Mitochondria in early mammalian development

Seminars in Cell & Developmental Biology 20 (2009) 354–364

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Seminars in Cell & Developmental Biology journal homepage: www.elsevier.com/locate/semcdb

Review

Mitochondria in early mammalian development Jonathan Van Blerkom a,b,∗ a b

Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80302, United States Colorado Reproductive Endocrinology Rose Medical Center, Denver, CO, United States

a r t i c l e

i n f o

Article history: Available online 24 December 2008 Keywords: Mitochondria Bioenergetics Spatial remodeling Regulatory roles Signaling

a b s t r a c t The role of mitochondria as central determinants of development competence of oocytes and preimplantation stage embryos is considered in the context of the diverse activities these organelles have in normal cell function. Stage- and cell-cycle-specific mitochondrial translocations and redistributions are described with respect to mechanisms of cytoplasmic remodeling that may establish domains of autonomous regulation of mitochondrial function and activity during early development. The functions of mitochondria as intracellular signaling elements, as regulators of signaling pathways, and oxygen sensors in differentiated cells are suggested to have similar capacities during mammalian oogenesis and early embryogenesis. Questions concerning the numerical size of the oocyte mitochondrial complement, the energy required to support normal preovulatory oogenesis and preimplantation embryogenesis, and the regulation of mitochondrial activity by extrinsic and intrinsic factors are addressed with respect the potential they may have for new investigational approaches to study the origin of the differential developmental competence of human oocytes and preimplantation stage embryos. © 2008 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondria in oocytes and early embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial numbers and bioenergetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disproportionate mitochondrial inheritance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bioenergetics and development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stage-specific changes in the spatial distribution of mitochondria during early development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence suggesting mitochondrial microzonation during early development and the relationship between  m, nitric oxide and oxygen . . Perspectives for future research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction From the earliest stages of a typical biology education curriculum, mitochondria are presented as the ‘powerhouses’ of the cell, and with more advanced cell biology and biochemistry courses, mitochondrial function is generally taught in terms of the details of enzymatic activities, respiratory cycles and the electron transport cascade involved in the generation of ATP. Whether it is unpleasant recollections of required memorization of the intermediately steps in these cycles and pathways, or the absence of direct relevance in

∗ Correspondence address: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80302, United States. E-mail address: [email protected]. 1084-9521/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2008.12.005

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daily clinical practice, it is not unusual to see the eyes of individuals engaged studies of early development, including human in vitro fertilization (IVF), glaze over when this type of information is presented in the context of developmental competence for oocytes and early embryos. Yet, the recognition of mitochondria as important determinants of the normality of oogenesis and preimplantation embryogenesis has become increasingly evident from both animal and clinical studies that have sought to correlate mitochondrial function and activity with developmental competence [1–6]. For the human, interest in mitochondria has focused primarily on their respiratory contribution to the bioenergetic capacity of the cytoplasm that supports developmentally critical processes associated with preovulatory nuclear and cytoplasmic maturation of the oocyte and morphodynamic activities such as compaction, cavitation, blastocyst expansion and hatching in preimplantation

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stage embryos. In particular, recent studies have sought to determine the role of mitochondria in early development by correlating ATP levels [6] with the normality of meiotic and mitotic spindle organization and chromosomal segregation during resumed meiosis in the oocyte and early cleavage in the embryo, respectively [7,8]. While the primary intent of these studies has been to determine threshold levels of ATP required to produce a chromosomally normal metaphase II oocyte (MII), or euploid (diploid) blastomere, other developmentally relevant issues in which a regulatory role for mitochondria has been indicated include certain aspects of fertilization [9], the normality of cytokinesis during cleavage [10], and their potential contribution to apoptotic cell death during the preimplantation stage [11]. Abnormalities in mitochondrial distribution are common in MII oocytes and early embryos and their occurrence in unusually large aggregates has been negatively correlated with developmental competence [12,13]. In clinical IVF, the detection of this cytoplasmic phenotype or ‘dysmorphism’ [14] at the light microscope level is often included in competence assessment schemes used to select oocytes for insemination [15,16]. The etiology of atypical mitochondrial aggregation (e.g., Fig. 1H, I, M, and N) is largely thought to result from aberrations in the organization of cytoskeletal elements that normally direct the movements of mitochondria within the cytoplasm ([17], see below), and as such, likely reflect underlying cytostructural defects that negatively influence developmental competence at several levels, of which mitochondrial organization is one [18,19]. What has become increasingly apparent from investigations of mitochondrial function in differentiated cells that may be relevant for investigation in the oocyte and early embryo is that in addition to their contribution to bioenergetic ability, they participate in diverse cellular activities such as the regulation of calcium homeostasis, oxygen sensing, fatty acid oxidation (␤-oxidation) and signal transduction [1,5,20,21]. As the principal source of reactive oxygen species (ROS) such as superoxide, mitochondrial damage associated with leakiness of the electron transport chain can initiate apoptotic pathways at high levels, whereas the lower levels of superoxide they normally produce are physiological and indeed necessary for important cellular functions [22]. The latter issue is a potentially important one in clinical IVF because the detection of ROS in human oocytes and early embryos with highly sensitive fluorescent probes, rather than precise identification of species and quantification of level, has been generally viewed as a negative factor for competence or indicative of developmentally premorbid condition [23] and has led some investigators to suggest that supplementation of culture medium with a combination of antioxidants may improve outcome after IVF by reducing the potential for ROS-induced cellular damage during prolonged embryo culture [24]. However, whether the benefits of this type of proactive approach are more apparent than real will require validation with outcome results from welldesigned prospective studies, and a clear demonstration that the levels of supplementation do not adversely affect signaling pathways in which ROS are normally involved [5,25,26]. The possibility of improving embryo performance by the manipulation of mitochondrial function or activity illustrates two emerging issues concerning the role of these organelles in early development and the possible implications they may have in clinical IVF. First, it is now generally accepted that mitochondrial function, activity and distribution are critical elements in the establishment and maintenance of developmental viability for both oocyte and embryo. Second, certain developmental dysfunctions assumed to be mitochondrial in origin, such as lethal levels of fragmentation, have been clinically treated by novel invasive methods. For example, the mechanical transfer of mitochondria [27] or cytoplasm derived from donor to patient oocytes [28] has been reported to be of benefit for certain infertile women by improving embryo

