- Email: [email protected]
Mitochondrial Fission Factor Drp1 Maintains Oocyte Quality via Dynamic Rearrangement of Multiple Organelles
Current Biology 24, 2451–2458, October 20, 2014 ª2014 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.cub.2014.08.060
Report Mitochondrial Fission Factor Drp1 Maintains Oocyte Quality via Dynamic Rearrangement of Multiple Organelles Osamu Udagawa,1,2 Takaya Ishihara,1 Maki Maeda,1,3 Yui Matsunaga,3 Satoshi Tsukamoto,3,4 Natsuko Kawano,5 Kenji Miyado,5 Hiroshi Shitara,6 Sadaki Yokota,7 Masatoshi Nomura,8 Katsuyoshi Mihara,9 Noboru Mizushima,3,10 and Naotada Ishihara1,3,* 1Department of Protein Biochemistry, Institute of Life Science, Kurume University, Kurume 839-0864, Japan 2National Institute for Environmental Studies, Center for Environmental Risk Research, Tsukuba 305-8506, Japan 3Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan 4Laboratory Animal Sciences Section, National Institute of Radiological Sciences, Chiba 263-8555, Japan 5National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan 6Laboratory for Transgenic Technology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan 7Pharmaceutical Sciences, Nagasaki International University, Sasebo 859-3298, Japan 8Department of Medicine and Bioregulatory Science, Kyushu University, Fukuoka 812-8582, Japan 9Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan 10Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan
Summary Mitochondria are dynamic organelles that change their morphology by active fusion and fission in response to cellular signaling and differentiation [1, 2]. The in vivo role of mitochondrial fission in mammals has been examined by using tissue-specific knockout (KO) mice of the mitochondria fission-regulating GTPase Drp1 [3, 4], as well as analyzing a human patient harboring a point mutation in Drp1 [5], showing that Drp1 is essential for embryonic and neonatal development and neuronal function. During oocyte maturation and aging, structures of various membrane organelles including mitochondria and the endoplasmic reticulum (ER) are changed dynamically [6, 7], and their organelle aggregation is related to germ cell formation and epigenetic regulation [8–10]. However, the underlying molecular mechanisms of organelle dynamics during the development and aging of oocytes have not been well understood. Here, we analyzed oocyte-specific mitochondrial fission factor Drp1-deficient mice and found that mitochondrial fission is essential for follicular maturation and ovulation in an agedependent manner. Mitochondria were highly aggregated with other organelles, such as the ER and secretory vesicles, in KO oocyte, which resulted in impaired Ca2+ signaling, intercellular communication via secretion, and meiotic resumption. We further found that oocytes from aged mice
*Correspondence: [email protected]
displayed reduced Drp1-dependent mitochondrial fission and defective organelle morphogenesis, similar to Drp1 KO oocytes. On the basis of these findings, it appears that mitochondrial fission maintains the competency of oocytes via multiorganelle rearrangement. Results and Discussion Drp1 in Oocytes Is Essential for Female Fertility, Ovulation, Follicular Maturation, and the Proliferation of Granulosa Cells around KO Oocytes To examine the contribution of mitochondrial fission to oocyte formation, mice bearing a Drp1flox allele were crossed to a transgenic line expressing ZP3-Cre, which is active in growing oocytes [11, 12]. To assess the fertility, we bred female Drp1flox/flox, ZP3-Cre mice, which we refer to here as KO mice, and control mice with stud males (Figure 1A). During a period of up to 2 months, mated control female mice gave an average of 6.7 pups per mating; however, KO mice gave 0.63 pups per mating, despite normal mating behavior and vaginal plug formation. Conversely, Drp1 KO males showed normal fecundity. Consistently, we could recover very few or no oocytes from the oviducts of KO mice after superovulation (Figure 1B), suggesting that the KO mice are defective for ovulation. Histological analyses of 8-week-old ovaries revealed that many immature follicles including primary follicles with a single layer of granulosa cells had accumulated in KO ovaries (Figures 1E–1G). Conversely, control ovaries had several secondary follicles with multiple layers of granulosa cells and several mature follicles with an antral cavity. Retarded follicular development was also observed in the ovaries of KO mice primed with PMSG (Figures 1E–1G). At 8 weeks old, we observed 9.3 oocytes in control and 14.7 oocytes in KO mice per ovary section, suggesting that oocytes were not eliminated from KO follicles. Next, we analyzed the expression of genes crucial for follicular maturation and ovulation using RT-PCR. The expression levels of activin-bB and FSHR, which are implicated in the regulation of granulosa cell proliferation and follicle growth [13–15], were low in KO ovaries (Figure 1C, see also Figures 3K–3M). The levels of follistatin, an endogenous inhibitor of activins [16], were also low. After PMSG injection, these defects were partly rescued, but were still observed (Figure 1C). The induction of LH/hCG target genes in granulosa cells [17] was also compromised (Figure 1D), consistent with the failure of follicular maturation and ovulation in KO mice even after superovulation. We then examined granulosa cell proliferation using the incorporation of bromodeoxyuridine (BrdU). In 8-week-old ovaries, 21.4% of granulosa cells were BrdU positive in the control ovaries (Figure 1H) compared with 8.0% in KO ovaries; thus, granulosa proliferation was severely blocked. Even after PMSG injection, BrdU incorporation was reduced in KO follicles (Figure 1H). The defects seen in the KO mice accumulated in an age-dependent manner, since follicular maturation (Figures 1E–1G) and BrdU incorporation (Figure 1H) were partly affected in 5-week-old KO ovaries. These data demonstrate that Drp1 ablation in oocytes affects follicular maturation and
Current Biology Vol 24 No 20 2452
Figure 1. Defective Follicular Maturation and Ovulation in Drp1 KO Oocytes (A) Control and Drp1 KO female mice (8–10 weeks old) were bred with stud males for up to 2 months. The average number of delivered pups per mouse with plug formation was recorded. Control (cont), n = 10; KO, n = 19; *p < 0.01. (B) Control and KO female 5-week-old mice were superovulated by PMSG and hCG stimulation. The number of oocytes collected from oviducts was counted. Cont, n = 6; KO, n = 6; *p < 0.01. (C and D) Control and KO ovaries from 8-week-old mice primed or not primed with PMSG (C) or from 3- to 4-week-old mice primed or not primed with PMSG and hCG (D) were analyzed by RT-PCR. GAPDH and b-actin were used as loading controls. Areg, amphiregulin; BTC, betacellulin; Has2, hyaluronan synthase 2; Tnfaip6, TNFa-induced protein 6; Ptgs2, prostaglandin synthase 2; LHR, LH receptor. (E and F) Ovaries from 5- and 8-week-old control and KO mice primed or not primed with PMSG were fixed, embedded, sectioned, and stained with hematoxylin and eosin. In the 8-week-old sections of (F), primary follicles (*), secondary follicles (**), and antral follicles (***) are indicated. (G) The number of developing follicles per section was counted (n = 2–3). (H) Top: BrdU was administered intraperitoneally for 2 hr before fixation, and BrdU in the ovarian sections was detected with an anti-BrdU antibody. Bottom: The number of BrdU-positive cells per follicles was counted. Data are presented as the mean 6 SEM.
the proliferation of surrounding granulosa cells, which might reflect the low abundance of FSHR (Figure 1C), leading to insufficient ovulation and female infertility. Abnormal Mitochondrial Morphology in Drp1 KO Oocytes We analyzed mitochondrial morphology in the oocytes of KO mice. Immunofluorescent staining of oocyte cryosections showed that mitochondria were distributed evenly throughout the cytoplasm of oocytes in control 8-week-old ovary sections (Figures 2A and S1A available online), as reported previously
[8, 18]. Many of the Drp1-positive dots were localized to mitochondria. In contrast, mitochondria were highly aggregated in KO oocytes (Figure 2A, arrowhead). As a result, mitochondriafree spaces in the cytoplasm of oocytes could sometimes be detected in many of the cryosections (Figure 2A, arrow). In the granulosa cells of KO mice, Drp1 was expressed normally, confirming that Drp1 was specifically depleted in oocytes. Altered mitochondrial morphology was also observed in oocytes isolated from KO mice (Figures 2B, 2D–2G, and S2A). As expected from our previous report [19], mtDNA nucleoids
Mitochondrial Fission Maintains Oocyte Quality 2453
Figure 2. Various Membrane Structures Are Clustered in Drp1 KO Oocytes (A) Cryosections of 8-week-old control and KO ovaries were stained with antibodies against Tom70 (green; mitochondria) and Drp1 (red). The outline of the oocyte is indicated by a white dotted circle in the merged images. Aggregated mitochondria are shown by the arrowhead, and the mitochondria-free area is shown by an arrow. Inset: magnified images. (B) Isolated oocytes were vitally stained with MitoTracker Red. (C) Electron micrographs of 8-week-old control and KO oocytes. Three magnified images are shown on the right. (D) Isolated oocytes were vitally costained with 100 nM MitoTracker Red and 10 mM ERTracker-DPX for 30 min. (E and F) Fixed oocytes were costained using antibodies against cytochrome c (cyt c) for mitochondria and Pmp70 for peroxisomes (E), or antibodies against Tom20 for mitochondria and GM130 for the Golgi apparatus (F). (G) Secretory vesicles were visualized with LCA-FITC.
were highly clustered in KO oocytes (Figure S2C). Electron microscopy (EM) observation showed that small rounded mitochondria with less developed cristae were distributed throughout the cytoplasm of control oocytes (Figures 2C and S1B). In contrast, elongated mitochondria with an irregular size were highly aggregated in KO oocytes. The average length of the major axis of mitochondria was 0.62 mm in control oocytes, whereas it was 1.12 mm in KO oocytes (Figure S1D). These data suggest that mitochondrial morphology is severely affected in Drp1 KO oocytes.
