/materials, setting, methods
Glucose metabolism trends were compared during final maturation between in vitro grown antral follicles and their in vivo controls. Follicles that failed to resume meiosis in vitro were also included.
Enzymatic spectrophotometric assays were used to measure glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and the antioxidant capacity in individual cell types. Pools of 5 oocytes and corresponding somatic cells were collected, from 3 independent experiments. Unpaired t-test was performed with significance when p < 0.05.
Main results and the role of chance
Important differences were detected between in vivo and in vitro conditions. GV to MII transition in in vivo follicles leads to a metabolic boost in CC as indicated by: i. significant increase in glycolysis, PPP and TCA cycle activity; ii. higher total antioxidant capacity (TAC) (p < 0.05) and small molecule antioxidant capacity (SMAC) (p < 0.01). After ovulation, the only significant change in oocytes was an increase in nicotinamide adenine dinucleotide phosphate (NADP+) level (p < 0.01), possibly due to increased reduced-NADP recycling.
Meiotic maturation triggered no significant differences in any of the metabolic pathways for in vitro grown oocytes. Contrary to their in vivo controls, in vitro CC showed significant upregulations limited to aconitase, lactate dehydrogenase (LDH) and glutathione-s-transferase (GST) activity (p < 0.05). In vitro GC showed increased G6PDH activity (p < 0.05), suggesting PPP upregulation.
Significant differences were detected between in vivo GV follicles and the in vitro failed-to-mature ones. Oocytes from impaired follicles have higher NADP+ levels (p < 0.0001) than their in vivo immature counterparts. CC showed higher phosphofructokinase (PFK), LDH, catalase activity and increased NADP + (p < 0.01), TAC and SMAC (p < 0.05) compared to in vivo GV CCs. GCs from failed-to-mature follicles have significantly higher LDH and superoxide dismutase (SOD) activity than in vivo GV GC (p < 0.05).
Limitations, reasons for caution
The altered metabolic patterns described here in in vitro follicles during oocyte GV to MII transition are probably the cumulative effects of both growth and maturation in vitro.
Wider implications of the findings: We explored extensively and directly, for the first time, several enzymes and metabolites involved in follicle glucose and redox metabolism in different cell types separately. Understanding of the follicle metabolic requirements is essential for the optimization of follicle culture systems and could lead to development of oocyte quality markers.