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developmental performance in vitro and outcome after transfer. However, an unintended consequence of cytoplasmic or mitochondrial transfer is the disproportionate expansion of the donor forms that creates a heteroplasmic condition in the cells of the offspring (see below, [29,30]). This unexpected outcome illustrates the need for unambiguous proof that idiopathic infertility associated with compromised developmental competence for the oocyte or embryo has an etiology that is mitochondrially based. One of the intents of this review is to discuss the current understanding of the role of mitochondria in early development and the extent to which dysfunctions or numerical deficiencies can be reasonably assumed to be proximate influences on oocyte competence and outcome. 2. Mitochondria in oocytes and early embryos All mitochondria in an individual are maternally inherited and during the preimplantation stages, their number/embryo should remain relatively constant because replication is not initiated until after implantation [31]. Consequently, the complement present in each mature oocyte (MII) at the time of fertilization represents a fixed number that is halved with each cell division during the preimplantation stages, assuming that segregation between daughter cells is largely numerically equivalent. Therefore, as the primary source of ATP, the number of mitochondria in the oocyte is likely an essential characteristic of the bioenergetic ability of the embryo to develop progressively and normally after fertilization [2–5]. This appears to be of particular significance in the human, where molecular quantification of mitochondrial DNA copy numbers (mtDNA) indicates (i) very significant differences exist between oocytes from the same and different cohorts, and (ii) subnormal mtDNA levels, if reflective of actual mitochondrial numbers, could be directly related to the capacity of the preovulatory oocyte to progress through meiotic maturation (to MII), or to develop normally after fertilization [32–35]. In other instances, defects in mitochondrial structure, such as organelle swelling and disruption of the cristae membranes, have been related to developmental incompetence for the oocyte, and their occurrence in the gametes of women of advanced maternal age in particular, has been suggested to be a significant factor contributing to the well-known age-related reduction in fertility [36]. The establishment of a direct correspondence between developmental competence and mitochondrial numbers, if related to levels of respiratory activity, offers the potential to identify a common etiology for diverse defects in developmental ability that are frequent in the human, such as abnormal or arrested cleavage [23], cytoplasmic and chromosomal abnormalities (e.g., aneuploidy) in oocytes and early embryos [37,38]. This notion is also appealing from an experimental standpoint because it may allow latent developmental defects that involve mitochondria, especially if negative effects manifest in the peri-implantation period, to be traced to the earliest stage of oogenesis, where an exponential expansion from <100 progenitor organelles in the primordial germ cell, to tens, if not hundreds of thousands of mitochondria, occurs by the cessation of oocyte growth ([39], see below). Indeed, mitochondrial expansion during oogenesis is a critical determinant of the severity with which certain inherited metabolic diseases (OXPHOS disease) are expressed. In these instances, mitochondria carrying pathogenic mtDNA mutations co-exist with normal forms (i.e., a heteroplasmic state); it is the type of genetic defect and the threshold levels at which these mutations occur (mutant load) that determines whether the condition is benign or toxic during development and after birth, debilitating or lethal for the affected individual [40,41]. In addition to genetic and functional aspects of mitochondria that can have important influences on the normality of early development, stage-specific changes in fine structural and spatial

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Fig. 1. Light and conventional epifluorescent microscopic images of maturing mouse oocytes (A–I) and two-cell embryos (J–N) stained with the mitochondria-specific fluorescent probe, rhodamine 123. At the outset of maturation, the germinal vesicle stage (A), mitochondria are largely uniformly distributed throughout the cytoplasm (B). Beginning around germinal vesicle breakdown and becoming more pronounced at the circular bivalent stage (CBV; C), mitochondria (M) are translocated to the perinuclear region along microtubular tracks that emanate from perinuclear microtubular organizing centers (MTOCs). In cross section, the perinuclear aggregation of mitochondria appears as a ring (arrows, C and D) that surrounds newly condensed chromosomes (cc; C), but in actuality, it occurs as a sphere. The perinuclear sphere persists through the formation of the first metaphase spindle (arrows, E and F) but assumes an elongated shape that conforms to the geometry of the perinuclear region as it prepares to segregate one diploid set of chromosomes into the first polar body at the completion of the first meiotic division. The arrest of meiosis at metaphase of the second meiotic division (MII) in the preovulatory oocyte is associated with a return to a more uniform cytoplasmic distribution of mitochondria (G), and in some oocytes, an apical cap of fluorescent organelles is detected in the newly released first polar body (arrow, G). Abnormal mitochondrial clustering and aggregation is common in mouse (H and I) oocytes matured in vitro and human oocytes obtained by follicular aspiration after ovarian hyperstimulation for in vitro fertilization. In the mouse, ancillary spheres of mitochondria form around MTOCs that failed to assume a perinuclear distribution (asterisks, H), while in the human, dense clusters of mitochondria persist in oocytes that prematurely arrest meiosis (i.e., prior to MII; arrows, I). Both phenotypes are inconsistent with developmental competence and indicative of underlying disorders in cytoskeletal and microtubular organization that likely result from focal abnormalities in cytoplasmic physiology. Perinuclear mitochondrial aggregation is a normal characteristic of cytoplasmic remodeling during cleavage. The arrows in (J–L) show cell-cycle-related perinuclear mitochondrial accumulations observed in the RITC (K) and FITC (L) channels. Common abnormalities in mitochondrial translocation that are frequent during cleavage in vitro include the persistence of dense clusters of mitochondria dispersed throughout the cytoplasm in mouse blastomeres (M, (M)) or highly localized around the nucleus in human blastomeres (N, (N)). With respect to competence, both are significant negatives for development. n, nucleolus; cc, newly condensed chromosomes, PB1, first polar body; PB2, second polar body.