Multiorganelle Aggregation in Drp1 KO Oocytes Drp1 is believed to be involved in the regulation of mitochondrial and peroxisomal morphology, although the evidence regarding whether Drp1 directly regulates ER morphology is obscure [20, 21]. As expected, small peroxisomes, which were dispersed in the cytoplasm of control oocytes, were clustered and partly overlapped with mitochondria in KO oocytes, as observed in ovary sections (Figure S1A) and isolated oocytes (Figure 2E). In ovary sections, most of the ER was clustered around mitochondrial clusters in KO oocytes
Current Biology Vol 24 No 20 2454
Figure 3. Mitochondrial Activity, Ca2+ Response, and GVBD in Drp1 KO Oocytes (A–D) Mitochondrial activity and mtDNA. (A) Immunoblot analyses of control and Drp1 KO oocytes using the indicated antibodies. A total of 20 control and 28 KO oocytes were used for each. (B) Five-week-old oocytes were stained with the membrane potential-dependent dye MitoTracker Red, and the integrated intensity of each oocyte was quantified. n = 2–7, *p < 0.05. CCCP was used as a negative control. (C) Five-week-old oocytes were subjected to ATP measurement. Oligomycin was used as a negative control. n = 3–5, *p < 0.01. (D) mtDNA copy number in single oocytes was calculated by qPCR and shown against oocyte diameter. (E–I) Ca2+ response. (E) Five-week-old oocytes were stained with Oregon green 488-BAPTA-AM, and the Ca2+ signals were recorded using time-lapse images and plotted. See also Movies S1 and S2. (F and G) Quantification of the frequency (F) and relative amplitude (G) of Ca2+ oscillations observed as in (E) are shown. n > 8, *p < 0.01. (H and I) Five-week-old oocytes were treated with EGTA (which blocks extracellular Ca2+ flux and Ca2+ oscillations) and then treated with thapsigargin. The increase of cytosolic Ca2+ was quantified as stored ER Ca2+ (I). Cont, n = 8; KO, n = 5; *p < 0.01. (J–M) Signaling for granulosa cell activation. (J) Schematic diagram of the experimental design of the coculture assay. (K) The indicated number of control oocytes was cocultured with KO follicles, and the follicles were analyzed by RT-PCR. Primary, secondary, and mature follicles collected from control mice were also examined as standards of follicular maturation. (legend continued on next page)
Mitochondrial Fission Maintains Oocyte Quality 2455
(Figure S1A). The partial overlap of mitochondria and the ER was also observed by vital staining of isolated oocytes (Figures 2D and S2A). In EM examination of control oocytes, most (81.5%) of the small spherical mitochondria were in close contact with ER tubules (Figures 2C, S1B, and S1C), as shown in previous reports [8, 22]. Long ER tubules were in contact with several mitochondria, forming a beaded structure of mitochondria. In contrast, the enlarged mitochondria in KO oocytes clustered with various membranous structures such as small vesicles and multivesicular structures. The induction of ER aggregation by Drp1 deficiency might be a cell-type-specific phenomenon, because Drp1 knockdown in HeLa cells and Drp1 KO in mouse embryonic fibroblasts did not induce ER accumulation [3]. Thus, oocyte-specific frequent and tight interactions between the mitochondria and ER might lead to their coaggregation in Drp1 KO oocytes. We also showed that treatment of isolated oocytes with a Drp1 inhibitor, mdivi-1, led to the formation of mild aggregates containing mitochondria and ER (Figure S2B), which also supports the above conclusion. There are several reports showing that mitochondria aggregate transiently in developing early germ cells. Intermitochondrial cement structures called nuages are important for epigenome regulation [9] and the clustering of mitochondria with the ER and Golgi apparatus to form Balbiani bodies, which are involved in mtDNA inheritance [8, 10, 23]. Here we found that the Golgi apparatus (Figure 2F) and cytoskeletal components, such as actin and tubulin (Figures S2D and S2E), were normal in KO oocytes. No ubiquitin signals were detected in the organelle aggregates (Figure S2E). These data exhibited the different characteristics of Balbiani bodies and aggresomes. Transzonal projections (TZPs), which are plasma membrane extensions from granulosa cells that traverse the zona pellucida, make contact with oocyte microvilli [24, 25]. In EM observation (Figure 2C) and immunofluorescent F-actin staining, which is a marker of TZPs (Figure S2E), we could not find any defects in TZP structures of Drp1 KO oocytes. Lens culinaris agglutinin (LCA) labels secretory vesicles in the cortical cytoplasm and plasma membrane of control oocytes [26]. In contrast, cytoplasmic clumps were highly labeled in Drp1-KO oocytes, indicating the accumulation of abnormal secretory vesicles (Figure 2G). This observation is consistent with a recent report showing aberrant synaptic vesicle structures in Drp1-deficient neurons [27]. Thus, Drp1 depletion influences not only mitochondrial morphology but also several other intracellular membrane structures. Calcium Response Is Affected in Drp1 KO Oocytes Next, we assessed mitochondrial function in Drp1 KO oocytes. First, the expression of several subunits of respiratory chain complexes was found to be normal in Drp1 KO oocytes (Figure 3A), although Drp1 protein was almost completely repressed. The intensity of the membrane-potential-dependent dye MitoTracker Red was comparable between control
and KO oocytes (Figure 3B), even though mitochondrial morphology was severely affected in KO oocytes (Figure 2B). ATP content analyzed by luminometric analysis was faintly affected in KO oocytes (Figure 3C). We then measured mtDNA copy number in isolated oocytes by qPCR. During oocyte growth in control mice, mtDNA copy number was highly increased (to approximately 4.0 3 105 copies) concomitantly with the enlargement of oocyte volume (Figure 3D), as reported previously [28]. However, we could not find a clear difference between the control and KO oocytes when they were the same diameter. Taken together, these findings indicate that mitochondrial fission is not essential for respiration or the increase of mtDNA copy number in oocytes. Ca2+ oscillations are known to be essential for oocyte development and maturation [29]; therefore, we measured spontaneous Ca2+ oscillations of GV-stage oocytes using Oregon Green 488-BAPTA-AM. Ca2+ oscillations occurred in 67% of fully grown GV-stage control oocytes, while oscillations were recorded in 55% of Drp1 KO oocytes (Figures 3E–3G and S3A and Movies S1 and S2). They had a similar frequency (interspike interval: 1.95 min in control oocytes and 2.11 min in KO oocytes) (Figures 3E and 3F), but KO oocytes had a significantly reduced amplitude (approximately 70% of control) (Figure 3G), suggesting that the Ca2+ response was deficient in Drp1 KO oocytes. Mitochondria are known to act as a Ca2+ store, so we checked the role of mitochondrial Ca2+ uptake in Ca2+ oscillations. When we pretreated the oocytes with RU360, an inhibitor of the mitochondrial Ca2+ uniporter, the amplitude was less affected (Figures S3B–S3D), in contrast to the decreased amplitude observed in KO oocytes (Figure 3G). Furthermore, the duration of the oscillations was partly but clearly increased by RU360 treatment, which was also inconsistent with the phenotypes seen in Drp1 KO oocytes (Figures S3B and S3E). Thus, mitochondrial Ca2+ uptake might not be the main cause of the Ca2+ signaling defect observed in Drp1 KO oocytes. In various cell types, Ca2+ is regulated via close contact of the ER with mitochondria, enabling events such as apoptosis and Ca2+ signaling [30, 31]. Next, we determined the contribution of Drp1 to the ER Ca2+ store in oocytes. In the presence of EGTA, to exclude the contribution of extracellular Ca2+ uptake, treatment with thapsigargin, an ER Ca2+ pump inhibitor, caused a large increase of Ca2+ in control oocytes, presumably by releasing Ca2+ from the ER (Figure 3H). However, this increase of Ca2+ was reduced in KO oocytes (Figure 3I), suggesting a decreased store of Ca2+ in the ER. Thus, the Ca2+ response via the ER is compromised in KO oocytes, implying the significance of mitochondria-derived interorganelle communication and cellular signaling. Follicular Arrest due to Defective Secretion from Drp1 KO Oocytes to Surrounding Granulosa Cells It is known that follicular maturation accompanied by granulosa cell proliferation requires communication between an oocyte and the surrounding niche via molecules, such as
(L) Ten naked oocytes isolated from control mice were precultured in medium, and then the medium was isolated. Drp1 KO follicles were cultured in the precultured medium and analyzed as in (K). (M) Ten naked oocytes isolated from control or KO mice were cocultured with KO arrested follicles and analyzed as above. (N and O) Meiotic resumption. Five-week-old oocytes were stained with MitoTracker Red in the presence of IBMX. After washout of IBMX, mitochondrial morphology (red fluorescence) associated with spontaneous meiotic resumption was monitored by time-lapse microscopy. Typical oocytes with aggregated mitochondria (‘‘mit-aggre’’) are shown in Figure S4. See also Movies S3 and S4. (N) The percentage of oocytes that completed GVBD was quantified. n = 2, *p < 0.05. (O) GVBD completion is indicated by a green arrow. GVs are enclosed by white dotted circles and indicated with a yellow arrow. Data are presented as the mean 6 SEM.
