Mitochondrial functions on oocytes and preimplantation embryos

Abstract

Oocyte quality has long been considered as a main limiting factor for in vitro fertilization (IVF). In the past decade, extensive observations demonstrated that the mitochondrion plays a vital role in the oocyte cytoplasm, for it can provide adenosine triphosphate (ATP) for fertilization and preimplantation embryo development and also act as stores of intracellular calcium and proapoptotic factors. During the oocyte maturation, mitochondria are characterized by distinct changes of their distribution pattern from being homogeneous to heterogeneous, which is correlated with the cumulus apoptosis. Oocyte quality decreases with the increasing maternal age. Recent studies have shown that low quality oocytes have some age-related dysfunctions, which include the decrease in mitochondrial membrane potential, increase of mitochondrial DNA (mtDNA) damages, chromosomal aneuploidies, the incidence of apoptosis, and changes in mitochondrial gene expression. All these dysfunctions may cause a high level of developmental retardation and arrest of preimplantation embryos. It has been suggested that these mitochondrial changes may arise from excessive reactive oxygen species (ROS) that is closely associated with the oxidative energy production or calcium overload, which may trigger permeability transition pore opening and subsequent apoptosis. Therefore, mitochondria can be seen as signs for oocyte quality evaluation, and it is possible that the oocyte quality can be improved by enhancing the physical function of mitochondria. Here we reviewed recent advances in mitochondrial functions on oocytes.

INTRODUCTION

Oocyte quality can be defined as its abilities to be fertilized, mature, and give rise to normal offspring (Duranthon and Renard, 2001). Good quality oocytes are exceedingly essential for fertilization, especially for successful in vitro maturation (IVM) and fertilization (IVF), which are two major assisted-reproductive technologies for female infertility. At present, the cellular mechanisms that impart oocyte quality have not been elucidated, although many researchers have studied the functions of mitochondria in oocytes (Table 1). Mitochondria are energy-supplying organelles, whose functional integrality is essential for cellular survival and development. Mitochondria in the oocyte can provide adenosine triphosphate (ATP) for fertilization and preimplantation embryo development (Torner et al., 2004) and they can act as stores of intracellular calcium (Ca) and proapoptotic factors as well.

Table 1.

Catalog of research progresses on mitochondrial function in oocytes and preimplantation embryos

Mitochondria	Referencesa
Mitochondrial inheritance	Cummins et al., 1997; Cox and Spradling, 2003; 2006
Mitochondrial distribution	Wilding et al., 2001; Torner et al., 2004; Nishi et al., 2003
Mitochondrial movement	van Blerkom, 1991; Cox and Spradling, 2003; 2006
mtDNA content	Reynier et al., 2001; Spikings et al., 2007
mtDNA mutation	Yesodi et al., 2002; Hsieh et al., 2004
Mitochondrial polarity	van Blerkom et al., 2003; 2006;van Blerkom and Davis, 2006; 2007
Function in calcium regulation	Machaty et al., 1997; Krisher, 2004; Whitaker, 2008
Mitochondrial dysfunction	Tarín, 1996; Eichenlaub-Ritter, 1998; Yin et al., 1998; Ottolenghi et al., 2004; Thouas et al., 2004; Zhang et al., 2006
Mitochondria transfer	Pinkert et al., 1997; Malter and Cohen, 2002; Kong et al., 2004; Yi et al., 2007

^aStudies involved in oocytes and embryos

Mitochondria have their own genetic materials, mitochondrial DNA (mtDNA) that is derived from maternal mtDNA exclusively, for the paternal mitochondria are not retained in the fertilized oocyte at the four-cell stage (Cummins et al., 1997). There are approximately 100 000 to 200 000 mtDNA copies in a mammalian oocyte (Reynier et al., 2001), which are divided among all daughter cells during the developmental progress of embryos. Because there is no mtDNA replication until postimplantation, this renders oocytes more susceptible to any kind of mitochondrial dysfunction (Spikings et al., 2007). Recent studies have shown that mitochondrial dysfunctions, such as the structural, spatial and genetic abnormalities in the oocyte, may influence normal embryo development, so mitochondrial characteristics and other mitochondrion-related changes can serve as signs of oocyte quality.

MITOCHONDRIA AND OOCYTE QUALITY AND MATURATION

Oocyte maturation includes nuclear maturation and cytoplasmic maturation, and both of them are essential for the fertilization and embryo development (Eppig, 1996). The nuclear maturation was characterized by the release of the first polar body (Fulka et al., 1998), while the cytoplasmic maturation is difficult to evaluate. But presently, it is reported that mitochondria may be used to estimate cytoplasmic maturation because oocyte maturation in vitro is accompanied by the distribution changes of active mitochondria in addition to distinct cumulus morphological changes (Torner et al., 2004).