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organization have also been implicated in establishment of developmental competence. Unlike their counterparts in differentiated cells, mitochondria in oocytes and newly fertilized eggs are structurally undeveloped and typically appear as small (≤1 m) circular forms with an electron dense matrix surrounded by truncated cristae that rarely penetrate or traverse the matrix [42,43]. However, while structurally undeveloped, they are functional and active in ATP generation by oxidative phosphorylation. During the cleavage and early blastocyst phases of preimplantation embryogenesis, mitochondria undergo changes in shape and structure that while species specific with respect to stage follow a similar pattern of transformation. During mid-cleavage (8–16 cells) in mouse, rabbit and human, mitochondrial geometry transitions from spherical to elliptical and the cristae become more numerous and able to traverse a matrix of lower electron density ([42–46], for review). By the expanded blastocyst stage, and for trophectodermal cells in particular, mitochondrial organization and morphology are usually similar to the highly active forms detected in differentiated cells, where numerous well-formed cristae completely traverse a matrix of low electron density. The replication of mitochondria occurs after they have fully developed but is not initiated until well after implantation, as demonstrated in the mouse, where genetic disruption of the expression of Tfam, a mitochondrial transcription factor essential for replication, results in the death of embryos homozygous for Tfam at the early gastrula stage [47]. The progressive, stage-specific development of mitochondria during the preimplantation stages is of physiological, developmental and clinical significance. Physiological significance is apparent because the number of organelles present in the oocyte is finite and after fertilization, this progenitor pool of non-replicating mitochondria is segregated between daughter cells at each division. Changes in organelle fine structure during early development are consistent with a progressive elevation in levels of respiratory activity, which would be expected to be compensatory with respect to diminished numbers/cell in maintaining ATP levels sufficient to meet the increasing energy demands of (i) developing blastomeres during cleavage and (ii) the fluid transporting activity of the trophectoderm at the blastocyst stage ([5], for review). However, the developmental relationship between the numerical size of the mitochondrial complement inherited from the oocyte, ATP generating capacity, and the actual bioenergetic requirements of the embryo to develop progressively is unclear. Indeed, subnormal mitochondrial numbers, if associated with a bioenergetic deficiency, could be a common etiology of diverse developmental defects observed in clinical IVF, which at present, have been suggested to include premature arrest of preovulatory maturation [34], abnormal organization and function of meiotic and mitotic spindles leading to chromosomal aneuploidy at MII [8], fertilization failure [35,48], chaotic chromosomal mosaicisms in early cleavage stage embryos [49] and arrested cell division during the preimplantation stage [10]. While an energetic deficit that can explain a wide variety of defects that affect human oocytes and early embryos is an appealing possibility, evidence in support of this notion needs to conclusively address the following fundamental questions: (i) does a numerical threshold exist for a mitochondrial complement that is consistent with competence to mature into a chromosomally normal oocyte which after fertilization, can develop normally and progressively through gestation to birth and (ii) are there definable ATP threshold levels required to support early developmental processes and how do they relate to mtDNA content or actual mitochondrial numbers? The apparent size of the mitochondrial population in fully grown, meiotically mature (MII) human oocytes has been based on quantitation of mtDNA copy numbers and the assumption that each mitochondrion contains one or two genomes; estimates of mitochondrial numbers have ranged from <50,000 to

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>1,000,000 [2,4,5,7,8,32,34,35,48]. If this range accurately reflects actual organelle numbers that can exist in the mature human oocyte at the time of fertilization, what is the relationship to competence, how are levels of ATP production regulated, and are there negative developmental consequences with respect to ROS generation if mitochondrial numbers occur in the high hundreds of thousands to well over a million? 3. Mitochondrial numbers and bioenergetics It would seem intuitive that a normal mitochondrial complement for a MII oocyte is one that is numerically consistent with preovulatory maturation, fertilization and sufficient to drive embryogenesis prior to the initiation of mitochondrial replication. At present, results from mtDNA copy number analyses of human MII oocytes are confusing because the range is so broad and associations with competence inconclusive [2,5]. For example, Zeng et al. [8] reported that the average mtDNA copy number of a presumably normal MII human oocyte, defined by the presence of a first polar body and well-organized meiotic spindle, was about 640 K (±238 K), and that this number corresponded to net cytoplasmic ATP of about 2 pmol, a level similar to the one suggested by Van Blerkom et al. [6] to be a competence-related threshold. Assuming that each organelle contains one or two genomes [39], the average number of mitochondria required to sustain this bioenergetic state should be between 320 K and 640 K organelles. In the same study, they detected immature oocytes, and MII oocytes with apparent spindle defects that resulted in embryos that developed abnormally or arrested cell division during the early cleavage stages. These oocytes had an average mtDNA copy number of 491 K (±153 K) and a net ATP content of 1.65 pmol. This suggests that the mitochondrial contribution to competence may require a threshold organelle level around 400 K in order to sustain a cytoplasmic ATP content of approximately 1.8–2.0 pmol. However, a very similar study by Santos et al. [48] found that the average mtDNA copy number of a normal, fertilized human oocyte was around 250 K, while the average complement of unfertilized siblings was approximately 165 K. Findings of this type are an example of the current controversy surrounding attempts to establish threshold levels for mitochondria or ATP because the average mtDNA copy number indicated by Santos et al. as normal is nearly 100 K lower than the mtDNA copy number reported by Zeng et al. to be associated with incompetent oocytes and abnormal embryogenesis. In this respect, both values are higher than the approximately 100 K level. Shoubridge and Wai [2] concluded was likely a normal value for the MII human oocyte. We have used two different approaches to address the question of complement size in normal appearing human oocytes; first, by assessing the number of copies of the mitochondrial gene ATPase 6 in intentionally uninseminated, normal appearing MII human oocytes [38] and second, by counting individual mitochondria in each cell of fully expanded and spontaneously hatched mouse and human blastocysts by (i) transmission electron microscopy (TEM, [46,50]) and (ii) fluorescent microscopy after staining with different mitochondria-specific probes [5]. Initially, the ATPase 6 results were considered suspect because copy numbers over a relatively wide range (80–250 K) were detected in morphologically equivalent oocytes derived from the same cohort; this suggested a technical artifact rather than a physiologically significant finding. Mitochondrial counts by TEM of 6-day 5.5 human embryos indicated about 150 organelles occurred in each cell (unpublished). After staining living embryos with mitochondria-specific probes and analysis by confocal and conventional epifluorescence microscopy, individual cells of expanded mouse and human blastocysts examined under slight compression [5], and spontaneously flattened trophectodermal and inner cell mass (ICM) cells during the first 24 h of outgrowth in vitro [5], showed that the typical ICM and trophectodermal cell of

