In vitro culture of oocytes from early preantral-stage mouse follicles: repair of interconnectivity with cumulus and role of glucose metabolism

An increasing proportion of young cancer survivors are using cryopreserved ovarian tissue (OTC) if they confront infertility due to gonadotoxic cancer treatment. Nevertheless, OTC is not suitable for a substantial group of cancer patients, as ovarian tissue transplantation might present the risk of reintroduction of malignant cells. In vitro oocyte growth and maturation may be an alternative strategy for fertility preservation in female patients that cannot benefit from conventional fertility preservation strategies. For some patients, reaching this goal would imply in vitro oocyte growth and maturation from early preantral follicle stages, whereas for others, only completing the final steps of in vitro maturation (IVM) of cumulus-oocyte-complexes (COCs) from small to mid-antral follicles retrieved transvaginally or from ovarian tissue (ex vivo) is needed.
To date, no in vitro follicle culture (IFC) system can successfully support the growth and maturation of human oocytes starting from early preantral stages up to fertilizable oocytes. The mechanisms and culture conditions regulating essential events in vitro such as primordial follicle activation, followed by synchronous follicle growth, are yet to be fully understood. In the context of female fertility preservation, and as a bioassay to explore the processes regulating in vitro oocyte development, our laboratory established a mouse secondary follicle culture system. Despite good maturation rates, in vitro grown oocytes display reduced developmental competence compared to in vivo grown and matured ones, indicating the need for further fine tuning of the culture conditions.
As a strategy for IFC optimization, an extensive glucose and redox metabolic characterization of in vivo and in vitro grown and matured mouse antral follicles was carried out within the individual follicle cell types, via enzymatic assays. Our results confirmed that in vivo, the somatic compartment exhibits intense glycolytic activity, with the oocytes mainly performing pentose phosphate pathway (PPP) activity. However, follicle maturation in vivo triggered a metabolic boost in cumulus cells (CCs), with increased pyruvate and lactate uptake, higher NADP+ levels, and upregulated tricarboxylic acid (TCA) cycle and small molecules antioxidant capacity (SMAC) activities, which was not identified in vitro, indicating altered metabolic functions of CCs that may affect oocyte competence acquisition. To explore potential metabolic alterations interfering with oocyte meiotic competence acquisition in vitro, follicles that failed to respond to nuclear maturation signals (meiotically blocked (MB)), were profiled and compared against their germinal vesicle (GV) and metaphase II (MII) controls. MB-CCs showed increased aconitase and glucose-6-phosphate dehydrogenase (G6PDH) activities with lower malate levels comparted to GV-CCs. These results suggest that in addition to their meiotic impairment the MB follicles might be trying to compensate by running high levels of TCA cycle and PPP activity in CCs, or that they might be unable to run adequate levels of aerobic metabolism given the suboptimal culture conditions.
Altogether the results indicate that oocyte competence could be improved in the IFC system by targeting the somatic cell metabolic pathways through culture condition refinements. Reducing O2 tension during IFC might have a positive impact on somatic cell differentiation and mitochondrial function while media supplementation with oocyte-secreted factors (OSF) could positively regulate CCs functionality.
In contrast to IFC, IVM has already been successfully introduced into the fertility clinic for selected infertile patients. IVM requires minimal to no hormonal stimulation, serving as an alternative treatment to ovarian stimulation (OS) in polycystic ovarian syndrome (PCOS) patients and for fertility preservation when OS and/or OTC are contraindicated. However, IVM yields suboptimal outcomes in terms of good quality embryo rates compared to conventional IVF. To improve in vitro oocyte competence acquisition, our laboratory previously developed a biphasic IVM system, capacitation (CAPA)-IVM, that uses c-type natriuretic peptide (CNP) for oocyte meiotic inhibition during pre-IVM which allows oocyte cytoplasmic maturation prior to the actual IVM step. One of the remaining limitations is that in a substantial proportion of human COCs retrieved for CAPAIVM, partially denuded (PD) oocytes, with poor blastocysts formation rates are recovered, likely due to mechanical stress during the oocyte pick-up.
Hence for testing the proof of concept for reversing the partial denudation in human COCs the mouse IFC system was used. Following the complete oocyte mechanical denudation at different folliculogenesis stages as well as the replacement in IFC of fully denuded in vivo grown oocytes at the antral stage, restoration of the TZPs network with conserved oocyte developmental competence was found. Furthermore, higher mRNA levels of Has2 were detected in CCs of reconnected COCs upon oocyte isolation at preantral stage. For better mimicking the human IVM condition, PD mouse COC were also cultured under controlled in vitro conditions using the mouse CAPA-IVM system. The PD COCs successfully restored the fully surrounded (FS) COCs morphology during CAPAIVM culture, with TZPs restoration and with unaltered oocyte developmental competence.
The results in the mouse model constitute positive premises for further testing of the concepts in human research. To improve oocyte competence, human PD COCs from PCOS and healthy volunteers could undergo CAPA-IVM, with culture medium supplemented with oocyte-secreted factors, to stimulate CC proliferation, and with unluteinized GCs isolated from follicular fluid.
In conclusion, both mouse in vitro systems proved their value as efficient tools to explore optimization strategies for of human fertility preservation and assisted reproductive technologies (ART).