Mitochondrial distribution and oocyte maturation

Homogeneousness and heterogeneousness are two major mitochondrial distribution patterns in oocytes. Homogeneous distribution of mitochondria throughout the cytoplasm is more common at germinal vesicle stage (Nishi et al., 2003), while heterogeneous distribution is more commonly observed in the oocyte of metaphase I or II (Torner et al., 2004). The increased level of mitochondrial aggregation around the nucleus indicates oocyte maturation, during which mitochondrial distribution changed from being homogeneous to heterogeneous. More precisely, in the heterogeneous state, the mitochondrial distribution changed from granulated aggregation to clustered aggregation. It is also observed that morphologically poor quality embryos are only characterized by the homogenous distribution of mitochondria (Wilding et al., 2001). Inappropriate culture conditions may inhibit mitochondrial movement to the inner cytoplasm and thus affect its cytoplasmic maturation (Torner et al., 2004). It was reported that mitochondrial redistribution in different cellular areas is mediated by cytoskeleton, especially microtubule network (van Blerkom, 1991). The abnormal distribution of mitochondria is related to the inappropriate formation of the cytoplasmic microtubule network, which can lead to the retardation or arrest of oocyte development and exert negative effects on the embryogenesis due to the abnormal ATP distribution (Nagai et al., 2006), because high energy supply around nucleus is very important during embryonic development. Therefore, proper distribution of active mitochondria during oocyte maturation is necessary for further development.

Calcium signaling in mitochondria

Mitochondrial redistribution in the oocyte may result from the high demand for ATP and calcium during cytoplasmic maturation, since the main functions of mitochondria are ATP synthesis and calcium supply (Krisher, 2004). Mitochondria form aggregates with the smooth endoplasmic reticulum (SER), and these aggregates are apparently involved in the energy production and deposition before fertilization (Torner et al., 2004). Mammalian oocyte maturation relies not only on a high level of ATP for nuclear envelope breakdown, but also on intracellular calcium, which is derived from the endoplasmic reticulum (ER) and mitochondria (Krisher, 2004).

Inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors were found to localize on the surface of the ER, which are active from the germinal vesicle stage to oocyte maturation (Machaty et al., 1997). These two receptors enable redundant calcium to release from the intracellular stores. Ca²⁺-induced Ca²⁺ release (CICR) is considered as a feedback process of IP3- and ryanodine-induced Ca²⁺ release (Chakraborti et al., 1999). Studies have shown that mitochondria have an enormous capacity to regulate Ca²⁺ and prevent cytoplasmic Ca²⁺ concentration from rising too high potently. When the Ca²⁺ in the matrix gets overloaded, mPT pore (mitochondrial permeability transition pore) will be formed for the Ca²⁺ release (Giacomello et al., 2007).

Mitochondria and ER are both important protectors involved in intracellular calcium homeostasis. Ca²⁺ enters mitochondria via the Ca²⁺ uniporter when Ca²⁺ concentration in the cytosol is over a certain threshold, and once the cytosolic Ca²⁺ has returned to its resting level, Ca²⁺ is pumped back into cytoplasm by the Na⁺/Ca²⁺ exchanger (Bootman et al., 2001). These two channels together with the Na⁺/H⁺ antiporter establish a transport cycle to mediate Ca²⁺ concentration in both cytosol and matrix. The cytosolic excessive Ca²⁺ is transported to the ER by sarco-endoplasmic reticulum Ca²⁺ ATPases (SERCAs) or removed from the cell through plasma membrane Ca²⁺-ATPase isoforms (PMCAs) and Na⁺/Ca²⁺ exchangers located in the plasma membrane to the extracellular medium. This cycle is quite crucial for intracellular Ca²⁺ homeostasis (Berridge et al., 2000; Smaili et al., 2000). Whitaker (2008) recently reviewed that calcium may perform an important function in embryonic cell-cycle transition and embryonic axis establishment.