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a mouse blastocyst contained ∼120 mitochondria and ∼180/cell in normally developed, fully expanded human blastocysts composed of 90–115 cells [5], with nuclear counts made with DNA-specific fluorescent probes to provide cell number. A similar number of mitochondria were detected in nascent trophectodermal outgrowths. If the assumption that the total number of mitochondria detectable at the blastocyst stage is largely reflective of the complement existing in the oocyte at fertilization, and that this number was consistent with competence because of the demonstrated ability of development to progress to the peri-implantation stage, the complement of the mouse oocyte is approximately 10,000 organelles while approximately 18,000 organelles occur in the human. Differences between actual mitochondrial numbers detected in blastocysts and complement estimates for the oocyte based on mtDNA content(s) are difficult reconcile if each mitochondrion contains one to two genomes. One possibility to account for these apparent differences is that early preimplantation development is accompanied by stage-specific loss, presumably by a degenerative process. A second possibility is that the intensity of mitochondriaspecific fluorescent probes is activity-related and some proportion of organelles in each cell is below a critical activity threshold. If a reduction in mitochondrial numbers involved degeneration (acute or progressive), damaged or deteriorating forms might be expected to be evident in electron microscopic images of mouse or human embryos examined during the preimplantation stages. However, extensive fine structural analyses of early embryogenesis in normally developing mouse and human preimplantation stage embryos [43–46,52] have not indicated that such forms occur. In contrast, mitochondrial damage or degeneration can be experimentally induced in early mouse embryos and the effects clearly detectable at the fine structural level and by performance in culture, where the adverse developmental consequences of diminished mitochondrial function are seen as arrested cell division, cytoplasmic degeneration, apoptosis, or pathological cell death [53,54]. In the absence of a naturally occurring mechanism that can affect a massive and stage-related loss of mitochondria that is so acute as to leave no microscopic or biochemical evidence, yet is consistent with embryogenesis progressing normally through the preimplantation stages, it seems more likely that the number of genomic copies in oocyte mitochondria is variable, and may often be greater than the currently suggested one or two copies/organelle. The possibility that some proportion of mitochondria in living cells was not counted because fluorescent staining properties or intensities may be activity related also seems unlikely; similar numbers were obtained with different stains that identified mitochondria regardless of activity level [5]. A third possibility is that the stagespecific elongation and functional transformation of mitochondria into highly active forms [44,45] involves organelle fusion. However, fine structural images of embryos between the cleavage and the expanded blastocyst stages in mouse and human show none of the features characteristic of mitochondrial fusion observed in other species, although at present, this possibility cannot be discounted entirely. Therefore, rather than numbering in the hundreds of thousands, as suggested from mtDNA copy estimates for the oocyte, a complement that is at least one order of magnitude lower is indicated. If confirmed, the results could have important clinical implications with respect to the normal size of the mitochondrial complement and its direct relationship to developmental competence [5]. 4. Disproportionate mitochondrial inheritance While the relative number of mitochondria that is consistent with developmental competence remains controversial, it is clear is that differences in mitochondrial inheritance exist between blastomeres during early cleavage, with developmental significance

