Mitochondrial unfolded protein response gene Clpp is required to maintain ovarian follicular reserve during aging, for oocyte competence, and development of pre‐implantation embryos

Summary Caseinolytic 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.

. Mitochondrial unfolded protein response (mtUPR) is activated in response to a variety of mitochondrial stress factors including accumulation of unfolded proteins in the mitochondrial matrix (Rath et al., 2012), imbalance between mitochondrial DNA (mtDNA)-encoded and nuclear-encoded electron transport chain (ETC) components (Nargund, Pellegrino, Fiorese, Baker & Haynes, 2012;Yoneda et al., 2004), and perturbation of mitochondrial physiology through inhibition of ETC function or accumulation of reactive oxygen species (ROS) (Nargund et al., 2012;Yoneda et al., 2004), and ensures mitochondrial proteostasis by inducing a vigorous transcriptional response that promotes folding, limits import, and reduces translation of mitochondrial proteins (reviewed in Hill & Van Remmen, 2014;Jensen & Jasper, 2014).

Mitochondrial unfolded protein response was first described in
Caenorhabditis elegans, where mitochondrial stress involving unfolded proteins upregulates the mitochondrial matrix caseinolytic peptidase P (CLPP), which cleaves misfolded proteins (Benedetti, Haynes, Yang, Harding & Ron, 2006;Haynes, Petrova, Benedetti, Yang & Ron, 2007). Cleaved proteins are then exported to cytosol, where they activate the stress activated transcription factor 1 (ATFS1). ATFS1 then enters the nucleus and activates Ubiquitin-like 5 (UBL5) to form a complex with DVE1 and to induce transcription of mitochondrial chaperones, such as heat shock protein 6 (HSP6) and HSP10 (Haynes, Yang, Blais, Neubert & Ron, 2010;Haynes et al., 2007). In addition, mtUPR induces coenzyme Q biosynthesis, glycolysis, and mitochondrial fission (Aldridge, Horibe & Hoogenraad, 2007), altering mitochondrial metabolism and dynamics to promote mitochondrial function and cell survival during stress. Mitochondrial unfolded protein response and the role of CLPP seem to be conserved in mammals (Benedetti et al., 2006;Zhao et al., 2002), where activation of JNK/c-JUN pathway leads to the expression of transcription factor C/EBP-homologous protein (CHOP), which, together with C/EBP, mediates the transcription of mtUPR genes (reviewed in Hill & Van Remmen, 2014). It is important that, in addition to inducing transcription of over 400 genes, mtUPR in yeast, C. elegans, and mammals are associated with phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a) by general control nonderepressible 2 (GCN2), resulting in global suppression of translation while mRNAs that contain upstream open reading frames (uORFs) are preferentially translated (Delaney et al., 2013;Rath et al., 2012). Transcriptional activation of mtUPR genes and translational suppression seem to be mediated by two parallel mechanisms, both requiring CLPP (Aldridge et al., 2007;Benedetti et al., 2006;Haynes et al., 2007;Zhao et al., 2002) and reviewed in (Jensen & Jasper, 2014;Schulz & Haynes, 2015). It is important that, recessive Clpp mutations have been identified in the human Perrault variant of ovarian failure and sensorineural hearing loss (Jenkinson et al., 2013), and global germline Clpp knockout mice display auditory deficits and complete female and male infertility, in addition to reduced pre/postnatal survival and marked ubiquitous growth retardation (Gispert et al., 2013).
Mitochondria structure, shape, number, and mtDNA copy number are tightly controlled during mouse and human oocyte and early embryo development (reviewed in Seli, 2016), and adenosine triphosphate (ATP) content of human oocytes correlates with embryo development and in vitro fertilization (IVF) outcome (Van Blerkom, Davis & Lee, 1995). Mature mouse and human oocytes contain somewhere between 50,000 and 550,000 mtDNA copies, with considerable degree of variability between samples (Pik o & Taylor, 1987;Steuerwald et al., 2000). In an interesting manner, despite drastic changes in mitochondria morphology observed during early pre-implantation embryo development, total number of mitochondria and mtDNA copy number seem to remain unchanged during cleavage divisions, making the oocyte the primary source of mitochondria for pre-implantation embryos (Piko & Matsumoto, 1976). Mitochondrial DNA replication resumes around the time of blastocyst formation and is first observed in trophectoderm (TE) cells (reviewed in St John, 2014), consistent with the significant increase in the energy needs of the embryo associated with rapid cell proliferation and implantation (Van Blerkom, 2011). Mitochondrial replication, in turn, starts after implantation (Murakoshi et al., 2013;Pik o & Taylor, 1987). Mitochondrial DNA copy number is higher in aneuploid blastocysts (which contain an abnormal chromosome number) and in euploid blastocysts that fail to implant (Fragouli et al., 2015), suggesting that higher mtDNA copy number reflects embryonic stress and is associated with lower reproductive potential.
In this study, we aimed to uncover the mechanisms leading to female infertility in mice with global germline deletion of Clpp (Gispert et al., 2013). We found that Clpp knockout (Clpp À/À ) mice generate lower number of mature oocytes and two-cell embryos and no blastocysts and that these deficiencies in oocyte and embryo development are associated with impaired mitochondrial function and dynamics. Clpp À/À mice ovaries showed accelerated depletion of follicular reserve, associated with mechanistic target of rapamycin (mTOR) pathway activation.

