Uncovering the important role of mitochondrial dynamics in oogenesis: impact on fertility and metabolic disorder transmission

Abstract

Oocyte health is tightly tied to mitochondria given their role in energy production, metabolite supply, calcium (Ca²⁺) buffering, and cell death regulation, among others. In turn, mitochondrial function strongly relies on these organelle dynamics once cyclic events of fusion and fission (division) are required for mitochondrial turnover, positioning, content homogenization, metabolic flexibility, interaction with subcellular compartments, etc. Importantly, during oogenesis, mitochondria change their architecture from an “orthodox” elongated shape characterized by the presence of numerous transversely oriented cristae to a round-to-oval morphology containing arched and concentrically arranged cristae. This, along with evidence showing that mitochondrial function is kept quiescent during most part of oocyte development, suggests an important role of mitochondrial dynamics in oogenesis. To investigate this, recent works have downregulated/upregulated in oocytes the expression of key effectors of mitochondrial dynamics, including mitofusins 1 (MFN1) and 2 (MFN2) and the dynamin-related protein 1 (DRP1). As a result, both MFN1 and DRP1 were found to be essential to oogenesis and fertility, while MFN2 deletion led to offspring with increased weight gain and glucose intolerance. Curiously, neither MFN1/MFN2 deficiency nor DRP1 overexpression enhanced mitochondrial fragmentation, indicating that mitochondrial size is strictly regulated in oocytes. Therefore, the present work seeks to discuss the role of mitochondria in supporting oogenesis as well as recent findings connecting defective mitochondrial dynamics in oocytes with infertility and transmission of metabolic disorders.

Introduction

In humans, the ovary is one of the first organs to fail with aging due to a progressive reduction of both follicle (the functional unit of the ovary) number and egg quality. Consequently, live birth rate declines from 43%, among women younger than 35 years, to 6%, among women aged 42 years or more (Baird et al. 2005; Schwartz and Mayaux, 1982; Van Noord-Zaadstra et al. 1991; Wright et al. 2008). While the decline in follicle number is thought to be explained by increased follicle atresia as a result of programed cell death (apoptosis), meiotic errors resulting in aneuploidy are the leading cause of poor egg quality (Schwartz and Mayaux, 1982; Van Noord-Zaadstra et al. 1991; Wright et al. 2008). The mitochondrion presents in this context as a pivotal organelle due to its key role in apoptosis and energy production for normal spindle assembly and chromosomal segregation (Al-Zubaidi et al. 2021; Babayev et al. 2016; Bentov et al. 2011; Johnson et al. 2007; May-Panloup et al. 2016; Pasquariello et al. 2019; Tilly and Sinclair, 2013; Van Blerkom et al. 1995, 2000; Yu et al. 2010). In addition, numerous metabolic processes regulated by mitochondria such as calcium (Ca²⁺) buffering, phospholipid biosynthesis, and reduction–oxidation state can affect egg formation (Al-Zubaidi et al. 2021; Carroll et al. 1994; May-Panloup et al. 2016; Spinelli and Haigis, 2018). In face of these findings, mitochondrial quality and number have been proposed as markers of egg quality. Indeed, there are several reports showing that mitochondrial DNA (mtDNA) copy number can be highly variable among oocytes (and, consequently, embryos) and associate with developmental potential, although further studies are needed to verify the extension and reliability of this association (Diez-Juan et al. 2015; Fragouli et al. 2015; Pasquariello et al. 2019; Santos et al. 2006; Seli 2016; Wai et al. 2010). Moreover, several attempts have been made to rescue poor quality eggs through supplementation with exogenous mitochondria, being their results a matter of constant debates (Cohen et al. 1997; Kristensen et al. 2017; Labarta et al. 2019a, b; Woods and Tilly, 2015).

The mitochondrial network can vary from hundreds to millions of mitochondria depending on the cell type and on the balance between mitochondrial fusion and fission (division). In turn, each mitochondrion is composed of two membranes, the inner and the outer, which delimitate two compartments, the mitochondrial lumen (matrix) and the intermembrane space; invagination of the inner membrane towards the matrix further determines the mitochondrial cristae (reviewed by (Pernas and Scorrano, 2016)). Within the mitochondrial matrix resides the enzymes taking part in the tricarboxylic acid cycle and β-oxidation, biochemical processes responsible for the oxidation of energetic substrates. Nicotinamide adenine dinucleotide (NAD)H and flavin adenine dinucleotide (FAD)H₂, generated from these oxidative processes, sustain the electron transport chain (ETC) through four enzymatic complexes in the cristae membrane, enabling reduction of oxygen (O₂) into water as well as the formation of a proton (H⁺) gradient across the inner mitochondrial membrane. Eventually, the H⁺ gradient is used by a fifth enzymatic complex (the F₁F₀-ATP synthase) in the cristae membrane to phosphorylate ADP into ATP, this latter representing the most abundant energy source for biological processes in the cell. In addition to ATP, reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals can be generated from the oxidative phosphorylation (OXPHOS) due to incomplete O₂ reduction. ROS can damage membranes, proteins, and nucleic acids, but the mitochondrion owns a highly effective mechanism for dealing with its production. Under certain circumstances, however, ROS production surpasses the mitochondrial oxidative defenses, with potential consequences to mitochondrial and cellular health (reviewed by (May-Panloup et al. 2016)).