Current Biology Vol 24 No 20 2456
GDF-9 and BMP15 secreted from oocytes, and the KIT ligand from granulosa cells [13–15]. To analyze the cause of abnormal follicular maturation in oocyte-specific Drp1 KO mice, we analyzed the communication between oocytes and granulosa cells, referring methods for a coculture system of isolated oocytes with follicles [32], a culture method for follicular differentiation [33], and RT-PCR (model in Figure 3J). Arrested immature follicles isolated from 8-week-old KO mice had a reduced number of activated granulosa cells and lower expression levels of mature follicle markers (Figure 3K), as described above (Figures 1C–1H). These immature KO follicles were cocultured with denuded granulosa-free 5-weekold control oocytes for 24 hr, and total RNA extracted from the KO follicles was examined by RT-PCR (Figure 3J). The increased expression of mature follicular markers suggests that coculturing with the control oocytes restored the activity of the granulosa cells in the KO follicles (Figure 3K). This recovery was caused by materials secreted from the oocytes, since media in which healthy oocytes had been precultured demonstrated follicle-stimulation activity (Figure 3L), as seen in the coculture experiments. In contrast, such a resumption of follicular growth was not detected by coculturing with denuded Drp1 KO oocytes (Figure 3M). As described above, staining of isolated oocytes with LCA showed the defective distribution of secretory vesicles (Figure 2G). Likewise, accumulated vesicles and multivesicular structures were observed in EM analysis of KO oocytes (Figure 2C, arrowheads). These results strongly suggest that the oocyte-derived secretion required for proper granulosa proliferation is impaired in Drp1 KO oocytes. In Drp1 KO oocytes, the zona pellucida, an extracellular matrix structure veiling oocytes, was formed (Figures S2D and S2E, arrow), which requires the secretion of glycoproteins such as ZP1 and ZP3, suggesting normal constitutive secretion; thus, we speculated that the Ca2+-dependent regulatory response might be impaired. Meiotic Resumption Is Defective in Drp1 KO Oocytes The germinal vesicle (GV) is a specialized nuclear structure of oocytes that is highly enlarged during oocyte maturation. At the resumption of meiotic maturation, GV break down (GVBD) occurs prior to polar body formation. During the GVBD stage, the dispersed mitochondria [6, 8] accumulate around the GV [34]. To elucidate further the relationship between the dynamics of mitochondria and other organelles in oocytes, we focused on GVBD. In our culture condition, GVBD was observed in 73.3% of control oocytes from 5-week-old mice between 2 and 4 hr of culture (Figure 3N). In contrast, GVBD was observed at a lower level in Drp1 KO oocytes (46.6%). This mild defect might be caused by the moderate phenotype in 5-week-old oocytes (see Figures 1E–1H), since GVBD was never observed in KO oocytes with solely aggregated mitochondria (Figures 3N and S4, ‘‘mit-aggre’’), suggesting that mitochondrial morphology affects the progression of GVBD. We analyzed mitochondrial dynamics further by live imaging. During GVBD, small fragmented mitochondria frequently and dynamically moved in the cytoplasm of control GV oocytes (Movie S3 and Figures 3O and S4). Before GVBD, a significant amount of mitochondria gathered to enwrap GVs. The ER also coaccumulated with mitochondria at the pre-GVBD stage in control oocytes (Figure S4). After GVBD, the mitochondria stopped their movement in the cytoplasm (Movie S3 and Figure 3O). In contrast, mitochondrial movement was restricted and GVBD was defective in KO oocytes with severe mitochondrial aggregation (Movie S4 and Figures 3O and S4).
These results demonstrate that mitochondrial dynamics are important for the rearrangement of multiple organelles during oocyte maturation. Deformed Mitochondrial Morphology and Drp1 Repression in Oocytes from Aged Mice During the long period of meiotic arrest in follicles, mitochondria are believed to be one of the leading determinants of oocyte quality [35]. Here we analyzed mitochondrial morphology and Drp1 in oocytes isolated from aged mice. In ovulated oocytes isolated from 8- to 14-week-old mice, small and short mitochondria were dispersed throughout the cytoplasm (Figures 4A and 4C) as described above (Figure 2). In contrast, in oocytes isolated from mice aged older than 11 months, which have reduced ovulation activity (4.6 oocytes per animal), the mitochondria and ER were aggregated more frequently (Figures 4A and 4C), confirming previous reports [36–38]. We further found that peroxisomes and secretory vesicles were also deformed in aged oocytes (Figure 4B). The secretory activity for stimulating granulosa cells was defective in aged oocytes (Figure 4D). Furthermore, immunoblot experiments revealed that the expression and phosphorylation of Drp1 was clearly and reproducibly repressed in aged oocytes compared with oocytes isolated from young mice (Figure 4E). Drp1 phosphorylation at Ser616 reportedly enhances mitochondrial fission [39]. These results suggest that mitochondrial morphology and Drp1-dependent mitochondrial fission are altered in aged oocytes, and we hypothesized that Drp1dependent mitochondrial fission might have a critical role in oocyte competency in mice (models in Figure 4G). Meanwhile, ATP content was partly decreased in aged oocytes (Figure 4F), inconsistent with Drp1 KO oocytes, suggesting that mitochondrial fission is not the sole causal defect in aged oocytes. It is noteworthy that the decreased ATP content caused neither mitochondrial nor ER aggregation in oocytes (Figure S2E), suggesting that Drp1 deficiency leads to multiorganelle deformation in aged oocytes, which might affect competency of oocytes as seen in Drp1 KO oocytes. In conclusion, the Drp1-dependent spatiotemporal regulation of mitochondria severely affects various types of membrane dynamics in oocytes, and thus, mitochondria act as a center for dynamic membrane remodeling during the maturation of oocytes. Our findings also indicate that oocyte-specific Drp1 KO mice are a potentially useful model for analyzing the cytoplasmic aging of oocytes. Experimental Procedures All mouse procedures and protocols were in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee at Kurume University. The female fertility test was carried out by mating with normal adult male mice for up to 2 months after checking for the presence of copulation plugs. Drp1flox/flox mice were generated as described previously [3]. ZP3-Cre Tg mice [11, 12] were used to produce the oocyte-specific Drp1-deficient mice. To isolate GV oocytes, ovarian follicular development was stimulated by an intraperitoneal injection of 5 IU PMSG to 5-week-old mice. Oocyte-cumulus cell complexes (OCCs) were collected from ovaries at 48 hr after PMSG priming, and cumulus cells surrounding the oocytes were removed completely by passing the OCCs several times through a hand-drawn small fine glass pipette. To isolate ovulated oocytes, mice were stimulated by an injection of 5 IU PMSG followed 48 hr later by 5 IU hCG. Oocytes were collected from the oviduct by gently teasing apart the ampulla to release OCCs into medium containing 0.3 mg/ml hyaluronidase and incubating OCCs at 37 C for 3– 5 min. For the analysis of aged oocytes, the injection of 10 IU PMSG was followed 48 hr later by the injection of 10 IU hCG for 20 hr. Cumulus-free
Mitochondrial Fission Maintains Oocyte Quality 2457
Figure 4. Drp1-Dependent Mitochondrial Morphogenesis Is Altered in Aged Oocytes (A) Subcellular distribution of the mitochondria and ER in young (8–14 weeks) and aged (44–77 weeks) oocytes. Superovulated oocytes were vitally costained with 200 nM MitoTracker Red and 10 mM ERTracker-DPX for 30 min in HTF medium. (B) The subcellular distribution of mitochondria, peroxisomes, and secretory vesicles in aged oocytes are shown. (C) Classification of the mitochondrial morphology of oocytes at each age. Oocytes with highly aggregated mitochondria were classified as ‘‘mit-aggre;’’ multiple types of milder or smaller aggregates were categorized as ‘‘mild-aggre.’’ Oocytes with dispersed mitochondria were categorized as ‘‘dispersion.’’ Young, n = 129; aged, n = 130; *p < 0.05, **p < 0.01. Data are presented as the mean 6 SEM. (D) Oocyte-derived signaling for granulosa cell activation in aged oocytes was analyzed as in Figures 3J–3M. (E) Immunoblot analyses of the young and aged oocytes were performed using antibodies against the indicated proteins. Two independent experiments are shown. A total of 17 young and 20 aged oocytes were used for each lane. (F) The ATP content of the aged oocytes was measured as in Figure 3C. (G) Schematic diagram of the present model. See details in the text. oocytes were used for immunoblotting. For coculture experiments, GV oocytes were isolated from ovaries. Other methods such as reagents, antibodies, oocyte culture, qPCR, immunofluorescence, primers, immunoblotting, staining, and the measurements of ATP, membrane potential, and Ca2+ are described in the Supplemental Information. Supplemental Information Supplemental Information includes four figures, Supplemental Experimental Procedures, and four movies and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2014.08.060. Author Contributions N.I., M.M., and M.N. contributed to the generation of the mice. O.U., T.I., M.M., Y.M., and S.T. analyzed the ovary phenotypes. O.U., N.K., and K. Miyado contributed to the isolated oocyte experiments. H.S. contributed to mtDNA quantification. S.Y. contributed to EM analysis. N.M. and K.
