Characterization of carbohydrate metabolism in in vivo- and in vitro-grown and matured mouse antral follicles†.

Establishing an ideal human follicle culture system for oncofertility patients relies mainly on animal models since donor tissue is scarce and often of suboptimal quality. The in vitro system developed in our laboratory supports the growth of prepubertal mouse secondary follicles up to mature oocytes. Given the importance of glucose in preparing the oocyte for proper maturation, a baseline characterization of follicle metabolism both in the culture system and in vivo was carried out. Markers of glucose-related pathways (glycolysis, tricarboxylic acid [TCA] cycle, pentose phosphate pathway [PPP], polyol pathway, and hexosamine biosynthetic pathway), as well as the antioxidant capacity, were measured in the different follicle cell types by both enzymatic activities (spectrophotometric detection) and gene expression (qPCR). This study confirmed that in vivo the somatic cells, mainly granulosa, exhibit intense glycolytic activity, while oocytes perform PPP. Throughout the final maturation step, oocytes in vivo and in vitro showed steady levels for all the key enzymes and metabolites. On the other hand, ovulation triggers a boost of pyruvate and lactate uptake in cumulus cells in vivo, consumes reduced nicotinamide adenine dinucleotide phosphate, and increases TCA cycle and small molecules antioxidant capacity activities, while in vitro, the metabolic upregulation in all the studied pathways is limited. This altered metabolic pattern might be a consequence of cell exhaustion because of culture conditions, impeding cumulus cells to fulfill their role in providing proper support for acquiring oocyte competence.

Glucose metabolism characterization during mouse in vitro maturation identifies alterations in cumulus cells†.

In vitro maturation (IVM) is an assisted reproduction technique with reduced hormone-related side-effects. Several attempts to implement IVM in routine practice have failed, primarily due to its relatively low efficiency compared with conventional in vitro fertilization (IVF). Recently, capacitation (CAPA)-IVM-a novel two-step IVM method-has improved the embryology outcomes through synchronizing the oocyte nuclear and cytoplasmic maturation. However, the efficiency gap between CAPA-IVM and conventional IVF is still noticeable especially in the numerical production of good quality embryos. Considering the importance of glucose for oocyte competence, its metabolization is studied within both in vivo and CAPA-IVM matured mouse cumulus-oocyte-complexes (COCs) through direct measurements in both cellular compartments, from transcriptional and translational perspectives, to reveal metabolic shortcomings within the CAPA-IVM COCs. These results confirmed that within in vivo COC, cumulus cells (CCs) are highly glycolytic, whereas oocytes, with low glycolytic activity, are deviating their glucose towards pentose phosphate pathway. No significant differences were observed in the CAPA-IVM oocytes compared with their in vivo counterparts. However, their CCs exhibited a precocious increase of glycolytic activity during the pre-maturation culture step and activity was decreased during the IVM step. Here, specific alterations in mouse COC glucose metabolism due to CAPA-IVM culture were characterized using direct measurements for the first time. Present data show that, while CAPA-IVM CCs are able to utilize glucose, their ability to support oocytes during final maturation is impaired. Future CAPA-IVM optimization strategies could focus on adjusting culture media energy substrate concentrations and/or implementing co-culture strategies.

Purging human ovarian cortex of contaminating leukaemic cells by targeting the mitotic catastrophe signalling pathway.

PURPOSE: Is it possible to eliminate metastasised chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) cells from ovarian cortex fragments by inhibition of Aurora B/C kinases (AURKB/C) without compromising ovarian tissue or follicles? METHODS: Human ovarian cortex tissue with experimentally induced tumour foci of CML, AML and primary cells of AML patients were exposed to a 24h treatment with 1 µM GSK1070916, an AURKB/C inhibitor, to eliminate malignant cells by invoking mitotic catastrophe. After treatment, the inhibitor was removed, followed by an additional culture period of 6 days to allow any remaining tumour cells to form new foci. Ovarian tissue integrity after treatment was analysed by four different assays. Appropriate controls were included in all experiments. RESULTS: Foci of metastasised CML and AML cells in ovarian cortex tissue were severely affected by a 24h ex vivo treatment with an AURKB/C inhibitor, leading to the formation of multi-nuclear syncytia and large-scale apoptosis. Ovarian tissue morphology and viability was not compromised by the treatment, as no significant difference was observed regarding the percentage of morphologically normal follicles, follicular viability, glucose uptake or in vitro growth of small follicles between ovarian cortex treated with 1 µM GSK1070916 and the control. CONCLUSION: Purging of CML/AML metastases in ovarian cortex is possible by targeting the Mitotic Catastrophe Signalling Pathway using GSK1070916 without affecting the ovarian tissue. This provides a therapeutic strategy to prevent reintroduction of leukaemia and enhances safety of autotransplantation in leukaemia patients currently considered at high risk for ovarian involvement.