The majority of the mitochondrial proteome (~ 1100 proteins) is encoded in the nucleus, translated in the cytoplasm, and imported into mitochondria (Rath et al. 2020). Nonetheless, mitochondria have their own genome (mtDNA), which in mammals is exclusively inherited from the mother, has ~ 16.5 kb, and encompasses 37 genes encoding for 13 polypeptides of the OXPHOS complexes (reviewed by (Stewart and Chinnery, 2020)). Additionally, recent works suggest a role for mtDNA in encoding mitochondrial derived peptides (MDPs) and small mitochondrial RNAs (Miller et al. 2020; Pozzi et al. 2019). Multiple mtDNA copies are present in each cell, being mtDNA copy number closely associated with the cell’s energy demand. One or more copies of mtDNA are packaged together along with proteins such as the mitochondrial transcription factor A (TFAM) to form a nucleoid, a likely platform for replication, transcription, and translation of mtDNA (reviewed by (Gustafsson et al. 2016)). Due to its relative short half-life, mtDNA is replicated at a correspondingly high rate, making it prone to replicative errors (Krasich and Copeland, 2017). As such, mutated mtDNA can coexist in a cell with wild-type molecules (referred to as heteroplasmy), potentially leading to devastating diseases (reviewed by (Craven et al. 2017)).

The present work aims to review the current knowledge regarding the role of mitochondria in supporting oocyte development. Several works encompassing morphofunctional characteristics of oocyte mitochondria are discussed, with an emphasis on recent findings connecting defective mitochondrial dynamics in oocytes with infertility and intergenerational transmission of metabolic disorders.

The unique characteristics of mitochondria in oocytes

Oogenesis commences with specification of primordial germ cells (PGCs) in the posterior epiblast base in pre-gastrulation post-implantation embryos. Thereafter, PGCs migrate to the gonadal ridges, differentiate into oogonia, and proliferate by mitosis prior to undergoing meiosis and giving rise to oocytes (reviewed by (Kobayashi and Surani, 2018)). Oocytes progress through the first stages of meiosis until reaching the late-diplotene resting stage referred to as dictyate; meiosis is resumed only after puberty, which in humans can take 40 years or more. At birth, the ovary is therefore populated with up to hundreds of thousands of oocytes surrounded by a single layer of squamous somatic granulosa cells (Fig. 1). These germ-soma structures, called primordial follicles, remain quiescent until they are recruited for development, which encompasses proliferation and differentiation of granulosa cells along with a ~ 100-fold increase in oocyte volume (Liu et al. 2014; Pedersen and Peters, 1968; Reddy et al. 2008; Rimon-Dahari et al. 2016; Zhang and Liu, 2015). During this period, known as folliculogenesis, the oocyte progresses through a series of changes and stockpiles several molecules and organelles, events that rely on the close interaction of the gamete with granulosa cells (Fig. 1). Eventually, the oocyte resumes meiosis and is ovulated at the metaphase II stage (Fig. 1), even though many oocytes can be eliminated through follicle atresia (reviewed by (Binelli and Murphy, 2010; Clarke, 2017)).

Fig. 1.