Mihara supervised work and jointly coordinated the project. N.I. and O.U. planned the project, analyzed the data, and wrote the manuscript. Acknowledgments We thank the members of our mouse facility and Drs Kuwano and Kida (Kurume University) for the use of the plate reader. This work was supported by a Funding Program for Next Generation World-Leading Researchers, by the Takeda Science Foundation, and by the Uehara Memorial Foundation to N.I. Received: May 10, 2014 Revised: July 25, 2014 Accepted: August 26, 2014 Published: September 25, 2014 References 1. McBride, H.M., Neuspiel, M., and Wasiak, S. (2006). Mitochondria: more than just a powerhouse. Curr. Biol. 16, R551–R560.
Current Biology Vol 24 No 20 2458
2. Ishihara, N., Otera, H., Oka, T., and Mihara, K. (2013). Regulation and physiologic functions of GTPases in mitochondrial fusion and fission in mammals. Antioxid. Redox Signal. 19, 389–399. 3. Ishihara, N., Nomura, M., Jofuku, A., Kato, H., Suzuki, S.O., Masuda, K., Otera, H., Nakanishi, Y., Nonaka, I., Goto, Y., et al. (2009). Mitochondrial fission factor Drp1 is essential for embryonic development and synapse formation in mice. Nat. Cell Biol. 11, 958–966. 4. Wakabayashi, J., Zhang, Z., Wakabayashi, N., Tamura, Y., Fukaya, M., Kensler, T.W., Iijima, M., and Sesaki, H. (2009). The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice. J. Cell Biol. 186, 805–816. 5. Waterham, H.R., Koster, J., van Roermund, C.W., Mooyer, P.A., Wanders, R.J., and Leonard, J.V. (2007). A lethal defect of mitochondrial and peroxisomal fission. N. Engl. J. Med. 356, 1736–1741. 6. Sundstro¨m, P., Nilsson, B.O., Liedholm, P., and Larsson, E. (1985). Ultrastructure of maturing human oocytes. Ann. N Y Acad. Sci. 442, 324–331. 7. Dalton, C.M., and Carroll, J. (2013). Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte. J. Cell Sci. 126, 2955–2964. 8. Pepling, M.E., Wilhelm, J.E., O’Hara, A.L., Gephardt, G.W., and Spradling, A.C. (2007). Mouse oocytes within germ cell cysts and primordial follicles contain a Balbiani body. Proc. Natl. Acad. Sci. USA 104, 187–192. 9. Watanabe, T., Chuma, S., Yamamoto, Y., Kuramochi-Miyagawa, S., Totoki, Y., Toyoda, A., Hoki, Y., Fujiyama, A., Shibata, T., Sado, T., et al. (2011). MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline. Dev. Cell 20, 364–375. 10. Wai, T., Teoli, D., and Shoubridge, E.A. (2008). The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes. Nat. Genet. 40, 1484–1488. 11. Tsukamoto, S., Kuma, A., Murakami, M., Kishi, C., Yamamoto, A., and Mizushima, N. (2008). Autophagy is essential for preimplantation development of mouse embryos. Science 321, 117–120. 12. de Vries, W.N., Binns, L.T., Fancher, K.S., Dean, J., Moore, R., Kemler, R., and Knowles, B.B. (2000). Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes. Genesis 26, 110–112. 13. Elvin, J.A., Yan, C., Wang, P., Nishimori, K., and Matzuk, M.M. (1999). Molecular characterization of the follicle defects in the growth differentiation factor 9-deficient ovary. Mol. Endocrinol. 13, 1018–1034. 14. Matzuk, M.M., Burns, K.H., Viveiros, M.M., and Eppig, J.J. (2002). Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science 296, 2178–2180. 15. Gilchrist, R.B., Lane, M., and Thompson, J.G. (2008). Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Hum. Reprod. Update 14, 159–177. 16. Sugino, K., Kurosawa, N., Nakamura, T., Takio, K., Shimasaki, S., Ling, N., Titani, K., and Sugino, H. (1993). Molecular heterogeneity of follistatin, an activin-binding protein. Higher affinity of the carboxyl-terminal truncated forms for heparan sulfate proteoglycans on the ovarian granulosa cell. J. Biol. Chem. 268, 15579–15587. 17. Hsieh, M., Lee, D., Panigone, S., Horner, K., Chen, R., Theologis, A., Lee, D.C., Threadgill, D.W., and Conti, M. (2007). Luteinizing hormonedependent activation of the epidermal growth factor network is essential for ovulation. Mol. Cell. Biol. 27, 1914–1924. 18. Wakai, T. (2012). Mitochondrial localization in mouse oocytes. J Mamm Ova Res 29, 155–160. 19. Ban-Ishihara, R., Ishihara, T., Sasaki, N., Mihara, K., and Ishihara, N. (2013). Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c. Proc. Natl. Acad. Sci. USA 110, 11863–11868. 20. Smirnova, E., Shurland, D.L., Ryazantsev, S.N., and van der Bliek, A.M. (1998). A human dynamin-related protein controls the distribution of mitochondria. J. Cell Biol. 143, 351–358. 21. Pitts, K.R., Yoon, Y., Krueger, E.W., and McNiven, M.A. (1999). The dynamin-like protein DLP1 is essential for normal distribution and morphology of the endoplasmic reticulum and mitochondria in mammalian cells. Mol. Biol. Cell 10, 4403–4417. 22. Bierkamp, C., Luxey, M., Metchat, A., Audouard, C., Dumollard, R., and Christians, E. (2010). Lack of maternal Heat Shock Factor 1 results in multiple cellular and developmental defects, including mitochondrial damage and altered redox homeostasis, and leads to reduced survival of mammalian oocytes and embryos. Dev. Biol. 339, 338–353.
23. Cox, R.T., and Spradling, A.C. (2003). A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis. Development 130, 1579–1590. 24. Buccione, R., Schroeder, A.C., and Eppig, J.J. (1990). Interactions between somatic cells and germ cells throughout mammalian oogenesis. Biol. Reprod. 43, 543–547. 25. Wigglesworth, K., Lee, K.B., O’Brien, M.J., Peng, J., Matzuk, M.M., and Eppig, J.J. (2013). Bidirectional communication between oocytes and ovarian follicular somatic cells is required for meiotic arrest of mammalian oocytes. Proc. Natl. Acad. Sci. USA 110, E3723–E3729. 26. Ducibella, T., Duffy, P., Reindollar, R., and Su, B. (1990). Changes in the distribution of mouse oocyte cortical granules and ability to undergo the cortical reaction during gonadotropin-stimulated meiotic maturation and aging in vivo. Biol. Reprod. 43, 870–876. 27. Li, H., Alavian, K.N., Lazrove, E., Mehta, N., Jones, A., Zhang, P., Licznerski, P., Graham, M., Uo, T., Guo, J., et al. (2013). A Bcl-xL-Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis. Nat. Cell Biol. 15, 773–785. 28. Cao, L., Shitara, H., Horii, T., Nagao, Y., Imai, H., Abe, K., Hara, T., Hayashi, J., and Yonekawa, H. (2007). The mitochondrial bottleneck occurs without reduction of mtDNA content in female mouse germ cells. Nat. Genet. 39, 386–390. 29. Carroll, J., Swann, K., Whittingham, D., and Whitaker, M. (1994). Spatiotemporal dynamics of intracellular [Ca2+]i oscillations during the growth and meiotic maturation of mouse oocytes. Development 120, 3507–3517. 30. Rizzuto, R., Duchen, M.R., and Pozzan, T. (2004). Flirting in little space: the ER/mitochondria Ca2+ liaison. Sci. STKE 2004, re1. 31. Kornmann, B. (2013). The molecular hug between the ER and the mitochondria. Curr. Opin. Cell Biol. 25, 443–448. 32. Sugiura, K., Su, Y.Q., Diaz, F.J., Pangas, S.A., Sharma, S., Wigglesworth, K., O’Brien, M.J., Matzuk, M.M., Shimasaki, S., and Eppig, J.J. (2007). Oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells. Development 134, 2593–2603. 33. Muruvi, W., Picton, H.M., Rodway, R.G., and Joyce, I.M. (2009). In vitro growth and differentiation of primary follicles isolated from cryopreserved sheep ovarian tissue. Anim. Reprod. Sci. 112, 36–50. 34. Van Blerkom, J. (1991). Microtubule mediation of cytoplasmic and nuclear maturation during the early stages of resumed meiosis in cultured mouse oocytes. Proc. Natl. Acad. Sci. USA 88, 5031–5035. 35. Tilly, J.L., and Sinclair, D.A. (2013). Germline energetics, aging, and female infertility. Cell Metab. 17, 838–850. 36. Selesniemi, K., Lee, H.J., Muhlhauser, A., and Tilly, J.L. (2011). Prevention of maternal aging-associated oocyte aneuploidy and meiotic spindle defects in mice by dietary and genetic strategies. Proc. Natl. Acad. Sci. USA 108, 12319–12324. 37. de Bruin, J.P., Dorland, M., Spek, E.R., Posthuma, G., van Haaften, M., Looman, C.W., and te Velde, E.R. (2004). Age-related changes in the ultrastructure of the resting follicle pool in human ovaries. Biol. Reprod. 70, 419–424. 38. Tarı´n, J.J., Pe´rez-Albala´, S., and Cano, A. (2001). Cellular and morphological traits of oocytes retrieved from aging mice after exogenous ovarian stimulation. Biol. Reprod. 65, 141–150. 39. Taguchi, N., Ishihara, N., Jofuku, A., Oka, T., and Mihara, K. (2007). Mitotic phosphorylation of dynamin-related GTPase Drp1 participates in mitochondrial fission. J. Biol. Chem. 282, 11521–11529.
