In vitro growth (IVG) and maturation (IVM) of oocytes have potential applications in folliculogenesis/oogenesis research and oncofertility. However, the normality and developmental competence of IVG oocytes, and resultant embryos is unknown. A previously validated, multi-phase, serum-free IVG system was optimised to support the development of sheep follicles as a model for human IVG.
Cortex mass and metabolism were quantified to support early preantral follicle growth over 30 days in situ within ovarian cortex, which was assessed histologically. In addition, 115 IVG preantral follicles (217.92±8.40µm) were dissected from cultured stroma (15 replicates) and 1.2% developed antral cavities post-dissection.
In vivo-derived preantral follicles (292.73±1.18µm, n=1,514) were isolated and grown to antral stage (739.87±4.97µm, n=234) for ≤27 days (21 replicates). IVG antral follicles underwent steroidogenic differentiation (72 hrs), meiotic maturation (24 hrs), followed by IVF insemination. Mean IVG oocyte MII rate (35.8±6.3%; n=16) was significantly lower (p>0.001) than controls, (87.5±4.0%; n=8). Median cleavage rate of IVG embryos (5.6±9.7%; n=12) was significantly lower (p>0.001) than in vivo controls (86.2±7.2%; n=12).
Expression patterns of 16 genes associated with somatic cell function and ≥80 genes associated with oocyte function were established within IVG follicles/oocytes and compared to size & stage matched in vivo-derived controls via qPCR. Significantly, differentially expressed genes were exhibited in 17.9% IVG preantral GV oocytes; 74.7% IVG antral GV oocytes; 78.5% IVG MII oocytes and 50.6% IVG 2-4 cell embryos relative to controls. Recurrent, significantly differentially expressed genes included CDC42, DNMT3A, EHMT2, KDM1B, PADI6, SMAD3, TET2, TLE6 and TXN. Furthermore, analysis of follicular somatic cells indicated that IVG follicles with greater developmental potential demonstrated subtle changes in AMH, FSHR, VCAN and KITLG expression.
In conclusion, mature sheep oocytes and early embryos can be obtained from a serum-free IVG system. However, gene expression profiles of IVG follicles, oocytes and embryos are distinct from in vivo grown controls and may indicate possible intraoocyte epigenetic and cytoplasmic dysregulation.