Unique characteristics of mitochondria in oocytes. Starting with puberty, a group of primordial follicles initiates development as represented by proliferation and differentiation of somatic granulosa cells as well as oocyte growth. This period, known as folliculogenesis, encompasses several stages (i.e., primary, secondary, and tertiary follicles) and eventually should result in the ovulation of a gamete competent for fertilization and embryogenesis. In turn, mitochondrial architecture changes dramatically during oocyte development. Initially, mitochondria display an “orthodox” configuration characterized by an elongated shape, (1.2–1.9 μm in length), numerous transversely oriented cristae, and a single large vacuole. However, the increased abundance of dumbbell-shaped mitochondria in early-developing oocytes indicates a change in mitochondrial dynamics that results, near ovulation, in mitochondria that are round (0.4–0.6 μm in diameter), vacuolated, and have arched and concentrically arranged cristae. Moreover, as oocytes develop, mitochondria, the smooth endoplasmic reticulum (ER), and lipid droplets become more frequently associated, pointing towards an important role of such organelle cluster (referred to as metabolic units) in late oogenesis. These changes in mitochondrial morphology, along with functional data, suggest that mitochondrial activity is downregulated through oogenesis. In agreement with this, granulosa cells generate multiple filopodia (arrowheads in the inset) that penetrate the zona pellucida (ZP) to reach the oocyte membrane and establish gap/adherens junctions. Apart from acting as a signaling hub, these cell junctions are used by granulosa cells to provide the gamete with energetic molecules such as ATP, pyruvate, nicotinamide adenine dinucleotide phosphate (NADP)H, cholesterol, and certain amino acids. The inset in “Ovulation” depicts an ovulated oocyte with mitochondria stained in green and visualized through confocal microscopy (scale bar = 25 μm)

While mitochondria in PGCs have a rounded shape (0.4–0.6 μm in diameter) and tubulo-vesicular cristae, these organelles get enlarged (0.8–1.0 μm in diameter) after differentiating into oogonia, besides owing sparse, lamellar cristae (Motta et al. 2000). In turn, during oocyte differentiation, mitochondria assume an “orthodox” (Fig. 1) configuration characterized by an elongated shape (1.2–1.9 μm in length) and numerous transversely oriented cristae, apart from a single large vacuole (Wassarman and Josefowicz, 1978). These morphological changes, which are suggestive of increased OXPHOS capacity, are, however, transient as dumbbell-shaped mitochondria become abundant in early developing oocytes (Fig. 1), eventually giving rise to round/oval vacuolated organelles displaying columnar-shaped, arched cristae (Wassarman and Josefowicz, 1978). Following, mitochondria in fully developed oocytes remain round/oval (0.4–0.6 μm in diameter), contain a large vacuole, and have arched/concentrically-arranged cristae (Fig. 1) (Trebichalská et al. 2021; Wassarman and Josefowicz, 1978). It is worth noting that in ruminants such as cows, mitochondria assume a singular hooded morphology in oocytes, with unclear functional consequences (Fair et al. 1997). Moreover, as oocytes develop, mitochondria, the smooth endoplasmic reticulum (ER), and lipid droplets become more frequently associated (Fig. 1), pointing towards an important role of such organelle cluster (referred to as metabolic units) in late oogenesis (Kruip et al. 1983; Motta et al. 2000; Trebichalská et al. 2021).

About one hundred thousand mitochondria are estimated in fully-grown oocytes, which represents a ~ 1000-fold increase compared to the number in PGCs (Cao et al. 2007; Jansen and De Boer, 1998; Motta et al. 2000; Wai et al. 2008; Wassarman and Josefowicz, 1978). Although this enormous population has been attributed to an essential role of the organelle (Harris et al. 2009), mitochondrial density decreases with folliculogenesis progression (Babayev et al. 2016). Moreover, growing oocytes have fragmented mitochondria and poorly developed mitochondrial cristae and are only slightly affected by PDHA1 deficiency (Johnson et al. 2007; Kruip et al. 1983; Motta et al. 2000; Tarazona et al. 2006; Trebichalská et al. 2021; Wassarman and Josefowicz, 1978), collectively suggesting that mitochondrial activity is likely downregulated in oocytes. Indeed, there are several reports showing that both pyruvate consumption (normalized by oocyte volume) and mitochondrial-coupled oxygen consumption decrease with oocyte development (Biggers et al. 1967; Eppig, 1976; Harris et al. 2009; Hashimoto et al. 2017; Magnusson et al. 1986; Porterfield et al. 1998; Thompson et al. 1996; Trimarchi et al. 2000). Therefore, it is believed that the gamete is kept at a relatively quiescent catabolic status due to the action of granulosa cells in providing the oocyte with several energetic molecules, including ATP, pyruvate, NAD phosphate (NADP)H, cholesterol, and certain amino acids (Fig. 1) (Biggers et al. 1967; Downs, 1995; Eppig et al. 2005; Johnson et al. 2007; Richani et al. 2021; Su et al. 2007, 2009; Sugiura et al. 2007). Accordingly, reprogramming of somatic cells into a pluripotent state is paralleled by a metabolic switch characterized by fragmentation of the mitochondrial network and decreased mitochondrial activity (Chen and Chan, 2017; Cho et al. 2006; Ma et al. 2015a; Prigione et al. 2014; Son et al. 2015). On the other hand, mitochondrial-coupled ATP formation is essential for resumption of meiosis, spindle formation, and chromosome segregation, likely due to end of granulosa cell-oocyte cooperation near ovulation (Abbassi et al. 2021; Al-Zubaidi et al. 2019; Brevini et al. 2005; Dalton et al. 2014; Downs, 1995; Downs et al. 2002; Krisher and Bavister, 1998; Kruip et al. 1983; Pasquariello et al. 2019; Stojkovic et al. 2001; Van Blerkom et al. 1995; Yu et al. 2010).