http://dx.doi.org/10.1016/j.cub.2014.08.060
Report Mitochondrial Fission Factor Drp1 Maintains Oocyte Quality via Dynamic Rearrangement of Multiple Organelles Osamu Udagawa,1,2 Takaya Ishihara,1 Maki Maeda,1,3 Yui Matsunaga,3 Satoshi Tsukamoto,3,4 Natsuko Kawano,5 Kenji Miyado,5 Hiroshi Shitara,6 Sadaki Yokota,7 Masatoshi Nomura,8 Katsuyoshi Mihara,9 Noboru Mizushima,3,10 and Naotada Ishihara1,3,* 1Department of Protein Biochemistry, Institute of Life Science, Kurume University, Kurume 839-0864, Japan 2National Institute for Environmental Studies, Center for Environmental Risk Research, Tsukuba 305-8506, Japan 3Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan 4Laboratory Animal Sciences Section, National Institute of Radiological Sciences, Chiba 263-8555, Japan 5National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan 6Laboratory for Transgenic Technology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan 7Pharmaceutical Sciences, Nagasaki International University, Sasebo 859-3298, Japan 8Department of Medicine and Bioregulatory Science, Kyushu University, Fukuoka 812-8582, Japan 9Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan 10Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan
Summary Mitochondria are dynamic organelles that change their morphology by active fusion and fission in response to cellular signaling and differentiation [1, 2]. The in vivo role of mitochondrial fission in mammals has been examined by using tissue-specific knockout (KO) mice of the mitochondria fission-regulating GTPase Drp1 [3, 4], as well as analyzing a human patient harboring a point mutation in Drp1 [5], showing that Drp1 is essential for embryonic and neonatal development and neuronal function. During oocyte maturation and aging, structures of various membrane organelles including mitochondria and the endoplasmic reticulum (ER) are changed dynamically [6, 7], and their organelle aggregation is related to germ cell formation and epigenetic regulation [8–10]. However, the underlying molecular mechanisms of organelle dynamics during the development and aging of oocytes have not been well understood. Here, we analyzed oocyte-specific mitochondrial fission factor Drp1-deficient mice and found that mitochondrial fission is essential for follicular maturation and ovulation in an agedependent manner. Mitochondria were highly aggregated with other organelles, such as the ER and secretory vesicles, in KO oocyte, which resulted in impaired Ca2+ signaling, intercellular communication via secretion, and meiotic resumption. We further found that oocytes from aged mice
*Correspondence: [email protected]
displayed reduced Drp1-dependent mitochondrial fission and defective organelle morphogenesis, similar to Drp1 KO oocytes. On the basis of these findings, it appears that mitochondrial fission maintains the competency of oocytes via multiorganelle rearrangement. Results and Discussion Drp1 in Oocytes Is Essential for Female Fertility, Ovulation, Follicular Maturation, and the Proliferation of Granulosa Cells around KO Oocytes To examine the contribution of mitochondrial fission to oocyte formation, mice bearing a Drp1flox allele were crossed to a transgenic line expressing ZP3-Cre, which is active in growing oocytes [11, 12]. To assess the fertility, we bred female Drp1flox/flox, ZP3-Cre mice, which we refer to here as KO mice, and control mice with stud males (Figure 1A). During a period of up to 2 months, mated control female mice gave an average of 6.7 pups per mating; however, KO mice gave 0.63 pups per mating, despite normal mating behavior and vaginal plug formation. Conversely, Drp1 KO males showed normal fecundity. Consistently, we could recover very few or no oocytes from the oviducts of KO mice after superovulation (Figure 1B), suggesting that the KO mice are defective for ovulation. Histological analyses of 8-week-old ovaries revealed that many immature follicles including primary follicles with a single layer of granulosa cells had accumulated in KO ovaries (Figures 1E–1G). Conversely, control ovaries had several secondary follicles with multiple layers of granulosa cells and several mature follicles with an antral cavity. Retarded follicular development was also observed in the ovaries of KO mice primed with PMSG (Figures 1E–1G). At 8 weeks old, we observed 9.3 oocytes in control and 14.7 oocytes in KO mice per ovary section, suggesting that oocytes were not eliminated from KO follicles. Next, we analyzed the expression of genes crucial for follicular maturation and ovulation using RT-PCR. The expression levels of activin-bB and FSHR, which are implicated in the regulation of granulosa cell proliferation and follicle growth [13–15], were low in KO ovaries (Figure 1C, see also Figures 3K–3M). The levels of follistatin, an endogenous inhibitor of activins [16], were also low. After PMSG injection, these defects were partly rescued, but were still observed (Figure 1C). The induction of LH/hCG target genes in granulosa cells [17] was also compromised (Figure 1D), consistent with the failure of follicular maturation and ovulation in KO mice even after superovulation. We then examined granulosa cell proliferation using the incorporation of bromodeoxyuridine (BrdU). In 8-week-old ovaries, 21.4% of granulosa cells were BrdU positive in the control ovaries (Figure 1H) compared with 8.0% in KO ovaries; thus, granulosa proliferation was severely blocked. Even after PMSG injection, BrdU incorporation was reduced in KO follicles (Figure 1H). The defects seen in the KO mice accumulated in an age-dependent manner, since follicular maturation (Figures 1E–1G) and BrdU incorporation (Figure 1H) were partly affected in 5-week-old KO ovaries. These data demonstrate that Drp1 ablation in oocytes affects follicular maturation and
Current Biology Vol 24 No 20 2452
Figure 1. Defective Follicular Maturation and Ovulation in Drp1 KO Oocytes (A) Control and Drp1 KO female mice (8–10 weeks old) were bred with stud males for up to 2 months. The average number of delivered pups per mouse with plug formation was recorded. Control (cont), n = 10; KO, n = 19; *p < 0.01. (B) Control and KO female 5-week-old mice were superovulated by PMSG and hCG stimulation. The number of oocytes collected from oviducts was counted. Cont, n = 6; KO, n = 6; *p < 0.01. (C and D) Control and KO ovaries from 8-week-old mice primed or not primed with PMSG (C) or from 3- to 4-week-old mice primed or not primed with PMSG and hCG (D) were analyzed by RT-PCR. GAPDH and b-actin were used as loading controls. Areg, amphiregulin; BTC, betacellulin; Has2, hyaluronan synthase 2; Tnfaip6, TNFa-induced protein 6; Ptgs2, prostaglandin synthase 2; LHR, LH receptor. (E and F) Ovaries from 5- and 8-week-old control and KO mice primed or not primed with PMSG were fixed, embedded, sectioned, and stained with hematoxylin and eosin. In the 8-week-old sections of (F), primary follicles (*), secondary follicles (**), and antral follicles (***) are indicated. (G) The number of developing follicles per section was counted (n = 2–3). (H) Top: BrdU was administered intraperitoneally for 2 hr before fixation, and BrdU in the ovarian sections was detected with an anti-BrdU antibody. Bottom: The number of BrdU-positive cells per follicles was counted. Data are presented as the mean 6 SEM.
the proliferation of surrounding granulosa cells, which might reflect the low abundance of FSHR (Figure 1C), leading to insufficient ovulation and female infertility. Abnormal Mitochondrial Morphology in Drp1 KO Oocytes We analyzed mitochondrial morphology in the oocytes of KO mice. Immunofluorescent staining of oocyte cryosections showed that mitochondria were distributed evenly throughout the cytoplasm of oocytes in control 8-week-old ovary sections (Figures 2A and S1A available online), as reported previously
[8, 18]. Many of the Drp1-positive dots were localized to mitochondria. In contrast, mitochondria were highly aggregated in KO oocytes (Figure 2A, arrowhead). As a result, mitochondriafree spaces in the cytoplasm of oocytes could sometimes be detected in many of the cryosections (Figure 2A, arrow). In the granulosa cells of KO mice, Drp1 was expressed normally, confirming that Drp1 was specifically depleted in oocytes. Altered mitochondrial morphology was also observed in oocytes isolated from KO mice (Figures 2B, 2D–2G, and S2A). As expected from our previous report [19], mtDNA nucleoids
Mitochondrial Fission Maintains Oocyte Quality 2453
Figure 2. Various Membrane Structures Are Clustered in Drp1 KO Oocytes (A) Cryosections of 8-week-old control and KO ovaries were stained with antibodies against Tom70 (green; mitochondria) and Drp1 (red). The outline of the oocyte is indicated by a white dotted circle in the merged images. Aggregated mitochondria are shown by the arrowhead, and the mitochondria-free area is shown by an arrow. Inset: magnified images. (B) Isolated oocytes were vitally stained with MitoTracker Red. (C) Electron micrographs of 8-week-old control and KO oocytes. Three magnified images are shown on the right. (D) Isolated oocytes were vitally costained with 100 nM MitoTracker Red and 10 mM ERTracker-DPX for 30 min. (E and F) Fixed oocytes were costained using antibodies against cytochrome c (cyt c) for mitochondria and Pmp70 for peroxisomes (E), or antibodies against Tom20 for mitochondria and GM130 for the Golgi apparatus (F). (G) Secretory vesicles were visualized with LCA-FITC.
were highly clustered in KO oocytes (Figure S2C). Electron microscopy (EM) observation showed that small rounded mitochondria with less developed cristae were distributed throughout the cytoplasm of control oocytes (Figures 2C and S1B). In contrast, elongated mitochondria with an irregular size were highly aggregated in KO oocytes. The average length of the major axis of mitochondria was 0.62 mm in control oocytes, whereas it was 1.12 mm in KO oocytes (Figure S1D). These data suggest that mitochondrial morphology is severely affected in Drp1 KO oocytes.