Interaction between ooplasmic mitochondria and cumulus cells

Communication between the oocyte and its surrounding cumulus cells is also important for oocyte development potential (Krisher, 2004) and therefore for the success of IVM and IVF (Mermillod et al., 2008). The cumulus cells are responsible for nutrition supply for oocytes in the final phase of oocyte maturation (Gilchrist et al., 2008). Morphological characters of cumulus cells can be used to value oocyte quality and its maturation (Torner et al., 2004). Compact and complete cumulus and bright and homogeneous ooplasm are considered as a sign of high-quality immature oocytes, whereas cumulus with no more than three layers or dark and heterogeneous ooplasm indicates low-quality immature oocytes (Blondin and Sirard, 1995).

It has been suggested that mitochondrial polarity is a determinant of ATP generation and calcium homeostasis (van Blerkom and Davis, 2007; van Blerkom, 2008), and van Blerkom et al.(2008) recently found that mitochondrial membrane potential (Δφ _m) in the subplasmalemmal domain of oocytes could be mediated by cumulus-derived nitric oxide (NO) during oocyte maturation. NO is important for ovulation and it can de-energize mitochondria by depressing the generation of ATP in the subplasmalemmal domain of oocytes, which is required for ovulatory process. Previous studies have shown that suppression of NO synthesis can prevent ovulation (Bu et al., 2003) and the effect of NO on oocyte maturation is related to its inhibition on estradiol production (Voznesenskaya and Blashkiv, 2005).

As we know, during meiotic progression, oocyte maturation also couples with cumulus expansion or apoptosis and the number of cumulus cells attached to matured oocytes decreases with age (Qiao et al., 2008). Torner et al.(2004) found that mitochondrial redistribution and their oxidative activity are essential to the degree of cumulus cell apoptosis during oocyte maturation. Mitochondrial fine homogeneous distribution is correlated to low oxidative activity and low levels of cumulus cell apoptosis, while granulated homogeneous distribution is correlated to high oxidative activity and high levels of cumulus cell apoptosis. It is probable that oocytes transport proapoptotic molecules from the activated ooplasmic mitochondria to the surrounding cumulus. Paracrine factors and gap junctions participate in the communication between oocytes and cumulus cells (Andreuccetti et al., 1999; Albertini et al., 2001). Recent studies have proven that oocytes may regulate the functions of cumulus itself instead of being controlled by cumulus (Gilchrist et al., 2008). Hussein et al.(2005) demonstrated that soluble paracrine signals from the oocyte may contribute to the levels of apoptosis in cumulus cells rather than gap junctions, because destroying the gap junctions between cumulus cells and oocytes has effects on apoptosis in all cumulus cell layers instead of the closest layer to the oocyte. Growth-differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are two key oocyte-secreted factors (OSFs), which are expressed in the ooplasm and function in cumulus cells. These two factors can activate the signaling pathways in cumulus cells to mediate the development of their neighboring oocytes. It is obvious that GDP9 and BMP15 play vital roles in oocyte maturation and quality determination (Gilchrist et al., 2008). Sharov et al.(2008) have detected the expression of GDP9 and BMP15 decreased in aged oocytes, which can be also used for oocyte quality evaluation.

MITOCHONDRIA AND OOCYTE AGING

Aging is the accumulation of function loss in diverse organs and tissues. Natural human fertility decreases with the maternal age, which may reduce the success rate of IVF. The success rate of IVF pregnancy in women older than 35 years old is markedly lower than that in younger ones (Hsieh et al., 2004). Previous investigations suggested that poor oocyte quality was responsible for the age-related decline in female fertility competence (Wu et al., 2000). For the aged oocytes, mitochondrial pathophysiology occurs in the ooplasm, which includes an increased density of mitochondrial matrix, increased mtDNA damage, and higher frequency of ruptured mitochondrial membranes (Ottolenghi et al., 2004), while Δφ _m, ATP synthesis and metabolic reactions in the electron transport chain decrease with age (Sharov et al., 2008). All these changes may link to the female infertility.

MtDNA and oocyte aging

Human mtDNA strictly derives from maternal inheritance (Kaneda et al., 1995; Cummins et al., 1997). The number of mtDNA copies varies in oocytes, and lower copy numbers may be caused by inadequate cytoplasmic maturation or mitochondrial biogenesis (Reynier et al., 2001). The mtDNA content in oocytes with normal fertilization capability is higher than that in abnormal oocytes (Reynier et al., 2001), and aging has been reported to exert a negative influence on the process of mitochondriogenesis (López-Lluch et al., 2008).