related to the extent of disproportionate segregation [3,19,55]. For the human, significant differences in mitochondrial inheritance have been detected at the two-cell stage, with adverse consequences ranging from slow or arrested cell division, to blastomere lysis in cells affected by a reduced ATP generating capacity that can be correlated with mitochondrial under-representation [55]. Disproportionate mitochondrial segregation at the two- or fourcell stage can also have latent effects on development that become evident during the latter stages of cleavage. In these instances, blastomeres derived from a progenitor cell with a sublethal mitochondrial complement become progressively less able to continue to divide and ultimately arrest cytokinesis as the level of ATP generation required to support normal cellular activities declines below a threshold level [55]. Instances of disproportionate segregation, which may be common in human embryos derived by IVF, demonstrate that a developmentally relevant association exists at the blastomere level between mitochondrial numbers and the bioenergetic status of the cytoplasm. However, a clear identification of fully grown human oocytes with subnormal numbers of mitochondria would go a long way in establishing a similar bioenergetic relationship for the female gamete [2]. 5. Bioenergetics and development Much of the current research noted above has attempted to establish a direct developmental correspondence between cytoplasmic energy levels (net ATP content), mitochondrial numbers, embryo performance in vitro, and outcome after uterine transfer (in clinical IVF). Reports of abnormal or arrested development after oocyte or early embryo mitochondria are damaged by photosensitization [53] or exposed to metabolic inhibitors or poisons in experimental systems such as the mouse [54] are often cited as ‘proof’ of their central importance in the establishment of developmental competence. However, the toxic developmental consequences of irreversible damage to mitochondria should come as no surprise because spontaneous or induced alterations that compromise the function or activity levels of other cellular components or essential molecular processes (e.g., genomic activation, transcription, autophagy, [56]) are lethal for early embryogenesis. Therefore, a subnormal biogenetic capacity that produces known pathologies in differentiated cells because of OXPHOS defects associated with advanced age or inherited pathogenic mtDNA mutations (myopathies or neuropathies) would likely have similar consequences for early development if due to numerical mitochondrial deficiencies in one or more cells of the early embryo. However, recent findings suggest that measurements of total cytoplasmic ATP content and mitochondrial or mtDNA copy numbers in the oocyte may not offer a complete picture of the bioenergetic requirements for early development [5]. For example, reducing the net cytoplasmic ATP content of the mouse oocyte by about 40% at the germinal vesicle stage does not inhibit spontaneous maturation in vitro to MII [6] or fertilization [57]. However, the resulting embryos rarely progress beyond the cleavage stages, indicating that if stage-specific energy threshold levels exist, they may be lower during early preimplantation development than at the late-cleavage-to-blastocyst stage. If the number of mitochondria present at the outset of fertilization is within the range suggested by numerical counts in blastocysts, rather than from mtDNA determinations in oocytes, as discussed above, then energy demands for morphodynamic processes after cleavage, such as cavitation and continuous fluid transport by the trophectoderm to form and maintain an expanding blastocyst cavity, may be satisfied by coincident mitochondrial development that enables increased levels of ATP generation by oxidative phosphorylation to occur against a background of organelle numbers/cell that naturally decrease with each division.

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Fine structural studies of human preimplantation stage embryos show that premature developmental arrest is often associated with a mitochondrial population that while seemingly normal in number, contains a high proportion of organelles that remain unchanged in form and organization from the state in which they occurred in the oocyte [46,52]. In contrast, small blastocyst-like structures containing as few as 10–15 cells can develop from in vitro fertilized human oocytes after 4 or 5 days of culture, and while abnormal as embryos, their ability to form and maintain a blastocyst cavity indicates sufficient ATP production to transport and internally accumulate fluid. In these instances, electron microscopic images show mitochondrial development that is stage appropriate for normal human blastocysts [50]. Although descriptive, these findings suggest that the normality of early embryogenesis in the human is closely aligned with the ability of a comparatively ‘small’ complement of inherited mitochondria to undergo changes in organization and structure that are consistent with upregulated ATP production. If confirmed, this raises fundamental and important questions of how mitochondrial development and activity are regulated during the preimplantation stages. Normal mitochondrial function is maintained by the import of cytoplasmic proteins from nuclear-encoded genes [31] and it may not be coincidental that the temporal sequence of their development during early embryogenesis, while species-specific, begins with the completion of embryonic genomic activation and increased levels of transcription and translation. Studies of oocyte competence intended to establish a cause-and-effect relationship that involves mitochondria should consider intrinsic defects in nuclear gene expression that may contribute to structural defects or respiratory chain deficiencies. Similarly, it will be important to determine whether the failure of mitochondria to undergo stagespecific structural transformations after fertilization is related to defects that pre-exist in the oocyte, or to the absence of critical proteins encoded by nuclear genes. Distinguishing between these possibilities may be particularly relevant in understanding the extent to which mitochondria contribute to infertility in general, and in particular, to the maternal-age-associated decline in oocyte and early embryo developmental competence. 6. Stage-specific changes in the spatial distribution of mitochondria during early development Contemporary views of mitochondria in differentiated cells often consider them as a homogenous population whose activity and function are coordinated by virtue of physical continuity or electrical coupling, which would allow these organelles to act coordinately in response to signals of extrinsic or intrinsic origin (e.g., changes in ambient free calcium levels). However, more recent studies demonstrate that mitochondria are morphologically and functionally heterogeneous in differentiated cells [57] and that functional heterogeneity is often spatially dependent [58]. Studies of mitochondrial distribution with mitochondria engineered to be fluorescent by the insertion of green fluorescent protein transgene, demonstrate ‘passive’ movements within the cytoplasm by means of normal intracellular circulation [59,60]. However, mitochondria are also actively translocated along cytoskeletal elements (e.g., microtubules, actin microfilaments, [61]) and their movements contribute to cytoplasmic remodeling that can occur in response to differential energy demands or the need to maintain calcium homeostasis. Diaz et al. [58] reported that the level of mitochondrial polarity, the potential difference across the inner mitochondrial membrane ( m, see below), undergoes dynamic changes at the margins of certain differentiated cells in response to the presence or absence of contact with neighboring cells. This finding indicates that mitochondrial activity can be influenced by location within the cytoplasm and whether they come under the