Multiorganelle Aggregation in Drp1 KO Oocytes Drp1 is believed to be involved in the regulation of mitochondrial and peroxisomal morphology, although the evidence regarding whether Drp1 directly regulates ER morphology is obscure [20, 21]. As expected, small peroxisomes, which were dispersed in the cytoplasm of control oocytes, were clustered and partly overlapped with mitochondria in KO oocytes, as observed in ovary sections (Figure S1A) and isolated oocytes (Figure 2E). In ovary sections, most of the ER was clustered around mitochondrial clusters in KO oocytes
Current Biology Vol 24 No 20 2454
Figure 3. Mitochondrial Activity, Ca2+ Response, and GVBD in Drp1 KO Oocytes (A–D) Mitochondrial activity and mtDNA. (A) Immunoblot analyses of control and Drp1 KO oocytes using the indicated antibodies. A total of 20 control and 28 KO oocytes were used for each. (B) Five-week-old oocytes were stained with the membrane potential-dependent dye MitoTracker Red, and the integrated intensity of each oocyte was quantified. n = 2–7, *p < 0.05. CCCP was used as a negative control. (C) Five-week-old oocytes were subjected to ATP measurement. Oligomycin was used as a negative control. n = 3–5, *p < 0.01. (D) mtDNA copy number in single oocytes was calculated by qPCR and shown against oocyte diameter. (E–I) Ca2+ response. (E) Five-week-old oocytes were stained with Oregon green 488-BAPTA-AM, and the Ca2+ signals were recorded using time-lapse images and plotted. See also Movies S1 and S2. (F and G) Quantification of the frequency (F) and relative amplitude (G) of Ca2+ oscillations observed as in (E) are shown. n > 8, *p < 0.01. (H and I) Five-week-old oocytes were treated with EGTA (which blocks extracellular Ca2+ flux and Ca2+ oscillations) and then treated with thapsigargin. The increase of cytosolic Ca2+ was quantified as stored ER Ca2+ (I). Cont, n = 8; KO, n = 5; *p < 0.01. (J–M) Signaling for granulosa cell activation. (J) Schematic diagram of the experimental design of the coculture assay. (K) The indicated number of control oocytes was cocultured with KO follicles, and the follicles were analyzed by RT-PCR. Primary, secondary, and mature follicles collected from control mice were also examined as standards of follicular maturation. (legend continued on next page)
Mitochondrial Fission Maintains Oocyte Quality 2455
(Figure S1A). The partial overlap of mitochondria and the ER was also observed by vital staining of isolated oocytes (Figures 2D and S2A). In EM examination of control oocytes, most (81.5%) of the small spherical mitochondria were in close contact with ER tubules (Figures 2C, S1B, and S1C), as shown in previous reports [8, 22]. Long ER tubules were in contact with several mitochondria, forming a beaded structure of mitochondria. In contrast, the enlarged mitochondria in KO oocytes clustered with various membranous structures such as small vesicles and multivesicular structures. The induction of ER aggregation by Drp1 deficiency might be a cell-type-specific phenomenon, because Drp1 knockdown in HeLa cells and Drp1 KO in mouse embryonic fibroblasts did not induce ER accumulation [3]. Thus, oocyte-specific frequent and tight interactions between the mitochondria and ER might lead to their coaggregation in Drp1 KO oocytes. We also showed that treatment of isolated oocytes with a Drp1 inhibitor, mdivi-1, led to the formation of mild aggregates containing mitochondria and ER (Figure S2B), which also supports the above conclusion. There are several reports showing that mitochondria aggregate transiently in developing early germ cells. Intermitochondrial cement structures called nuages are important for epigenome regulation [9] and the clustering of mitochondria with the ER and Golgi apparatus to form Balbiani bodies, which are involved in mtDNA inheritance [8, 10, 23]. Here we found that the Golgi apparatus (Figure 2F) and cytoskeletal components, such as actin and tubulin (Figures S2D and S2E), were normal in KO oocytes. No ubiquitin signals were detected in the organelle aggregates (Figure S2E). These data exhibited the different characteristics of Balbiani bodies and aggresomes. Transzonal projections (TZPs), which are plasma membrane extensions from granulosa cells that traverse the zona pellucida, make contact with oocyte microvilli [24, 25]. In EM observation (Figure 2C) and immunofluorescent F-actin staining, which is a marker of TZPs (Figure S2E), we could not find any defects in TZP structures of Drp1 KO oocytes. Lens culinaris agglutinin (LCA) labels secretory vesicles in the cortical cytoplasm and plasma membrane of control oocytes [26]. In contrast, cytoplasmic clumps were highly labeled in Drp1-KO oocytes, indicating the accumulation of abnormal secretory vesicles (Figure 2G). This observation is consistent with a recent report showing aberrant synaptic vesicle structures in Drp1-deficient neurons [27]. Thus, Drp1 depletion influences not only mitochondrial morphology but also several other intracellular membrane structures. Calcium Response Is Affected in Drp1 KO Oocytes Next, we assessed mitochondrial function in Drp1 KO oocytes. First, the expression of several subunits of respiratory chain complexes was found to be normal in Drp1 KO oocytes (Figure 3A), although Drp1 protein was almost completely repressed. The intensity of the membrane-potential-dependent dye MitoTracker Red was comparable between control
and KO oocytes (Figure 3B), even though mitochondrial morphology was severely affected in KO oocytes (Figure 2B). ATP content analyzed by luminometric analysis was faintly affected in KO oocytes (Figure 3C). We then measured mtDNA copy number in isolated oocytes by qPCR. During oocyte growth in control mice, mtDNA copy number was highly increased (to approximately 4.0 3 105 copies) concomitantly with the enlargement of oocyte volume (Figure 3D), as reported previously [28]. However, we could not find a clear difference between the control and KO oocytes when they were the same diameter. Taken together, these findings indicate that mitochondrial fission is not essential for respiration or the increase of mtDNA copy number in oocytes. Ca2+ oscillations are known to be essential for oocyte development and maturation [29]; therefore, we measured spontaneous Ca2+ oscillations of GV-stage oocytes using Oregon Green 488-BAPTA-AM. Ca2+ oscillations occurred in 67% of fully grown GV-stage control oocytes, while oscillations were recorded in 55% of Drp1 KO oocytes (Figures 3E–3G and S3A and Movies S1 and S2). They had a similar frequency (interspike interval: 1.95 min in control oocytes and 2.11 min in KO oocytes) (Figures 3E and 3F), but KO oocytes had a significantly reduced amplitude (approximately 70% of control) (Figure 3G), suggesting that the Ca2+ response was deficient in Drp1 KO oocytes. Mitochondria are known to act as a Ca2+ store, so we checked the role of mitochondrial Ca2+ uptake in Ca2+ oscillations. When we pretreated the oocytes with RU360, an inhibitor of the mitochondrial Ca2+ uniporter, the amplitude was less affected (Figures S3B–S3D), in contrast to the decreased amplitude observed in KO oocytes (Figure 3G). Furthermore, the duration of the oscillations was partly but clearly increased by RU360 treatment, which was also inconsistent with the phenotypes seen in Drp1 KO oocytes (Figures S3B and S3E). Thus, mitochondrial Ca2+ uptake might not be the main cause of the Ca2+ signaling defect observed in Drp1 KO oocytes. In various cell types, Ca2+ is regulated via close contact of the ER with mitochondria, enabling events such as apoptosis and Ca2+ signaling [30, 31]. Next, we determined the contribution of Drp1 to the ER Ca2+ store in oocytes. In the presence of EGTA, to exclude the contribution of extracellular Ca2+ uptake, treatment with thapsigargin, an ER Ca2+ pump inhibitor, caused a large increase of Ca2+ in control oocytes, presumably by releasing Ca2+ from the ER (Figure 3H). However, this increase of Ca2+ was reduced in KO oocytes (Figure 3I), suggesting a decreased store of Ca2+ in the ER. Thus, the Ca2+ response via the ER is compromised in KO oocytes, implying the significance of mitochondria-derived interorganelle communication and cellular signaling. Follicular Arrest due to Defective Secretion from Drp1 KO Oocytes to Surrounding Granulosa Cells It is known that follicular maturation accompanied by granulosa cell proliferation requires communication between an oocyte and the surrounding niche via molecules, such as
(L) Ten naked oocytes isolated from control mice were precultured in medium, and then the medium was isolated. Drp1 KO follicles were cultured in the precultured medium and analyzed as in (K). (M) Ten naked oocytes isolated from control or KO mice were cocultured with KO arrested follicles and analyzed as above. (N and O) Meiotic resumption. Five-week-old oocytes were stained with MitoTracker Red in the presence of IBMX. After washout of IBMX, mitochondrial morphology (red fluorescence) associated with spontaneous meiotic resumption was monitored by time-lapse microscopy. Typical oocytes with aggregated mitochondria (‘‘mit-aggre’’) are shown in Figure S4. See also Movies S3 and S4. (N) The percentage of oocytes that completed GVBD was quantified. n = 2, *p < 0.05. (O) GVBD completion is indicated by a green arrow. GVs are enclosed by white dotted circles and indicated with a yellow arrow. Data are presented as the mean 6 SEM.
