P-689 Mutated MDR-1 impairs chromosome alignment and reduces oocyte quality

Abstract

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