Over 150 mtDNA mutations have been identified (Yesodi et al., 2002), among which the 4977-bp deletion named as ΔmtDNA⁴⁹⁷⁷, is the most common one. The ΔmtDNA⁴⁹⁷⁷ accumulation with aging suggests that it should be a marker of aging (Meißner et al., 1997). ΔmtDNA⁴⁹⁷⁷ results from the removal of structural genes including ATPases 6, ATPase 8, cytochrome oxidase III, and NADH-CoQ oxidoreductase subunits 3, 4L, 4 and 5 (Wang and Lü, 2009). The accumulated mtDNA mutations induce insufficient amounts of active mitochondria and a decline of oxidative phosphorylation efficiency, all of which may contribute to poor oocyte fertilization (Hsieh et al., 2004). In addition, mtDNA mutation in aged cells may generate a specific peptide for triggering cytochrome-c release (Dubec et al., 2008). At present, a study showed another mtDNA deletion, ΔmtDNA⁵²⁸⁶, that was a non-age-related marker for impaired oocyte quality in IVF (Yesodi et al., 2002).

The production of reactive oxygen species (ROS) is the main reason for oxidative damages of DNA. 8-oxodeoxyguanosine (8-oxo-dG) is the most common base modification in mutagenic damage, which is usually used as a biomarker of oxidative stress (Haghdoost et al., 2005). Previous observations suggested that oxidative damage and a lack of DNA repair mechanism may contribute to mtDNA mutation accumulation. Anson et al.(1998) demonstrated that there was a DNA repair pathway in the mitochondrion by detecting lesion ratio between nuclear DNA and mtDNA. A common sense is that the level of 8-oxo-dG is much higher in mtDNA than in nuclear DNA, if there is no DNA repair capability in the mitochondrion. But the result that the ratio was a constant suggested the existence of DNA repair capability. Vermulst et al.(2008) also proved the existence of a homology-directed double-stranded break (DSB) repair mechanism in mammalian mitochondria. Kujoth et al.(2005) found that accumulation of mtDNA mutations increases in the incidence of apoptosis with aging instead of responding to oxidative stress. It was also found that mtDNA mutation was not correlated with increased ROS production (Trifunovic et al., 2005). Therefore, it may be a phenotypic expression, which differs from mtDNA mutation accumulation that causes cellular aging (Vermulst et al., 2008).

Mitochondrial polarity, ATP content, and oocyte aging

The embryo development is strongly correlated with the activity of mitochondria in oocytes, which is characterized by Δφ _m (Wilding et al., 2001). High-polarized mitochondria are commonly seen in the pericortical cytoplasm of oocytes and early embryos, which may be associated with their functions in metabolic regulation including ATP production and calcium homostasis (van Blerkom et al., 2003). Intracellular high-polarized mitochondria can reflect the embryo ability to reactivate development in the diapausing state (van Blerkom and Davis, 2006). The high-polarized mitochondrial domain in an oocyte may also have an effect on sperm penetration and cortical granule exocytosis (van Blerkom and Davis, 2007). Low-polarized mitochondria are associated with abnormal embryos (Wilding et al., 2001). Δφ _m may be involved in material transport and energy transition, so a decrease of Δφ _m may lead to mitochondrial dysfunction. Studies on embryo fragmentation showed that large number of fragmentation resulted in the residual cell without any high-polarized mitochondria in subplasmalemmal domain, while studies on 2-cell embryos with unequal blastomeres showed that the daughter cells without high-polarized mitochondria in subplasmalemmal domain stopped dividing (van Blerkom and Davis, 2006). van Blerkom et al.(2006) demonstrated that Δφ _m was related to intercellular contact and communication instead of morphological difference and cell specificity. In addition, studies have shown that maternal age had an effect on oocyte mitochondrial activity. The age-related decline of Δφ _m was observed in oocytes and preimplantation embryos (Wilding et al., 2001). Cyclosporine A (CsA) is an inhibitor of mPT pore opening, and it can restore Δφ _m decease caused in aged cells (Rottenberg and Shaolong, 1997), which suggests that the opening of mPT pore may have an effect on Δφ _m. Fan et al.(1998) showed that the expression of adenine nucleotide translocase (ANT), an important component in mPT pore, decreased in aged cells, which may be a reason for Δφ _m decease. Δφ _m decline is a signal of mitochondrial membrane permeability transition, and continuous membrane permeability transition represents the abolished proton gradient across the inner mitochondrial membrane, which blocks ATP synthesis. Reduced ATP level may limit the energy supply for the oocytes or preimplantation embryos (Thouas et al., 2004) and high ATP content may support high Δφ _m effectively (van Blerkom et al., 2006). However, embryos are contained in the hypoxic uterine lumen, which is not beneficial for ATP synthesis (Lonergan et al., 2007). So high ATP level can also limit embryo development.