Current Biology Vol 24 No 20 2456
GDF-9 and BMP15 secreted from oocytes, and the KIT ligand from granulosa cells [13–15]. To analyze the cause of abnormal follicular maturation in oocyte-specific Drp1 KO mice, we analyzed the communication between oocytes and granulosa cells, referring methods for a coculture system of isolated oocytes with follicles [32], a culture method for follicular differentiation [33], and RT-PCR (model in Figure 3J). Arrested immature follicles isolated from 8-week-old KO mice had a reduced number of activated granulosa cells and lower expression levels of mature follicle markers (Figure 3K), as described above (Figures 1C–1H). These immature KO follicles were cocultured with denuded granulosa-free 5-weekold control oocytes for 24 hr, and total RNA extracted from the KO follicles was examined by RT-PCR (Figure 3J). The increased expression of mature follicular markers suggests that coculturing with the control oocytes restored the activity of the granulosa cells in the KO follicles (Figure 3K). This recovery was caused by materials secreted from the oocytes, since media in which healthy oocytes had been precultured demonstrated follicle-stimulation activity (Figure 3L), as seen in the coculture experiments. In contrast, such a resumption of follicular growth was not detected by coculturing with denuded Drp1 KO oocytes (Figure 3M). As described above, staining of isolated oocytes with LCA showed the defective distribution of secretory vesicles (Figure 2G). Likewise, accumulated vesicles and multivesicular structures were observed in EM analysis of KO oocytes (Figure 2C, arrowheads). These results strongly suggest that the oocyte-derived secretion required for proper granulosa proliferation is impaired in Drp1 KO oocytes. In Drp1 KO oocytes, the zona pellucida, an extracellular matrix structure veiling oocytes, was formed (Figures S2D and S2E, arrow), which requires the secretion of glycoproteins such as ZP1 and ZP3, suggesting normal constitutive secretion; thus, we speculated that the Ca2+-dependent regulatory response might be impaired. Meiotic Resumption Is Defective in Drp1 KO Oocytes The germinal vesicle (GV) is a specialized nuclear structure of oocytes that is highly enlarged during oocyte maturation. At the resumption of meiotic maturation, GV break down (GVBD) occurs prior to polar body formation. During the GVBD stage, the dispersed mitochondria [6, 8] accumulate around the GV [34]. To elucidate further the relationship between the dynamics of mitochondria and other organelles in oocytes, we focused on GVBD. In our culture condition, GVBD was observed in 73.3% of control oocytes from 5-week-old mice between 2 and 4 hr of culture (Figure 3N). In contrast, GVBD was observed at a lower level in Drp1 KO oocytes (46.6%). This mild defect might be caused by the moderate phenotype in 5-week-old oocytes (see Figures 1E–1H), since GVBD was never observed in KO oocytes with solely aggregated mitochondria (Figures 3N and S4, ‘‘mit-aggre’’), suggesting that mitochondrial morphology affects the progression of GVBD. We analyzed mitochondrial dynamics further by live imaging. During GVBD, small fragmented mitochondria frequently and dynamically moved in the cytoplasm of control GV oocytes (Movie S3 and Figures 3O and S4). Before GVBD, a significant amount of mitochondria gathered to enwrap GVs. The ER also coaccumulated with mitochondria at the pre-GVBD stage in control oocytes (Figure S4). After GVBD, the mitochondria stopped their movement in the cytoplasm (Movie S3 and Figure 3O). In contrast, mitochondrial movement was restricted and GVBD was defective in KO oocytes with severe mitochondrial aggregation (Movie S4 and Figures 3O and S4).
These results demonstrate that mitochondrial dynamics are important for the rearrangement of multiple organelles during oocyte maturation. Deformed Mitochondrial Morphology and Drp1 Repression in Oocytes from Aged Mice During the long period of meiotic arrest in follicles, mitochondria are believed to be one of the leading determinants of oocyte quality [35]. Here we analyzed mitochondrial morphology and Drp1 in oocytes isolated from aged mice. In ovulated oocytes isolated from 8- to 14-week-old mice, small and short mitochondria were dispersed throughout the cytoplasm (Figures 4A and 4C) as described above (Figure 2). In contrast, in oocytes isolated from mice aged older than 11 months, which have reduced ovulation activity (4.6 oocytes per animal), the mitochondria and ER were aggregated more frequently (Figures 4A and 4C), confirming previous reports [36–38]. We further found that peroxisomes and secretory vesicles were also deformed in aged oocytes (Figure 4B). The secretory activity for stimulating granulosa cells was defective in aged oocytes (Figure 4D). Furthermore, immunoblot experiments revealed that the expression and phosphorylation of Drp1 was clearly and reproducibly repressed in aged oocytes compared with oocytes isolated from young mice (Figure 4E). Drp1 phosphorylation at Ser616 reportedly enhances mitochondrial fission [39]. These results suggest that mitochondrial morphology and Drp1-dependent mitochondrial fission are altered in aged oocytes, and we hypothesized that Drp1dependent mitochondrial fission might have a critical role in oocyte competency in mice (models in Figure 4G). Meanwhile, ATP content was partly decreased in aged oocytes (Figure 4F), inconsistent with Drp1 KO oocytes, suggesting that mitochondrial fission is not the sole causal defect in aged oocytes. It is noteworthy that the decreased ATP content caused neither mitochondrial nor ER aggregation in oocytes (Figure S2E), suggesting that Drp1 deficiency leads to multiorganelle deformation in aged oocytes, which might affect competency of oocytes as seen in Drp1 KO oocytes. In conclusion, the Drp1-dependent spatiotemporal regulation of mitochondria severely affects various types of membrane dynamics in oocytes, and thus, mitochondria act as a center for dynamic membrane remodeling during the maturation of oocytes. Our findings also indicate that oocyte-specific Drp1 KO mice are a potentially useful model for analyzing the cytoplasmic aging of oocytes. Experimental Procedures All mouse procedures and protocols were in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee at Kurume University. The female fertility test was carried out by mating with normal adult male mice for up to 2 months after checking for the presence of copulation plugs. Drp1flox/flox mice were generated as described previously [3]. ZP3-Cre Tg mice [11, 12] were used to produce the oocyte-specific Drp1-deficient mice. To isolate GV oocytes, ovarian follicular development was stimulated by an intraperitoneal injection of 5 IU PMSG to 5-week-old mice. Oocyte-cumulus cell complexes (OCCs) were collected from ovaries at 48 hr after PMSG priming, and cumulus cells surrounding the oocytes were removed completely by passing the OCCs several times through a hand-drawn small fine glass pipette. To isolate ovulated oocytes, mice were stimulated by an injection of 5 IU PMSG followed 48 hr later by 5 IU hCG. Oocytes were collected from the oviduct by gently teasing apart the ampulla to release OCCs into medium containing 0.3 mg/ml hyaluronidase and incubating OCCs at 37 C for 3– 5 min. For the analysis of aged oocytes, the injection of 10 IU PMSG was followed 48 hr later by the injection of 10 IU hCG for 20 hr. Cumulus-free
Mitochondrial Fission Maintains Oocyte Quality 2457
Figure 4. Drp1-Dependent Mitochondrial Morphogenesis Is Altered in Aged Oocytes (A) Subcellular distribution of the mitochondria and ER in young (8–14 weeks) and aged (44–77 weeks) oocytes. Superovulated oocytes were vitally costained with 200 nM MitoTracker Red and 10 mM ERTracker-DPX for 30 min in HTF medium. (B) The subcellular distribution of mitochondria, peroxisomes, and secretory vesicles in aged oocytes are shown. (C) Classification of the mitochondrial morphology of oocytes at each age. Oocytes with highly aggregated mitochondria were classified as ‘‘mit-aggre;’’ multiple types of milder or smaller aggregates were categorized as ‘‘mild-aggre.’’ Oocytes with dispersed mitochondria were categorized as ‘‘dispersion.’’ Young, n = 129; aged, n = 130; *p < 0.05, **p < 0.01. Data are presented as the mean 6 SEM. (D) Oocyte-derived signaling for granulosa cell activation in aged oocytes was analyzed as in Figures 3J–3M. (E) Immunoblot analyses of the young and aged oocytes were performed using antibodies against the indicated proteins. Two independent experiments are shown. A total of 17 young and 20 aged oocytes were used for each lane. (F) The ATP content of the aged oocytes was measured as in Figure 3C. (G) Schematic diagram of the present model. See details in the text. oocytes were used for immunoblotting. For coculture experiments, GV oocytes were isolated from ovaries. Other methods such as reagents, antibodies, oocyte culture, qPCR, immunofluorescence, primers, immunoblotting, staining, and the measurements of ATP, membrane potential, and Ca2+ are described in the Supplemental Information. Supplemental Information Supplemental Information includes four figures, Supplemental Experimental Procedures, and four movies and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2014.08.060. Author Contributions N.I., M.M., and M.N. contributed to the generation of the mice. O.U., T.I., M.M., Y.M., and S.T. analyzed the ovary phenotypes. O.U., N.K., and K. Miyado contributed to the isolated oocyte experiments. H.S. contributed to mtDNA quantification. S.Y. contributed to EM analysis. N.M. and K.