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influence of external forces associated with intercellular contact and communication, such as ionic fluxes through gap junctions. A similar change in mitochondrial potential dynamics was reported for cleavage stage mouse embryos, where focal changes in polarity were related to the presence or absence of intercellular contact [61]. For differentiated cells, current evidence indicates that mitochondria are not usually in direct physical contact with one and another, which as Collins et al. [57] note, allows them to have distinct and individual functional properties, undergo spatial remodeling, or activity-related changes in the magnitude of  m in response to differential intracytoplasmic conditions, energy demands or external signals. While mitochondria are usually considered spatially static elements whose primary function is an energetic one, this notion has changed considerably as important regulatory, redox; signaling and oxygen sensing functions have been identified [25,26,62–64]. In contrast to differentiated cells, the progenitor population of mitochondria in the fully grown mammalian oocyte is morphologically homogenous, undeveloped, and usually uniformly distributed within the cytoplasm (e.g., mouse germinal vesicle stage, Fig. 1A and B). However, stage-specific changes in their spatial organization during meiotic maturation and early embryogenesis suggest regulatory roles that may be similar to their counterparts in differentiated cells. For example, transmission and scanning electron microscopic images of mouse and human oocytes show mitochondria in direct contact with cisternae of the smooth-surfaced endoplasmic reticulum (SER, [46]) and in particular, small clusters of mitochondria surround compact assemblages of SER in the pericortical and subplasmalemmal cytoplasm [62]. Cross talk that is mediated by calcium fluxes between these two organellar systems can focally upregulate levels of mitochondrial ATP production [1,20,21] and in this context, may be indicative of a type of localized functional heterogeneity for oocyte mitochondria that may be an important regulatory factor in early development [1,5,20], as discussed below. Mitochondrial movements (circulation) and directed translocations are another cytoplasmic dynamic that has common features in oocytes, early embryos and differentiated cells. Mitochondria are not stationary nor do they have a permanent morphology, but rather change shape and position within differentiated cells. These mitochondrial behaviors were first described nearly a century ago [65], a fact that often comes as a surprise to contemporary biologists. The molecular and cellular mechanisms and mechanics of mitochondrial translocation are now well understood to involve cytostructural elements such as microfilaments (actin), intermediate filaments, and microtubules. Vectored mitochondrial movement has been studied in detail with regard to the interaction of specific mitochondrial proteins and cytostructural elements, and for microtubules in particular, the involvement and function of motor proteins such as dyenin and kinesins, whose activities are driven by the hydrolysis of ATP ([17], for review), has been long established. For example, the inward movement and cytoskeletal-mediated translocation of mitochondria from cortical to more central or perinuclear regions is thought to be regulated by dyenin running on tracks of cytoskeletal microtubules towards the centrally located minus end of the structure(s), while lateral or circumferential movements may be facilitated by motor proteins (e.g., myosin) moving along actin cables, intermediate filaments and microtubules (dyenin and kinesin). Studies of intracellular circulation, perfusion and convection have shown how these normal intracellular processes can locally influence metabolic homeostasis, compartmentalize enzymatic activities, and integrate diverse cellular and molecular activities within specific cytoplasmic domains [60,66]. Dynamic changes in the spatial distribution of mitochondria that increase or decrease their density within discrete regions of the cytoplasm are detectable

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expressions of a fundamental mechanism employed by cells to maintain metabolic homeostasis or respond intracytoplasmic changes in energy demands, metabolite concentrations, and free calcium levels that are focal and transient [66]. Directed mitochondrial translocations in which clusters or aggregates of organelles arise and disperse can also be auto-regulatory with respect to levels of respiratory activity, ROS biogenesis and the local regulation of calcium homeostasis, and can also affect focal alterations in cytoplasmic physiology (e.g., cytoplasmic redox potential, pH) that in turn, influence enzymatic and other cellular activities [66]. It has been suggested that stage-specific redistributions of mitochondria detected in maturing oocytes, pronuclear eggs and early cleavage stage embryos may have local physiological and regulatory influences similar to those described above for differentiated cells [4,5]. In particular, mitochondrial translocations to the perinuclear region have been shown to be vectored along microtubular tracks originating from perinuclear foci [18,19,55] and in some species, result in a pronounced spherical accumulation around chromosomes at the circular bivalent and metaphase I (MI; [67]; Fig. 1E and F) stages in the oocyte, and at pronuclear stages (Fig. 2J–L) and early cleavage stages in the embryo (Fig. 1J–L). While similar perinuclear accumulations have been detected in maturing oocytes and early embryos of several mammals [68–70], including the human [55], the extent to which this is a common aspect of development in mammals is unknown and interpretations of physiological or developmental significance need to consider the conditions in which maturation and embryo development occur. In order to distinguish between developmental processes that are normally occurring from those which may be adaptive and influenced by culture, in vivo studies of cytoplasmic remodeling or organelle redistributions in oocytes and early embryos should be undertaken in parallel, whenever possible. In this respect, stage-specific mitochondrial redistributions during oocyte maturation in vivo and in vitro in the mouse are similar [67,70]. At present, it appears likely that the inward movement and perinuclear accumulation of mitochondria in oocytes, pronuclear eggs, cleavage stage blastomeres and differentiated cells utilize a common cytostructural pathway(s). Whether stage-specific spatial remodeling of mitochondria in maturing oocytes and early embryos by redistribution, translocation and aggregation has functional, physiological or regulatory influences similar to those reported for differentiated cells is currently under investigation. In this regard, Van Blerkom and Runner [67] first proposed that mitochondrial translocation to the perinuclear region in the maturing mouse oocyte, which begins just prior to germinal vesicle breakdown and is coincident with the evolution of microtubular arrays from perinuclear microtubule organizing centers (MTOCs, [18]), served to elevate ambient ATP levels to meet locally higher energy demands that were presumed to be required to (i) promote or support kinetic activities associated with nuclear maturation (development of meiotic spindles, chromosomal segregation, polar body formation) and (ii) significantly increase cytoplasmic motility, especially in the perinuclear cytoplasm [71]. This possibility was supported by reversibly inhibiting normal microtubular dynamics (assembly or disassembly) at different stages of maturation, which arrested the progression of mitochondrial translocation and meiotic maturation. Time-lapse analysis showed that disassembly of perinuclear microtubules was accompanied by a reduction or cessation in intracytoplasmic motility that was reversed when normal culture conditions were restored [71]. During formation of the MII spindle under normal culture conditions, the dense perinuclear accumulation of mitochondria progressively disperses such that at the completion of MII, mitochondrial distribution is largely uniform (Fig. 1G). In the mouse, mitochondrial dispersion is coincident with the spontaneous disassembly of MTOC-derived arrays, but dispersion could be arrested

