SummaryCaseinolytic peptidase P mediates degradation of unfolded mitochondrial proteins and activates mitochondrial unfolded protein response (mtUPR) to maintain protein homeostasis. Clpp −/− female mice generate a lower number of mature oocytes and two‐cell embryos, and no blastocysts. Clpp −/− oocytes have smaller mitochondria, with lower aspect ratio (length/width), and decreased expression of genes that promote fusion. A 4‐fold increase in atretic follicles at 3 months, and reduced number of primordial follicles at 6–12 months are observed in Clpp −/− ovaries. This is associated with upregulation of p‐S6, p‐S6K, p‐4EBP1 and p‐AKT473, p‐mTOR2481 consistent with mTORC1 and mTORC2 activation, respectively, and Clpp −/− oocyte competence is partially rescued by mTOR inhibitor rapamycin. Our findings demonstrate that CLPP is required for oocyte and embryo development and oocyte mitochondrial function and dynamics. Absence of CLPP results in mTOR pathway activation, and accelerated depletion of ovarian follicular reserve.

Mitochondria change their shape through fusion and fission in order to adapt to their metabolic milieu. Mitofusin-2 (MFN2) is a key regulatory protein in this process, mediating mitochondrial fusion and interaction with endoplasmic reticulum. Targeted deletion of Mfn2 in oocytes resulted in mitochondrial dysfunction and female subfertility associated with impaired oocyte maturation and follicle development. Oocytes lacking MFN2 showed shortened telomeres and increased apoptosis, resulting in compromised oocyte quality and accelerated follicular depletion, consistent with a reproductive aging phenotype.

Mitochondria are dynamic organelles that continually adapt their structure through fusion and fission in response to changes in their bioenergetic environment. Targeted deletion of mitochondrial fusion protein mitofusin1 (MFN1) in oocytes resulted in female infertility associated with failure to achieve oocyte maturation. Oocyte-granulosa cell communication was impaired, and cadherins and connexins were downregulated, resulting in follicle developmental arrest at the secondary follicle stage. Deletion of MFN1 in oocytes resulted in mitochondrial dysfunction and altered mitochondrial dynamics, as well as accumulation of ceramide, which contributed to increased apoptosis and a reproductive phenotype that was partially rescued by treatment with ceramide synthesis inhibitor myriocin. Absence of MFN1 and resulting apoptotic cell loss also caused depletion of ovarian follicular reserve, and a phenotype consistent with accelerated female reproductive aging.

Treatment with the RANKL/RANK pathway inhibitor RANK-Fc significantly decreased human leiomyoma cell proliferation and tumor growth. This suggests that the RANKL/RANK pathway could serve as a potential target for the prevention and treatment of uterine leiomyoma.

OBJECTIVE: To determine whether metabolic imaging using fluorescence lifetime imaging microscopy (FLIM) identifies metabolic differences between normal oocytes and those with metabolic dysfunction. DESIGN: Experimental study. SETTING: Academic research laboratories. PATIENT(S): None. MAIN INTERVENTIONS: Oocytes from mice with global knockout of Clpp (caseinolytic peptidase P; n=52) were compared to wild type (WT) oocytes (n=55) as a model of severe oocyte dysfunction. Oocytes from old mice (1-year old; n=29) were compared to oocytes from young mice (12-week-old; n=35) as a model of mild oocyte dysfunction. MAIN OUTCOME MEASURE(S): FLIM was used to measure the naturally occurring NADH and FAD autofluorescence in individual oocytes. Eight metabolic parameters were obtained from each measurement (4 per fluorophore): short (τ1) and long (τ2) fluorescence lifetime, fluorescence intensity (I), and fraction of the molecule engaged with enzyme (F). ROS levels and blastocyst development rates were measured to assess illumination safety. RESULTS: In Clpp-knockout oocytes compared to WT, FAD τ1 and τ2 were longer (p<0.0001) and I was higher (p<0.01), NADH τ2 was longer (p<0.0001), and F was lower (p<0.0001). In older oocytes compared to young ones, FAD τ1 was longer (p<0.001) and I was lower (p<0.01), while NADH τ1and τ2were shorter (p<0.0001 for both), I and F were lower (p<0.0001 and p<0.05, respectively). FLIM did not affect ROS levels or blastocyst development rates. CONCLUSIONS: FLIM parameters exhibit strong differentiation between Clpp-knockout vs WT, and old vs young oocytes. FLIM could potentially be used as a non-invasive tool to assess mitochondrial function in oocytes.