Main effectors of mitochondrial dynamics

A large body of evidence has implicated mitochondria in a range of complex biological processes encompassing cellular signaling, differentiation, migration, stemness, autophagy, senescence, inflammation, immune response, and cell death, apart from their long-standing role in energy production. Additionally, mitochondria interact with each other and with subcellular compartments such as the ER, lysosomes, peroxisomes, endosomes, melanosomes, lipid droplets, the plasma membrane, and the nuclear membrane (reviewed by (Gordaliza‐Alaguero et al. 2019)). To efficiently play these functions, mitochondria are constantly adapting their morphology and localization in the cell, which is collectively known as mitochondrial dynamics (Fig. 2). As such, the mitochondrial network can range in shape from isolated, rounded organelles to long interconnected tubules. In addition, mitochondria can distribute evenly across the cytosol or form clusters to meet local demands, events that are cell specific and can vary throughout the cell cycle and in response to metabolic and cellular clues (reviewed by (Pernas and Scorrano, 2016)).

Fig. 2.

Main effectors of mitochondrial dynamics. Cyclic events of organelle fusion and fission (division) driving, respectively, elongation and fragmentation of the mitochondrial network are responsible for determining mitochondrial dynamics. Towards that, mitofusins 1 (MFN1) and 2 (MFN2) as well as the optic atrophy protein 1 (OPA1) are the main effectors of fusion. MFN1 and MFN2 are both located in the outer mitochondrial membrane, whereas OPA1 resides in the inner mitochondrial membrane. While MFN1 and OPA1 have a central role in the fusion reaction, MFN2 is a key regulator of the oxidative metabolism and has been implicated on mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) formation. OPA1 is also central to mitochondrial cristae remodeling. In turn, mitochondrial fission is primarily carried out by the dynamin-related protein 1 (DRP1), which locates in the cytosol and acts at sites of mitochondria-ER membrane juxtaposition

Mitochondrial dynamics is essential for maintenance of health organelles as repeated cycles of organelle fusion and fission are required for homogenization of the mitochondrial network (Ban-Ishihara et al. 2013; Chen et al. 2003, 2005; Ishihara et al. 2015; Twig et al. 2008). Furthermore, mitochondrial dynamics regulates organelle transport, inheritance, and selective removal (Kleele et al. 2021; Lieber et al. 2019; Misko et al. 2012; Twig et al. 2008), with defects in this machinery leading to a range of diseases in humans (Table 1). Mitofusins 1 (MFN1) and 2 (MFN2) as well as the optic atrophy protein 1 (OPA1), all belonging to the dynamin-related family of large GTPases, are the main effectors responsible for mitochondrial fusion in mammals (Chen et al. 2003, 2005, 2007, 2010). MFN1 and MFN2 are both located in the outer mitochondrial membrane, whereas OPA1 resides in the inner mitochondrial membrane (Fig. 2). While MFN1 and OPA1 have a central role in the fusion reaction, the exact role of MFN2 in fusion remains unclear. OPA1 is also central to mitochondrial cristae remodeling, being its function under the control of multiple enzymes such as YME1L1, OMA1, and PARL (reviewed by (Pernas and Scorrano, 2016)). More recently, mitoguardins 1 (MIGA1) and 2 (MIGA1), which act downstream of mitofusins, have also been shown to regulate mitochondrial fusion by interacting with mitochondrial phospholipase D (MitoPLD) to facilitate MitoPLD dimer formation (Zhang et al. 2016a). On other hand, mitochondrial fission is primarily carried out by the dynamin-related protein 1 (DRP1), which locates in the cytosol and is attracted to the mitochondrial outer membrane by receptors such as the mitochondrial fission factor (MFF), the mitochondrial fission 1 protein (FIS1), and the mitochondrial dynamics proteins of 49 kDa (MID49) and 51 kDa (MID51). Once bound to these receptors, DRP1 is capable of oligomerizing and driving mitochondrial division (Fig. 2) (Giacomello et al. 2020; Ishihara et al. 2009; Kleele et al. 2021).

Table 1.