by acute treatment with agents (e.g., taxol) that inhibit disassembly [18]. After fertilization, mitochondria re-accumulate to form a relatively dense peripronuclear sphere (Fig. 2J–L, rhodamine 123 staining, imaged by scanning laser confocal microscopy), which normally disperses prior to the first cleavage division, but then reforms around each nucleus in the nascent two-cell embryo ([67], Fig. 1J–L). When detected, stage-specific changes in mitochondrial distribution in the maturing oocyte and early embryo are coincident with the presence or absence of perinuclear microtubular arrays whose biogenesis appears to be cell-cycle-related and indicative of coordinated activities that balance local ATP supply and demand. The notion that mitochondrial reorganizations balance local energy demand and supply is supported by the studies of Dumollard et al. [1,20], who reported that local changes in free calcium levels focally up- or downregulate levels of mitochondrial respiration in the mouse oocyte. These investigators proposed that differential energy demands within in the oocyte could be satisfied locally by stimulating spatially distinct aggregates of mitochondria that co-localize with calcium storage elements (e.g., SER), and in this manner, provide a mechanism by which discrete cytoplasmic regions respond autonomously to changing energy demands [5,20,61]. In this regard, Dumollard et al. [20] suggested that transient changes in levels of ATP production that are differentially localized within the ooplasm have the virtue of not requiring the participation of the entire mitochondrial complement and therefore, should not increase net cytoplasmic levels of reactive oxygen species produced by ‘leakiness’ of the mitochondrial electron transport chain [22]. The possibility that transient domains of differential respiratory activity exist within the oocyte is an attractive notion because it is not only consistent with how mitochondria behave in differentiated cells, but in the largest cell in the body, could afford a high degree of autonomous regulation of ATP production that is responsive to local changes in ionic or physiological conditions. Mitochondrial translocation and aggregation may also be auto-regulatory because clustering, by altering proximal pH (by ATP hydrolysis), can influence levels of mitochondrial participation in calcium homeostasis, owing to their ability to sequester and release this cation [63]. Indeed, focal changes in cytoplasmic physiology (intracytoplasmic pH, pHi) have been shown to upregulate respiratory rates because metabolite uptake and utilization can increase when mitochondrial clustering reduces ambient pHi [66]. Dynamic spatial remodeling of mitochondria by translocation and clustering is a normal cellular process, which forms ‘microzones’ of differential physiology (microzonation) that in turn, can compartmentalize certain enzymatic and other cytoplasmic activities without requiring enclosure by a membrane (functional compartmentalization, [66]). Therefore, it seems reasonable to propose that cell-cycle-related mitochondrial aggregation in the oocyte and early embryo might have a similar function. Recently, Van Blerkom [5] suggested that a similar phenomenon of mitochondria-induced microzonation and functional compartmentalization may occur in the maturing oocyte and early embryo, and could be auto-regulatory with respect to stage-specific changes in mitochondrial organization. He suggested that the increased density of perinuclear mitochondria, if accompanied by a coincident increase in ATP hydrolysis, could locally reduce pHi to levels that favor microtubular disassembly and become permissive for the dispersal of mitochondria from the perinuclear region. In the same respect, a reduction in high mitochondrial density could promote the reappearance of cytoplasmic conditions that favor microtubular array formation, which would promote directed mitochondrial movements to the cell center. In this manner, by episodically influencing ambient pHi, mitochondria may be capable of a degree of self-regulation of their respiratory activity and able to mediate their stage- or cell-cycle-specific spatial distribution during early

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Fig. 2. Mouse (A–C) and human (D–L) oocytes and early embryos show a subplasmalemmal domain of high potential (polarized) mitochondria (HPM) after staining with fluorescent potentiometric stains such as JC-1. HPM ( m, ≥−140 mV) emit a red signal in the RITC channel (A) while their lower potential counterparts ( m <−100 mV) fluoresce green. Under conventional epifluorescent optics, HPM appear as orange emitting organelles in the FITC channel (e.g., D–G). (B) and (C) are scanning laser confocal pseudo-color images showing that perinuclear HPM (B) can be distinguished from the more abundant but lower potential forms in the cytoplasm (C). (D–I) show the subplasmalemmal localization and distribution of HPM at the GV (D), MI (E), MII (F) and pronuclear stages (PN; G). The subplasmalemmal domain is stable and inherited after fertilization in each blastomere as a numerically declining population of organelles (HPM, (H) and (I); four-cell and six-cell, respectively). (J–L) are rhodamine 123 stained images of a human pronuclear (PN) embryo imaged in the FITC channel (J), and in pseudocolor for relative intensity (K and L), by scanning laser confocal microscopy. Typically, a dense and transient perinuclear accumulation of mitochondria (M) is observed in a section series (K) and its largely spherical geometry clearly evident in fully compiled images (L). As in the oocyte, mitochondria translocate to the perinuclear region along microtubules that emanate from perinuclear foci. This cytoplasmic phenotype is characteristic of the pronuclear stage; its geometry is an important determinant of the equivalence of mitochondrial inheritance between blastomeres, and as such, is indicative of normal developmental competence.

