MDR-1 is a transmembrane ATP-dependent effluxer present in organs that transport a variety of xenobiotics and byproducts. Previous findings by our group demonstrated that this transporter is also present in the oocyte mitochondrial membrane and that its mutation led to abnormal mitochondrial homeostasis. Considering the importance of these organelles in the female gamete, we assessed the impact of MDR-1 dysfunction on mouse oocyte quality, with a particular focus on the meiotic spindle organization, aneuploidies, Ca2+ homeostasis, ATP production and mtDNA mutations. Our results demonstrate that young Mdr1 mutant mice produce oocytes characterized by lower quality, with a significant delay in the germinal vesicle (GV) to germinal vesicle breakdown (GVBD) transition, an increased percentage of symmetric divisions, chromosome mis-alignments and a severely altered meiotic spindle shape compared to the wild types. Mutant oocytes exhibit 7000 more single nucleotide polymorphisms (SNPs) in the exomic DNA and twice the amount of mitochondrial DNA SNPs compared to the wild-type ones. Ca2+ analysis revealed the inability of MDR-1 mutant oocytes to manage Ca2+ storage content and oscillations in response to several stimuli and ATP quantification shows that mutant oocytes trend towards lower ATP levels compared to wild types. Finally, 1-year-old mutant ovaries express a lower amount of Sirt1, Sirt3, Sirt5, Sirt6 and Sirt7 compared to wild type levels. These results, together emphasize the importance of MDR-1 in mitochondrial physiology and highlight the influence of MDR-1 on oocyte quality and ovarian aging.

Study question With this study we aim to assess the link between oocyte mitochondrial physiology and spindle formation with respect to Multidrug Resistance transporter 1 (MDR-1) function. Summary answer Our most recent data suggest that oocyte quality is compromised in young and old Mdr1 mutant mice. What is known already MDR-1 is a transmembrane ATP-dependent effluxer present in different organs that transport a variety of xenobiotics and byproducts from one side of the membrane to the other. Previous findings by Clark et al. 2019 demonstrated that this transporter is also present in the oocyte mitochondrial membrane and that its mutation led to abnormal mitochondrial homeostasis. Mitochondria are essential for normal oocyte physiology, quality and competence for fertilization and blastocyst development (Chappel, 2013; Dumollard et al., 2007). Therefore, any alteration of their bioenergetics and functions affects the quality of the egg, eventually compromising fertility. Study design, size, duration To carry out the experiments we used Mdr1mutant female mice, expressing an aberrant MDR-1 protein. We focused our analysis on ovaries and oocytes of young (2-3 months) and old (9-12 months) mutant mice, comparing the results with age-matched wild types. Participants/materials, setting, methods In these conditions, we performed ATP quantification and we utilized oocyte in vitro maturation (IVM), immunofluorescence analysis and Ca2+ stimulation analysis. Main results and the role of chance Clark et al. 2019 previously observed that young mutant animals ovulate more eggs compared to wild-types. However, oocytes were fragile and lysed easily and didn’t yield more pups (Clark et al 2019). Conversely, we observed that old Mdr1 mutant mice ovulated less eggs compared to age-matched controls (P < 0.01, n = 4), suggesting diminished ovarian reserve which is associated with poor oocyte quality. Our preliminary data concerning oocyte ATP concentration suggest that mutant germinal vesicle (GV) oocytes present lower ATP levels compared to wild types. Immunofluorescence analysis on metaphase I (MI) oocyte spindle revealed that mutant oocytes present a higher percentage of misaligned chromosomes compared to wild type oocytes (34% and 15%, respectively; P < 0.05, n = 3). Lastly, considering that mitochondria are fundamental for Ca2+ homeostasis, which in turn is necessary for the fertilization process, Ca2+ waves released from mutant vs wild-type metaphase II (MII) oocytes were analyzed using stimulation with strontium, ionomycin, thapsigargin and PLCZ. In all conditions, mutant MII oocytes had altered Ca2+ response (n=∼10 cells per genotype). Limitations, reasons for caution Although these findings demonstrate the importance of MDR-1 for proper maturation and subsequent oocyte development, further studies are necessary to elucidate the specific role and localization of this transporter in the oocyte’s mitochondrial membranes. Wider implications of the findings Our mouse model indicates the importance of mitochondrial homeostasis in infertility. Moreover, our study will provide enhanced understanding of the molecular mechanisms defining oocyte developmental competence, providing insights into human fertility studies. Trial registration number not applicable