Main effectors of mitochondrial dynamics and their role in diseases

Gene	Main role	Disease/symptoms	References
Mitofusin 1 (MFN1)	Outer mitochondrial membrane protein that mediates mitochondrial fusion	Unknown	Chen et al. (2003); Chen et al. (2005); Chen et al. (2007); Chen et al. (2010)
Mitofusin 2 (MFN2)	Outer mitochondrial membrane protein that mediates mitochondrion-mitochondrion interaction and juxtaposition of mitochondria with the endoplasmic reticulum	Charcot-Marie-Tooth disease type 2A (CMTD2A) Hereditary motor and sensory neuropathy (HMSN) Multiple symmetric lipomatosis (MSL) Canine fetal-onset neuroaxonal dystrophy	Brockman et al. (2008); Chen et al. (2003); Chen et al. (2007); Chen et al. (2005); Chen et al. (2010); Fyfe et al. (2011); Rouzier et al. (2012); Sawyer et al. (2015); Zhu et al. (2005); Züchner et al. (2004)
Optic atrophy 1 protein (OPA1)	Inner mitochondrial membrane protein that mediates mitochondrial fusion	Autosomal dominant optic atrophy (ADOA) Mitochondrial DNA depletion syndrome Behr syndrome Syndromic Parkinson disease and dementia	Alexander et al. (2000); Amati-Bonneau et al. (2008); Carelli et al. (2015); Delettre et al. (2000); Hudson et al. (2008); Schaaf et al. (2011); Spiegel et al. (2016)
Yeast mitochondrial escape 1-like 1 (YME1L1)	Inner mitochondrial membrane peptidase that mediates OPA1 processing	Intellectual disability, motor development delay, expressive speech delay, optic nerve atrophy associated with visual impairment, and hearing impairment	Anand et al. (2014); Hartmann et al. (2016)
OMA1 zinc metalloprotease	Inner mitochondrial membrane peptidase that mediates OPA1 processing	Unknown	Anand et al. (2014)
Rbd1p orthologue presenilin-associated rhomboid-like protein (PARL)	Inner mitochondrial membrane peptidase that mediates OPA1 processing	Unknown	Saita et al. (2017)
Dynamin-related protein 1 (DRP1)	Cytoplasmic protein that mediates mitochondrial fission	Encephalopathy Autosomal dominant optic atrophy Microcephaly and pain insensitivity Intractable forms of epilepsy Neonatal lethality	Fahrner et al. (2016); Gerber et al. (2017); Ishihara et al., (2009); Kleele et al., (2021); Losón et al. (2013); Sheffer et al. (2016); Vanstone et al. (2016); von Spiczak et al. (2017); Waterham et al. (2007)
Mitochondrial fission factor (MFF)	Outer mitochondrial membrane receptor that recruits DRP1 for organelle fission	Leigh-like encephalopathy, optic atrophy, and peripheral neuropathy Encephalopathy due to defective mitochondrial and peroxisomal fission	Kleele et al., (2021); Koch et al. (2016); Losón et al. (2013); Palmer et al. (2013); Shamseldin et al. (2012)
Mitochondrial fission 1 protein (FIS1)	Outer mitochondrial membrane receptor that recruits DRP1 for organelle fission	Unknown	Kleele et al., (2021); Losón et al. (2013); Palmer et al. (2013); Shen et al. (2014);
Mitochondrial dynamics protein of 49 kDa (MID49)	Outer mitochondrial membrane receptor that recruits DRP1 for organelle fission	Unknown	Losón et al. (2013); Palmer et al. (2013)
Mitochondrial dynamics protein of 51 kDa (MID51)	Outer mitochondrial membrane receptor that recruits DRP1 for promoting organelle fission	Unknown	Losón et al. (2013); Palmer et al. (2013)

Prior to mitochondrial fission, the smooth ER wraps around mitochondria to possibly drive, with the aid of actin filaments, initial organelle constriction (Fig. 2). Actin polymerization further supports DRP1 oligomerization and GTP hydrolysis-mediated membrane constriction (Chakrabarti et al. 2018; Korobova et al. 2013; Manor et al. 2015). It is noteworthy that MFF and DRP1 often colocalizes with nucleoids, thus favoring maintenance of small and fragmented nucleoids, cristae structure, and the pro-apoptotic status of mitochondria (Ban-Ishihara et al. 2013; Lewis et al. 2016). Mitochondria-ER contact sites (MERCs) serve, therefore, as fission platforms, apart from acting as important hubs for lipid trafficking, Ca²⁺ signaling, ER stress, apoptosis, and macroautophagy. Several protein complexes such as VAPB-PTPIP51, IP3R1/2/3-GRP75-VDAC1, and BAP31-FIS1 have been implicated in mitochondria-ER juxtaposition. The same holds true for MFN2 (Fig. 2), which is also present in the ER membrane where from it can homo-oligomerize and hetero-oligomerize with either MFN2 or MFN1 in mitochondria to tether both organelle membranes (de Brito and Scorrano, 2008; Gordaliza‐Alaguero et al. 2019).