Mihara supervised work and jointly coordinated the project. N.I. and O.U. planned the project, analyzed the data, and wrote the manuscript. Acknowledgments We thank the members of our mouse facility and Drs Kuwano and Kida (Kurume University) for the use of the plate reader. This work was supported by a Funding Program for Next Generation World-Leading Researchers, by the Takeda Science Foundation, and by the Uehara Memorial Foundation to N.I. Received: May 10, 2014 Revised: July 25, 2014 Accepted: August 26, 2014 Published: September 25, 2014 References 1. McBride, H.M., Neuspiel, M., and Wasiak, S. (2006). Mitochondria: more than just a powerhouse. Curr. Biol. 16, R551–R560.
Current Biology Vol 24 No 20 2458
2. Ishihara, N., Otera, H., Oka, T., and Mihara, K. (2013). Regulation and physiologic functions of GTPases in mitochondrial fusion and fission in mammals. Antioxid. Redox Signal. 19, 389–399. 3. Ishihara, N., Nomura, M., Jofuku, A., Kato, H., Suzuki, S.O., Masuda, K., Otera, H., Nakanishi, Y., Nonaka, I., Goto, Y., et al. (2009). Mitochondrial fission factor Drp1 is essential for embryonic development and synapse formation in mice. Nat. Cell Biol. 11, 958–966. 4. Wakabayashi, J., Zhang, Z., Wakabayashi, N., Tamura, Y., Fukaya, M., Kensler, T.W., Iijima, M., and Sesaki, H. (2009). The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice. J. Cell Biol. 186, 805–816. 5. Waterham, H.R., Koster, J., van Roermund, C.W., Mooyer, P.A., Wanders, R.J., and Leonard, J.V. (2007). A lethal defect of mitochondrial and peroxisomal fission. N. Engl. J. Med. 356, 1736–1741. 6. Sundstro¨m, P., Nilsson, B.O., Liedholm, P., and Larsson, E. (1985). Ultrastructure of maturing human oocytes. Ann. N Y Acad. Sci. 442, 324–331. 7. Dalton, C.M., and Carroll, J. (2013). Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte. J. Cell Sci. 126, 2955–2964. 8. Pepling, M.E., Wilhelm, J.E., O’Hara, A.L., Gephardt, G.W., and Spradling, A.C. (2007). Mouse oocytes within germ cell cysts and primordial follicles contain a Balbiani body. Proc. Natl. Acad. Sci. USA 104, 187–192. 9. Watanabe, T., Chuma, S., Yamamoto, Y., Kuramochi-Miyagawa, S., Totoki, Y., Toyoda, A., Hoki, Y., Fujiyama, A., Shibata, T., Sado, T., et al. (2011). MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline. Dev. Cell 20, 364–375. 10. Wai, T., Teoli, D., and Shoubridge, E.A. (2008). The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes. Nat. Genet. 40, 1484–1488. 11. Tsukamoto, S., Kuma, A., Murakami, M., Kishi, C., Yamamoto, A., and Mizushima, N. (2008). Autophagy is essential for preimplantation development of mouse embryos. Science 321, 117–120. 12. de Vries, W.N., Binns, L.T., Fancher, K.S., Dean, J., Moore, R., Kemler, R., and Knowles, B.B. (2000). Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes. Genesis 26, 110–112. 13. Elvin, J.A., Yan, C., Wang, P., Nishimori, K., and Matzuk, M.M. (1999). Molecular characterization of the follicle defects in the growth differentiation factor 9-deficient ovary. Mol. Endocrinol. 13, 1018–1034. 14. Matzuk, M.M., Burns, K.H., Viveiros, M.M., and Eppig, J.J. (2002). Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science 296, 2178–2180. 15. Gilchrist, R.B., Lane, M., and Thompson, J.G. (2008). Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Hum. Reprod. Update 14, 159–177. 16. Sugino, K., Kurosawa, N., Nakamura, T., Takio, K., Shimasaki, S., Ling, N., Titani, K., and Sugino, H. (1993). Molecular heterogeneity of follistatin, an activin-binding protein. Higher affinity of the carboxyl-terminal truncated forms for heparan sulfate proteoglycans on the ovarian granulosa cell. J. Biol. Chem. 268, 15579–15587. 17. Hsieh, M., Lee, D., Panigone, S., Horner, K., Chen, R., Theologis, A., Lee, D.C., Threadgill, D.W., and Conti, M. (2007). Luteinizing hormonedependent activation of the epidermal growth factor network is essential for ovulation. Mol. Cell. Biol. 27, 1914–1924. 18. Wakai, T. (2012). Mitochondrial localization in mouse oocytes. J Mamm Ova Res 29, 155–160. 19. Ban-Ishihara, R., Ishihara, T., Sasaki, N., Mihara, K., and Ishihara, N. (2013). Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c. Proc. Natl. Acad. Sci. USA 110, 11863–11868. 20. Smirnova, E., Shurland, D.L., Ryazantsev, S.N., and van der Bliek, A.M. (1998). A human dynamin-related protein controls the distribution of mitochondria. J. Cell Biol. 143, 351–358. 21. Pitts, K.R., Yoon, Y., Krueger, E.W., and McNiven, M.A. (1999). The dynamin-like protein DLP1 is essential for normal distribution and morphology of the endoplasmic reticulum and mitochondria in mammalian cells. Mol. Biol. Cell 10, 4403–4417. 22. Bierkamp, C., Luxey, M., Metchat, A., Audouard, C., Dumollard, R., and Christians, E. (2010). Lack of maternal Heat Shock Factor 1 results in multiple cellular and developmental defects, including mitochondrial damage and altered redox homeostasis, and leads to reduced survival of mammalian oocytes and embryos. Dev. Biol. 339, 338–353.
23. Cox, R.T., and Spradling, A.C. (2003). A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis. Development 130, 1579–1590. 24. Buccione, R., Schroeder, A.C., and Eppig, J.J. (1990). Interactions between somatic cells and germ cells throughout mammalian oogenesis. Biol. Reprod. 43, 543–547. 25. Wigglesworth, K., Lee, K.B., O’Brien, M.J., Peng, J., Matzuk, M.M., and Eppig, J.J. (2013). Bidirectional communication between oocytes and ovarian follicular somatic cells is required for meiotic arrest of mammalian oocytes. Proc. Natl. Acad. Sci. USA 110, E3723–E3729. 26. Ducibella, T., Duffy, P., Reindollar, R., and Su, B. (1990). Changes in the distribution of mouse oocyte cortical granules and ability to undergo the cortical reaction during gonadotropin-stimulated meiotic maturation and aging in vivo. Biol. Reprod. 43, 870–876. 27. Li, H., Alavian, K.N., Lazrove, E., Mehta, N., Jones, A., Zhang, P., Licznerski, P., Graham, M., Uo, T., Guo, J., et al. (2013). A Bcl-xL-Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis. Nat. Cell Biol. 15, 773–785. 28. Cao, L., Shitara, H., Horii, T., Nagao, Y., Imai, H., Abe, K., Hara, T., Hayashi, J., and Yonekawa, H. (2007). The mitochondrial bottleneck occurs without reduction of mtDNA content in female mouse germ cells. Nat. Genet. 39, 386–390. 29. Carroll, J., Swann, K., Whittingham, D., and Whitaker, M. (1994). Spatiotemporal dynamics of intracellular [Ca2+]i oscillations during the growth and meiotic maturation of mouse oocytes. Development 120, 3507–3517. 30. Rizzuto, R., Duchen, M.R., and Pozzan, T. (2004). Flirting in little space: the ER/mitochondria Ca2+ liaison. Sci. STKE 2004, re1. 31. Kornmann, B. (2013). The molecular hug between the ER and the mitochondria. Curr. Opin. Cell Biol. 25, 443–448. 32. Sugiura, K., Su, Y.Q., Diaz, F.J., Pangas, S.A., Sharma, S., Wigglesworth, K., O’Brien, M.J., Matzuk, M.M., Shimasaki, S., and Eppig, J.J. (2007). Oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells. Development 134, 2593–2603. 33. Muruvi, W., Picton, H.M., Rodway, R.G., and Joyce, I.M. (2009). In vitro growth and differentiation of primary follicles isolated from cryopreserved sheep ovarian tissue. Anim. Reprod. Sci. 112, 36–50. 34. Van Blerkom, J. (1991). Microtubule mediation of cytoplasmic and nuclear maturation during the early stages of resumed meiosis in cultured mouse oocytes. Proc. Natl. Acad. Sci. USA 88, 5031–5035. 35. Tilly, J.L., and Sinclair, D.A. (2013). Germline energetics, aging, and female infertility. Cell Metab. 17, 838–850. 36. Selesniemi, K., Lee, H.J., Muhlhauser, A., and Tilly, J.L. (2011). Prevention of maternal aging-associated oocyte aneuploidy and meiotic spindle defects in mice by dietary and genetic strategies. Proc. Natl. Acad. Sci. USA 108, 12319–12324. 37. de Bruin, J.P., Dorland, M., Spek, E.R., Posthuma, G., van Haaften, M., Looman, C.W., and te Velde, E.R. (2004). Age-related changes in the ultrastructure of the resting follicle pool in human ovaries. Biol. Reprod. 70, 419–424. 38. Tarı´n, J.J., Pe´rez-Albala´, S., and Cano, A. (2001). Cellular and morphological traits of oocytes retrieved from aging mice after exogenous ovarian stimulation. Biol. Reprod. 65, 141–150. 39. Taguchi, N., Ishihara, N., Jofuku, A., Oka, T., and Mihara, K. (2007). Mitotic phosphorylation of dynamin-related GTPase Drp1 participates in mitochondrial fission. J. Biol. Chem. 282, 11521–11529.