| Clpp is essential for female fertility, oocyte maturation, and embryo development
Male and female Clpp +/À mice appeared phenotypically normal, and intercrossing of the heterozygous mice produced homozygous Clppdeficient mice with a normal male-to-female ratio. This indicated that the targeted disruption of Clpp gene did not cause a significant selective disadvantage with regard to sex. Clpp-deficient female mice (3-month-old) were viable; however, they were significantly smaller compared to wild-type (WT). Their uteri and ovaries were also significantly smaller (n = 4 for each genotype; Supporting Information Figure S1). Metabolic status of Clpp-deficient female mice was assessed in 3 to 9 months of age. Serum glucose, cholesterol, and phospholipids showed no significant difference between Clpp-deficient and WT mice at any time point, while triglycerides were significantly lower in 9 months old Clpp À/À mice (p < 0.01; Supporting Information Figure S2). To confirm the reported infertility of Clpp À/À female mice (Gispert et al., 2013), we conducted a continuous mating study using sexually mature female mice (n = 5 for each genotype) and WT male mice of proven fertility. After 12 weeks of mating, there were no pregnancies or deliveries observed in Clpp À/À female mice.
Wild-type females exhibited normal fertility.

| Mitochondrial dysfunction is associated with
increased ROS and mtDNA, decreased ATP generation, and impaired mitochondrial dynamics and mtUPR pathway gene expression in Clpp À/À oocytes Next we compared the mitochondrial function of Clpp À/À oocytes to WT. Clpp À/À GV stage oocytes had higher ROS levels (74.6 AE 4.6 vs. 41.4 AE 2.2 pixel intensity, p < 0.001; Figure 2a The expression of UPR mt pathway genes, Hspd1, Hspe1, and Dnaja3, were also significantly lower in Clpp À/À oocytes (Supporting Information Figure S5). EM showed that Clpp À/À oocyte mitochondria were smaller in size (0.125 AE 0.002vs 0.143 AE 0.005 lm 2 , p < 0.05) and had a smaller aspect ratio (length/width; 1.32 AE 0.01 vs. 1.38 AE 0.007; p < 0.01) with a more round contour (Figure 2h-j). This was associated with a decreased expression of Mfn1, Mfn2, and Opa1 (fusion genes) without a change in Drp1 (fission gene; Figure 2k). In total, these data suggest that mitochondrial function and dynamics are severely affected in Clpp À/À oocytes.

| Targeted deletion of Clpp results in accelerated depletion of ovarian follicular reserve
We assessed follicle development in the ovaries of unstimulated Clpp +/+ and Clpp À/À mice at 3, 6, 9, and 12 months of age. At 3 months, the number of primordial (which represent ovarian follicular reserve), primary, secondary, and antral follicles did not differ between Clpp +/+ and Clpp À/À , while Clpp À/À ovaries had a 4-fold higher number of atretic follicles (n = 4 mice for each genotype; Figure 3a-b). By 6 months, Clpp À/À mice ovaries had significantly lower number of primordial and primary follicles (n = 4 mice for each genotype), in addition to higher number of atretic follicles (Figure 3c-d).
Apoptosis and proliferation of granulosa cells at different stage of folliculogenesis were assessed by TUNEL and Ki67 immunofluorescent staining, respectively, in 3-and 6-month-old Clpp +/+ and Clpp À/À mice ovaries. Apoptotic rate of granulosa cells was significantly higher at antral follicle stage in 3-month-old Clpp À/À mice