Regulation of mitochondrial dynamics in oocytes and implications to fertility

Given that oocyte mitochondria have a fragmented morphology, are vacuolated, and contain arched and concentrically arranged cristae, it is tempting to suppose that these characteristics are coordinated by specific regulation of mitochondrial dynamics in oocytes. To investigate this, Udagawa et al. (2014) performed oocyte-specific deletion of DRP1 in mice, finding that oocyte mitochondria are fusion competent as fission deficiency resulted in elongated organelles (Fig. 3a). This conclusion is further supported by the presence of profusion proteins MFN1, MFN2, and OPA1 in oocytes (Liu et al. 2016a; Wakai et al. 2014). Yet, overexpression of either MFN1 or MFN2 did not lead to mitochondrial tubulation (Wakai et al. 2014), supporting in oocytes an imbalance of mitochondrial dynamics towards fission. Alternatively, this result might be attributed to the short period (≤ 15 h) of mitofusin overexpression or restricted to late stages of oogenesis as mitochondrion-mitochondrion contact sites were clearly increased following mitofusin overexpression (Wakai et al. 2014). In addition, mitofusin overexpression led to markedly aggregated mitochondria, particularly around the nucleus/chromosomes (Wakai et al. 2014), a phenotype that relied on MFN1 GTPase activity and associated with disrupted spindle/metaphase plate organization (Wakai et al. 2014).

Fig. 3.

Mitochondrial dynamics in oocytes is essential to fertility and links mitochondrial-endoplasmic reticulum (ER) dysfunction with metabolic disorder transmission. Oocyte-specific deletion of dynamin-related protein 1 (DRP1) results in oocytes with elongated mitochondria, depleted endoplasmic reticulum (ER) calcium (Ca²⁺) stores, multiorganelle (i.e., mitochondria, ER, peroxisomes and vesicles) aggregation, and defective secretory vesicle release (a). In turn, mitofusin 1 (MFN1) knockout strongly impairs mitochondrial dynamics as evidenced by organelle swelling, clustering, abnormal cristae, and inner membrane vesiculation, although mitochondrial shape remains unchanged (b). These defects are paralleled by decreased expression of oxidative phosphorylation subunits, decreased mitochondrial membrane potential, decreased mitochondrial DNA (mtDNA) copy number, increased flavin adenine dinucleotide (FAD) levels, and increased ROS levels. As a result, MFN1-deficient oocytes have decreased ATP content, increased apoptosis, and impaired PI3K-AKT signaling. Despite these differences, the lack of either MFN1 or DRP1 results in infertility due impaired granulosa cell-oocyte communication and arrested folliculogenesis. On the other hand, oocytes lacking mitofusin 2 (MFN2) have a pronounced transcriptomic change with 943 differentially expressed genes (DEGs) associated with pathways regulating, among others, ER function, the phagosome, endocrine resistance, and mitophagy (c). These oocytes also present dysregulated mitochondria-ER contact sites (MERCs) and mitochondrial dysfunction characterized by mitochondrial aggregation and swelling, decreased mitochondrial membrane potential, lower mtDNA copy number, lower nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) levels, increased FAD levels, and decreased ATP content. Unexpectedly, cristae structure is well developed in MFN2-knockout oocytes, better than in wild-type mice. As a result, oocyte-specific MFN2 deficiency results in offspring with increased weight gain and glucose intolerance, abnormalities that associate with slight liver mitochondrial dysfunction, increased insulinemia, and impaired insulin signaling