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development. However, it is also known that cell-cycle-associated defects in mitochondrial reorganization occur in the oocyte and early embryo, and that these defects are inconsistent with normal meiotic maturation or are developmentally lethal during the preimplantation stages. For example, the asterisks in Fig. 1H denote spheres of mitochondria that have formed around MTOCs in mouse oocytes that arrest meiotic progression prior to the circular bivalent stage. In these instances, maturational arrest is associated with the failure of several MTOCs to assume a normal perinuclear distribution, and the microtubular arrays they elaborate serve to anchor and translocate mitochondria from the adjacent cytoplasm (as they would have functioned in a perinuclear location, [18]). Premature arrest of meiosis in fully grown human and mouse oocytes is often accompanied by normal mitochondrial perinuclear aggregation, but the aggregate fails to disperse at MII (e.g., Fig. 1I). A similar situation occurs in blastomeres of mouse and human embryos that arrest cytokinesis at the two- or four-cell stage. In these instances, large mitochondrial clusters develop and persist throughout the cytoplasm (Fig. 1M), or fail to disperse (Fig. 1N) from a normal perinuclear distribution (Fig. 2J–L) that develops shortly after cell division. It has been suggested [5] that these developmentally significant defects may constitutively alter local cytoplasmic conditions (e.g., pHi) in ways that are inconsistent with normal enzymatic activities and the functioning of regulatory signaling pathways. 7. Evidence suggesting mitochondrial microzonation during early development and the relationship between  m, nitric oxide and oxygen One of the more intriguing findings to come from the study of mitochondria in early mouse and human development is the occurrence of a subplasmalemmal domain, or microzone, of high potential ( m) organelles [5,21,61] that are detectable in living cells with mitochondria-specific, potentiometric fluorescent probes, such as JC-1 (HPM, Fig. 2A–I). While morphologically identical to mitochondria located in other regions of the oocyte (Fig. 2A–G) or cleavage stage blastomeres (Fig. 2H and I), these organelles are comparatively unique because of their location immediately beneath the oolemma and a  m that is markedly higher than their cytoplasmic counterparts. High potential mitochondria localized to subplasmalemmal cytoplasm and imaged by scanning laser confocal and conventional fluorescence microscopy after JC-1 staining are shown in Fig. 2A–C and D–I, respectively. We have reported that this domain contains <5% of the total mitochondrial complement [10], but more recent morphometric analysis indicates that ≤∼3% may be more accurate. Their contribution to the net cytoplasmic ATP content appears to be relatively marginal [9]. The domain is inherited in its entirety after fertilization, with organelle numbers in the subplasmalemmal cytoplasm reduced with each cleavage division [10]. Moreover, studies of mitochondrial reorganization in the maturing mouse oocyte show that these high potential mitochondria remain spatially stable while the more abundant lower potential forms undergo translocation and perinuclear aggregation [10]. Centrifugation of human and mouse oocytes slightly displaces the domain but it reforms rapidly and with the same mitochondria [10]. Either partial or total loss of these high potential mitochondria from a human blastomere can result from spontaneous fragmentation that is minor in extent and does not decrease cell volume or the bioenergetic status of the cytoplasm. Yet, the failure of affected cells to divide after spontaneous elimination of most of the domain in early cleavage stage human embryos suggests they have a specialized role in early development [10]. This possibility was supported by experimentally manipulating the magnitude of  m in the subplasmalemmal domain of mouse oocytes, which blocked sperm penetration (but not attachment to

the oolemma) when  m was reduced, but was permissive when levels returned to normal [9]. It has recently been reported that the magnitude of  m in the intact oocyte may be regulated by the relative levels of oxygen and nitric oxide (NO) that occur at the surface of the oolemma and extend into the subplasmalemmal cytoplasm [72]. These investigators proposed that competition between dissolved intrafollicular oxygen that diffuses into the follicle from the perifollicular vasculature, and NO produced by cells of the cumulus oophorus and corona radiata that directly surround the preovulatory oocyte, likely occurs at the level of cytochrome C oxidase, the final step in electron transport, localized to the inner mitochondrial membrane. Upregulation of  m in the subplasmalemmal domain of intact oocytes occurs at the terminal stages of preovulatory maturation (MII) and was suggested to lead to the establishment of a ‘permissive window’ for fertilization as the depressive effect of NO on  m was reduced [72]. It has also been suggested that high potential subplasmalemmal mitochondria may be more active than lower potential forms in ATP generation, which supplies phosphate for the phosphorylation of proteins and lipids located in the adjacent cytoplasm [5,9,10]. In this context, preliminary findings indicate that the stage-specific upregulation of  m in the subplasmalemmal domain is coincident with the phosphorylation of lipids that enter the oolemma, and after incorporation, appear to promote molecular conditions permissive for sperm penetration and cortical granule exocytosis (Van Blerkom, unpublished).  m is a fundamental electrochemical property of mitochondria, and differences in the magnitude of this transmembrane potential directly influence diverse mitochondrial functions, including ATP generation, cytoplasmic protein import and processing, maintenance of organelle volume (volume homeostasis), and levels of mitochondrial participation in the regulation of calcium homeostasis and signal transduction (especially those signaling pathways whose expression requires superoxide; for review, [5]). It has been reported that differences in mitochondrial potential may influence the differentiative capacity of cells in late preimplantation stage embryos [51], and in embryonic stem cells [5,73]. For the oocyte and early embryo, the adverse developmental consequences associated with spontaneous elimination of high polarized mitochondria, their failure to be replaced from an abundant population of lower potential organelles [10], and the reversible,  m-related inhibition of sperm penetration and cortical granule exocytosis [9], suggests that by virtue of their high potential, they are functionally compartmentalized within the cytoplasm and in this capacity, may coordinately regulate diverse developmental activities co-localized within the same subplasmalemmal microzone. Continued investigation of the relationship between NO, oxygen and  m, and mitochondrial function and activity, has the potential to provide new insights into mechanisms of regulation during early development in the mammal, and may be especially relevant if signal transduction pathways known to involve mitochondria and which are  m sensitive in differentiated cells are identified in the oocyte and early embryo [74–77]. 8. Perspectives for future research The findings presented here indicate that the function of mitochondria in the oocyte and early embryo likely extends beyond their long known respiratory role, although their bioenergetic activity and capacity are clearly central to developmental success. It is also likely that the relationship between competence and bioenergetics for oocytes and preimplantation stage embryos may be more complex than apparent differences in mtDNA content or organelle numbers suggest. Therefore, questions concerning spatial differences in energy production, distribution, and utilization at the cellular level may be more relevant for understanding how bioen-

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