| Gene expression is altered in Clpp À/À oocytes
To delineate the gene pathways affected by the absence of Clpp, a comprehensive genomewide transcriptomic investigation was conducted. Unsupervised hierarchical clustering of the differentially expressed genes partitioned into two distinct clusters to separate Clpp À/À and Clpp +/+ GV oocytes from 3-month-old mice (Figure 5a).
A total of 124 genes were significantly differentially expressed (p < 0.05) in Clpp À/À oocytes compared to WT (73 upregulated and 51 downregulated; Figure 5c,i, Supporting Information Table S2); top 10 upregulated and downregulated annotated genes in 3-month-old Clpp À/À mice oocytes are listed (Figure 5b). Gene ontology (GO) cluster analysis indicated significant over-representation of elements involved in regulation of cell death, development, meiosis, and embryonic development (Figure 5d). To note, TNFR1/2 signaling pathway and Protein Kinase A signaling pathway were affected in Clpp À/À oocytes (Figure 5j).
Hierarchical clustering of the differentially expressed genes also partitioned into two distinct clusters to separate Clpp À/À and Clpp +/+ GV oocytes from 6-month-old mice (Figure 5e). A total of 239 genes were significantly differentially expressed in Clpp À/À oocytes compared to WT (151 upregulated and 88 downregulated; Figure 5g,i, Supporting Information Table S3), top 10 upregulated and downregulated annotated genes in Clpp À/À oocytes 6 months are listed ( Figure 5f). Gene ontology cluster analysis indicated significant overrepresentation of elements involved in regulation of apoptosis, oxidative stress, and oocyte and embryonic development (Figure 5h).
After establishing that CLPP is required for oocyte and early embryo development, we assessed how CLPP deficiency affects mitochondrial function and dynamics in Clpp À/À mice oocytes. We first focused on parameters reflecting the efficiency of oocyte energy metabolism and found Clpp À/À mice oocytes to have higher amount of ROS, with decreased membrane potential and ATP production (Figure 2a-e), as well as lower expression of genes coding for ETC proteins (Figure 2g). Similar to that observed in human embryos that are aneuploid or fail to implant (Fragouli et al., 2015), Clpp À/À mice oocytes had significantly higher mtDNA copy number (Figure 2f). We then assessed whether the environment with impaired energy metabolism affects mitochondrial dynamics in Clpp À/À oocytes. EM analysis revealed Clpp À/À mice oocytes to have smaller and shorter mitochondria, suggesting decreased fusion (Figure 2h-j). We also found transcripts coding for fusion proteins (Mfn1, Mfn2, Opa1) to be downregulated, while the expression of Drp1, which mediates fission, was unchanged (Figure 2k). Overall, F I G U R E 6 Mechanistic target of rapamycin (mTOR) signaling is activated in Clpp À/À oocytes. (a and b) Western blot analysis for phosphorylated S6 (pS6), total S6, pAKT473, and total AKT in 3-month-old Clpp +/+ and Clpp À/À mouse ovaries. Band densities were normalized to corresponding S6 and AKT. HSP90 was used as a loading control. (c and d) Western blot analysis for pS6, total S6, pS6K, total S6K, p4EBP1, total 4EBP1, pAKT473, total AKT, p-mTOR2481, and total mTOR2481 in 6-month-old Clpp +/+ and Clpp À/À mouse ovaries. Band densities were normalized to corresponding S6, S6K, 4EBP1, AKT, and mTOR2481. HSP90 and b-ACTIN were used as loading controls. (e) Bar chart showing p-S6 and p-AKT immunofluorescence intensity in oocytes of 3-month-old Clpp +/+ and Clpp À/À mice. (f and g) Representative micrographs of pS6 and pAKT473 immunofluorescence in 3-month-old Clpp +/+ and Clpp À/À mouse oocytes. (h) Bar chart showing pS6 and pAKT immunofluorescence intensity in oocytes of 6-month-old Clpp +/+ and Clpp À/À mice. (i and j) Representative micrographs of pS6 and pAKT immunofluorescence in 6-month-old Clpp +/+ and Clpp À/À mouse oocytes. Data represent means AE SEM. *p < 0.05. **p < 0.01. ***p < 0.001. Significance was determined by t test [Correction added on 6 June 2018, after first online publication: Figure 6 has been corrected in this current version.] both cellular energy metabolism and mitochondrial dynamics seem to be severely affected in Clpp À/À mice oocytes.