Targeted deletion of DRP1 during oocyte development resulted in female infertility due to impaired folliculogenesis, a result attributed to depleted ER Ca²⁺ stores, multiorganelle (i.e., mitochondria, ER, peroxisomes and vesicles) aggregation, and defective secretory vesicle release (Fig. 3a) (Udagawa et al. 2014). Curiously, neither mitochondrial membrane potential nor mtDNA copy number was altered in DRP1-deficient oocytes, although nucleoids were highly clustered (Udagawa et al. 2014). The apparent lack of effect on mitochondrial function contrasts with reports showing that fission is critical for mitochondrial network homogenization and turnover (Ban-Ishihara et al. 2013; Ishihara et al. 2015; Ota et al. 2020; Twig et al. 2008), a finding that might be secondary to the unique characteristics of mitochondria in oocytes. In turn, targeted deletion of MFN1 strongly prevented mtDNA accumulation during oocyte growth as well as led to evident mitochondrial dysfunction characterized by organelle swelling, abnormal expression of OXPHOS subunits, decreased membrane potential, increased FAD level, increased ROS level, and decreased ATP content (Fig. 3b). MFN1-deficient oocytes also contained highly clustered mitochondria with severe ultrastructure abnormalities such as inner membrane vesicles and decreased number of cristae (Carvalho et al. 2020; Hou et al. 2019; Zhang et al. 2019a). These findings are consistent with previous reports showing that MFN1 is required for mitochondrial health, mtDNA stability, and OPA1 activity (Chen et al. 2003, 2005, 2007, 2010; Cipolat et al. 2004), thus confirming the important role of fusion in oocytes as evidenced for MIGA1 and MIGA2 knockouts (Liu et al. 2016b). Nonetheless, neither MFN1 deficiency nor DRP1 overexpression enhanced mitochondrial fragmentation (Carvalho et al. 2020; Hou et al. 2019; Wakai et al. 2014; Zhang et al. 2019a), indicating that mitochondrial size is strictly regulated in oocytes. This finding contrasts, however, with that of oocytes lacking the mitochondrial unfolded protein response CLPP, which did display smaller mitochondria in addition to decreased expression of profusion genes Mfn1, Mfn2, and Opa1 (Wang et al. 2018a).

Apart from an evident mitochondrial dysfunction, MFN1-deficient oocytes also presented increased apoptosis, disruption of adherens/gap junctions, and impaired communication with granulosa cells (Fig. 3b) (Carvalho et al. 2020; Zhang et al. 2019a). Remarkably, females with MFN1-deficient oocytes never ovulated due to a folliculogenesis arrest at the secondary-to-antral follicular stage, a phenotype that was shown to worsen with aging and led to accelerated follicular depletion (Carvalho et al. 2020; Hou et al. 2019; Zhang et al. 2019a). Zhang et al. (2019a) associated these effects of MFN1 deletion to accumulation of ceramide in oocytes, a membrane sphingolipid and an important inducer of apoptosis and cell cycle arrest (Fig. 3b). In turn, Carvalho et al. (2020) found that the additional knockout of MFN2 mitigated the impact of MFN1 deficiency on folliculogenesis, enabling enhanced mitochondrial health, oocyte growth, and ovulation in double-knockout females (Carvalho et al. 2020). Moreover, while the single loss of MFN1 altered the expression of 161 genes, the double loss of MFN1 and MFN2 impacted the expression of 474 genes when compared to wild-type oocytes. Enrichment analysis of transcriptomic differences linked the effects of single MFN1 deletion to disrupted PI3K-Akt signaling (Fig. 3b), a pathway that is known to have a fundamental role in oogenesis, being required for granulosa cell-oocyte communication (Dong et al. 1996; Liu et al. 2014; Reddy et al. 2008). In keeping with this, previous reports have shown that MFN2 inhibits the PI3K-AKT signaling (Chen et al. 2004, 2014; de Brito and Scorrano, 2009; Ma et al. 2015b), suggesting that the phenotype of MFN1-knockout oocytes may be explained, at least in part, by a deleterious action of MFN2. Indeed, Mfn1 expression in wild-type oocytes was fivefold higher than that of Mfn2, and the treatment of MFN1-deficient oocytes with an agonist of the PI3K-AKT signaling partially rescued oogenesis/folliculogenesis (Carvalho et al. 2020). Therefore, mitofusins seem to play distinct, nonredundant roles in oocytes, with MFN1 acting downstream of MFN2 to counter its activity.

Role of mitochondrial dynamics in oocyte-linked transmission of metabolic disorders

Although environmental and genetic factors are the main determinants of metabolic disorders, female over-nutrition permanently programs the metabolism of offspring (Agarwal et al. 2018; Akamine et al. 2010; Jungheim et al. 2010; Mdaki et al. 2016; Murrin et al. 2012; Oestreich and Moley 2017; Rattanatray et al. 2010; Reynolds et al. 2013; Ruager-Martin et al. 2010; Shankar et al. 2008; Wyman et al. 2008). Accordingly, maternal obesity has been found to cause lipid accumulation in granulosa cells and oocytes, thereby resulting in oocytes with dysfunctional mitochondria as evidenced by their ultrastructural defects, aggregation, lower membrane potential, increased ROS, and increased Ca²⁺ (Andreas et al. 2019; Boots et al. 2016; Boudoures et al. 2017; Ferey et al. 2019; Marei et al. 2020; Reynolds et al. 2013; Wu et al. 2010, 2015). In addition, obesity leads to MERC disruption, ER stress, and activation of the unfolded protein response (UPR) in oocytes (Marei et al. 2019, 2020; Wu et al. 2015; Zhao et al. 2017). Thus, besides impacting oocyte health and fertility, these abnormalities, likely underpinned by abnormal mitochondria and epigenetic marks (Agarwal et al. 2018; Keleher et al. 2018; Ou et al. 2019; Ruebel et al. 2017; Sirard, 2019; Wang et al. 2018b), can be passed down the maternal lineage and contribute with the onset of metabolic disorders in the following generations.

