TARGETED DELETION OF MITOFUSIN 1 AND MITOSUFIN 2 CAUSES FEMALE INFERTILITY AND LOSS OF FOLLICULAR RESERVE

Abstract

Mitofusin 1 (MFN1) and Mitofusin 2 (MFN2) are transmembrane GTPases that regulate mitochondrial fusion and are required for the maintenance of cellular homeostasis. The aim of the study was to determine the role of mitofusins in female reproductive competence and senescence using a mouse model with oocyte-specific deletion of Mfn1 and Mfn2. Mice with oocyte-specific double deletion of Mfn1 and Mfn2 were generated by mating Mfn1 flox/flox or Mfn2 flox/flox with Zp3-Cre Mfn1 and Mfn2 males. In all experiments, adult (8-weeks-old) Mfn1-/-/Mfn2-/- female mice were compared to wild-type (WT). To assess fertility, female mice from each group (n=5) were mated with adult WT males of proven fertility for 12 weeks. The ability to generate oocytes (germinal vesicle [GV] and metaphase II [MII]) was assessed after injection with PMSG (10IU) or PMSG and hCG (10IU). Unfertilized MII oocytes were collected 14 hours after hCG injection. Follicle development was assessed in ovaries from Mfn1-/-/Mfn2-/- and WT mice after fixation, paraffin embedding, and sectioning, followed by hematoxylin and eosin (H&E) staining. ATP levels were determined by bioluminescent assay. Mitochondrial DNA (mtDNA) copy number was measured in individual oocytes by cloning of mitochondria specific gene (Cox3) as a standard, followed by quantitative real-time PCR (qPCR). Expression levels of mitochondrial respiratory chain genes was evaluated with qRT-PCR. Student’s t-test and Chi-Square analysis were applied. Mature female Mfn1-/-/Mfn2-/- were infertile compared to WT females (0 ± 0 vs ± 5.62 ± 1.64 pups per litter; p < 0.0001). Mfn1-/-/Mfn2-/- mice showed a significant decrease in GV oocyte number (12.75 ± 1.70 vs 40.25 ± 3.5; p < 0.0001) and generated no MII oocytes (0 ± 0 vs 31.01 ± 3.5; p < 0.0001) in comparison to WT. Eight-week-old Mfn1-/-/Mfn2-/- mice ovaries had similar number of primordial, primary, and secondary follicles compared to WT. However, the number of early antral and antral follicles numbers was significantly decreased (17.2 ± 10.2 vs 55.3 ± 9.50; p < 0.03, and 2.3 ± 1.5 vs 16.3 ± 5.8; p < 0.04). At 6 months, Mfn1-/-/Mfn2-/- mice had a lower number of primordial, early antral, and antral follicles compared to WT. At 12 months, all follicle stages were found to be significantly lower in Mfn1-/-/Mfn2-/- mice. Decreased ATP production (0.62 ± 0.1 vs 1.23 ± 0.12; p = 0.002) and mtDNA copy number (446033 ± 53978 vs 706672 ± 72470; p = 0.003) were also observed in Mfn1-/-/Mfn2-/-oocytes compared to WT. Similarly, the expression of mitochondrial chain respiratory genes Cox1, Sdhb, Uqcrc2 and Atp5a1 were decreased in Mfn1-/-/Mfn2-/- oocytes compared to WT. Our findings demonstrate that lack of MFN1 and MFN2 results in infertility and accelerated loss of follicular reserve consistent with a diminished ovarian reserve (DOR) phenotype.