A general decline in mitochondrial function occurs as organisms age (reviewed in Lopez-Otin, Blasco, Partridge, Serrano & Kroemer, 2013). In a paradoxical way, in yeast (Delaney et al., 2013), worms (Dillin et al., 2002), flies (Owusu-Ansah, Song & Perrimon, 2013), and mice (Liu et al., 2005), suppression of mitochondrial ETC function increases lifespan (Dell'agnello et al., 2007;Durieux, Wolff & Dillin, 2011). While counterintuitive, these findings are supported by reports demonstrating that upregulation of mitochondrial stress response contributes to enhanced longevity in the long-lived mitochondrial mutants (Durieux et al., 2011;Kirchman, Kim, Lai & Jazwinski, 1999). A link between mtUPR and longevity was first revealed in two long-lived C. elegans mitochondrial ETC mutants (isp-1 and clk-1; Durieux et al., 2011). RNAi knockdown of either UBL5 or DVE1 (mediators of mtUPR) reversed lifespan extension in both mutants. Similar to that, increased longevity by muscle-specific disruption of ETC Complex I in Drosophila was dependent on mtUPR (Owusu-Ansah et al., 2013), and Surf1 knockout mice deficient for ETC Complex IV had increased expression of mtUPR genes (Dell'agnello et al., 2007;Pulliam et al., 2014). It is important that, a number of other prolongevity models, such as NAD+/Sirtuin1 or rapamycin in C. elegans, also require mtUPR (Houtkooper et al., 2013;Owusu-Ansah et al., 2013). It is likely that CLPP deficiency in oocytes results in a compromised mitochondrial stress response contributing to accumulation of damaged proteins, reduced oxidative phosphorylation, increased reactive oxidative stress production, and culminates in oocyte dysfunction and accelerated follicular depletion.
After characterizing the developmental and metabolic changes that occur in CLPP-deficient oocytes and histomorphometric findings consistent with accelerated ovarian follicular depletion, we adopted an unbiased approach to identify genes and pathways affected by CLPP and performed RNAseq analysis in 3 and 6 months Clpp À/À GV stage oocytes, compared to WT ( Figure 5).
Mechanistic target of rapamycin is a serine/threonine protein kinase of the phosphatidylinositol-3-OH-kinase (PI(3)K)-related family that functions as a master regulator of cellular growth and metabolism in response to nutrient and hormonal cues (reviewed in Johnson, Rabinovitch & Kaeberlein, 2013). Mechanistic target of rapamycin functions in two different complexes: mTORC1 and mTORC2. Rapamycin, which inhibits the mTORC1, significantly extends lifespan in a number of model systems including mice (Harrison et al., 2009). In addition, mTORC1 inhibits autophagy in both yeast and mammalian cells (reviewed in Wei, Zhang, Cai & Xu, 2015), while both mTORC1 and mTORC2 regulate growth and proliferation (reviewed in Johnson et al., 2013;Saxton & Sabatini, 2017). We therefore assessed pS6 (downstream mediator of mTORC1) and pAKT473 (downstream mediator of mTORC2) activity at 3-and 6-month-old Clpp À/À mice ovaries and oocytes compared to WT ( Figure 6). We observed a significant upregulation of pS6 at 6 months. pAKT 473 was also increased at 6 months, but to a lesser extent. We further assessed pS6K, p4EBP1 (downstream mediators of mTORC1), and p-mTOR2481 (downstream mediator of mTORC2) activity at 6-month-old Clpp À/À mice ovaries; they were similarly upregulated. Then, we performed rescue experiments with rapamycin, an mTOR inhibitor. Rapamycin treatment has been reported to prolong ovarian lifespan (Dou et al., 2017), and inhibition of mTORC1 or mTORC1/2 within ovaries during chemotherapy co-treatment resulted in preservation of primordial follicle counts (Goldman et al., 2017). In the current study, oocyte competence was significantly improved both in vivo and in vitro by rapamycin treatment (Figure 7). These findings collectively suggest that increased mTOR activation in Clpp À/À mice is at least partially responsible for their reproductive phenotype. It is also possible for rapamycin treatment to rescue the increased follicular atresia and accelerated follicular depletion phenotype as has recently been suggested for the Fmr1 knockout mice (Mok-Lin et al., 2018). These interactions as well as the impact of rapamycin treatment on fertilization, pre-implantation embryo development, and fertility remain to be investigated.
In this study, we have two important and potentially related findings regarding CLPP's role on female reproduction. First, we find that Clpp-deficiency results in female infertility due to impaired oocyte and early embryo development. Second, we observe that targeted deletion of Clpp results in accelerated follicular depletion, which could represent a phenotype reminiscent of premature ovarian aging in Clpp À/À mice, especially within the context of mTOR activation and chromosome misalignment on oocyte spindles. Individual contributions of granulosa/cumulus and oocyte dysfunction to infertility and follicular depletion and the molecular mechanisms leading to observed changes in gene expression remain to be studied using cell-specific knockout models.