It is currently unclear the precise mechanism by which obesity affects mitochondria and the ER in oocytes. However, it is well known that nutrient abundance can overload the ETC with highly charged electrons, resulting in excessive ROS generation and, eventually, oxidative stress. ROS can damage mitochondria, leading to mitochondrial fragmentation and dysfunction, further aggravating the oxidative stress (reviewed by (Kakimoto and Kowaltowski, 2016; Zorzano et al. 2015)). In this context, MFN2 has been shown to exert an important role in adapting the mitochondrial metabolism according to nutrient availability. MFN2 acts through regulation of both mitochondrion-mitochondrion and mitochondrion-ER interactions, besides determining mitochondrial interaction with other subcellular compartments such as lipid droplets and lysosomes (reviewed by (Benador et al. 2019; Gordaliza‐Alaguero et al. 2019)). There is also a bunch of evidence connecting decreased MFN2 expression with metabolic inflexibility, mitochondrial dysfunction, ER stress, impaired insulin signaling, leptin resistance, glucose intolerance, obesity, and diabetes (Bach et al. 2003; Mahdaviani et al. 2017; Mourier et al. 2015; Muñoz et al. 2014; Ngoh et al. 2012; Pich et al. 2005; Schneeberger et al. 2013; Sebastian et al. 2012; Sebastián et al. 2016; Tubbs et al. 2014).

Given the key role of MFN2 in regulating the energetic metabolism, Garcia et al. (2020) have investigated whether mouse dams with oocyte-specific deletion of MFN2 deliver offspring that are prone to metabolic disorders. Earlier reports have shown that MFN2 is not essential for oocyte development, with little, if any, impact on fertility (Carvalho et al. 2020; Hou et al. 2019; Zhang et al. 2019b). In spite of this, MFN2-deficient oocytes displayed mitochondrial dysfunction, dysregulated MERCs, and an overt transcriptomic change encompassing 943 differentially expressed genes (Garcia et al. 2020). Enrichment analysis of the transcriptomic data revealed an over-representation of genes related to ER function, phagosome, endocrine resistance, and mitophagy (Garcia et al. 2020). These findings are in keeping with previous reports in which MFN2 was targeted through either oocyte-specific deletion or RNA interference, thereby leading to mitochondrial dysfunction, shortened telomeres, increased apoptosis, and, in turn, poor quality oocytes and accelerated follicular depletion (Liu et al. 2016a; Zhang et al. 2016b, 2019b). Likewise, MFN2 overexpression, which increased mitochondria-ER juxtaposition in oocytes, led to alterations in the ER such as a disintegrated network, clusterization, and loss of Ca²⁺ stores (Wakai et al. 2014). Consequently, offspring derived from MFN2-deficient oocytes presented increased weight gain and glucose intolerance, a phenotype that was linked to decreased insulinemia and defective insulin signaling (Garcia et al. 2020). Accordingly, Zhao et al. (2017) have reported that obesity affects MERCs in oocytes, an effect that is prevented by PACS2 downregulation. Furthermore, Saben et al. (2016) have shown that maternal metabolic syndrome leads to persistent transmission of impaired mitochondrial fission and decreased OPA1 expression across three generations. Altogether, these findings support a role for mitochondrial dynamics in regulating mother-to-offspring transmission of metabolic disorders. Further studies should focus on characterizing a possible underlying mechanism linking maternal obesity with abnormal mitochondrial dynamics.

Concluding remarks

A large body of evidence supports that mitochondria are kept under a relatively quiescent state in oocytes, likely to prevent the gamete from oxidative stress once somatic granulosa cells are responsible for supplying most of the energetic molecules needed for oogenesis. As such, mitochondrial health is inextricably linked to mitochondrial dynamics, which regulates in oocytes several aspects of the mitochondrial biology including organelle morphofunctional characteristics, mobility, interaction with subcellular compartments, signal transduction, and apoptosis. Thereby, malfunctioning of mitochondrial dynamics can disturb oocyte formation, ultimately leading to infertility and disease transmission.

Declarations

Ethical approval

This article does not contain any studies with human participants or animals performed by the author.

Conflict of interest

The author declares no competing interests.