Mitochondria and apoptosis in oocytes

Previous IVM studies showed that apoptotic rate of immature oocytes from old patients is significantly higher than that from young ones, which suggested that age-related apoptosis in poor-quality oocyte might be one of the reasons for the female infertility (Wu et al., 2000). Excessive ROS produced in aged cells has been reported to provoke the opening of mPT pores and subsequently the release of cytochrome-c, which activates caspase cascade pathway that can induce cellular degradation (Thouas et al., 2004).

The mPT pore is a nonselective and high-conductance channel. It is responsible for the mitochondrial membrane permeability transition, which is a critical control point in apoptosis. Induction of the mPT pore can lead to mitochondrial swelling and cell death. It is believed that mPT pore opening is a consequence of apoptosis, not the reason for it (Kinnally and Antonsson, 2007). Excessive ROS generation, Ca²⁺ overload, or the expression of Bcl-2 family proteins can lead to the formation of the mPT pore, followed the release of cytochrome-c and the activation of caspase cascade.

Several proteins have been thought to form the mPT pore, which include ANT, voltage-dependent anion channel (VDAC), and cyclophilin-D (Crompton, 1999). ANT is located in the inner mitochondrial membrane and can exchange adenosine diphosphate (ADP) for ATP between the mitochondrial matrix and the cytosol. Active ANT is a dimer that can alternate between two conformations: ANT-c (cytosol) and ANT-m (matrix) (Vyssokikh and Brdiczka, 2003). When induced by atracyloside or pyridoxal phosphate, the ADP/ATP-binding site of ANT would face the cytosol in a way that it can interact with VDAC in the outer membrane. In this state, the mPT pore is opened up. But when induced by bongkrekate, the ADP/ATP-binding site faces the matrix without interaction with VDAC, and the mPT pore is inhibited (Crompton, 1999).

VDAC is a highly conserved protein in the outer mitochondrial membrane and belongs to the porin family that can form aqueous channels, which allow for the passive diffusion of metabolites less than 5000 kDa (Vieira et al., 2000). VDAC is also a dimer, which can also change between VDAC-t (tensed) and VDAC-r (relaxed) (Vyssokikh and Brdiczka, 2003). The VDAC-t can interact with the ANT-c, while the VDAC-r is not bound to ANT, only existing as a monomer. On the surface of VDAC, there are some specific binding sites for proteins including hexokinase, glycerol kinase, and Bax. Studies have shown that hexokinase and Bax can compete for the same binding site (Pastorino et al., 2002). When this binding site is occupied by hexokinase, the permeability transition caused by ANT-c will be prevented potently (Beutner et al., 1998). In the mitochondrion, there is an octamer of creatine kinase that can inhibit the interaction between VDAC and ANT, for it binds to VDAC from the inner surface of the outer membrane, and the octamer reduces the affinity of VDAC for both Bcl-2 and Bax (Vyssokikh and Brdiczka, 2003). So the octamer has been suggested to be the mitochondrial target of the pro-apoptotic proteins, Bax and Bcl-2, which would allow VDAC to form a cytochrome-c release channel.

Cyp D belongs to the cyclophilin family. It possesses peptidyl-prolyl cis/trans isomerases (PPIase) activity, which is crucial for protein folding (Galat and Metcalfe, 1995). Cyp D is localized in the mitochondrial matrix normally. When activated, its PPIase activation may induce a conformational change of ANT and then result in the opening of the mPT pore. PPIase is inhibited by CsA, which is another member of the cyclophilin family (Tsujimoto and Shimizu, 2007). Ca²⁺ and adenine nucleotides are the regulators of the affinity between Cyp D and CsA. Ca²⁺ can reduce the binding affinity while the adenine nucleotides promote it (Crompton, 1999). In addition to the CsA-sensitive and Ca²⁺-dependent mPT pore, there exists a CsA-insensitive mPT pore (He and Lemasters, 2002), although its mechanism is totally unknown.

Mitochondria are the major sources of ROS, which can do some oxidative damage to themselves (Takahashi et al., 2004). It has been recently suggested that mPT pore opening is the result of coactions of Ca²⁺ overload and ROS generation (Mather and Hagai Rottenberg, 2000), and Ca²⁺ overload can also enhance ROS generation in mitochondria (Grijalba et al., 1999). However, the mPT pore opening does not destine cellular apoptosis. In a low conductance state, the opening is reversible, and the excitable state may contribute to the generation of Ca²⁺ waves. The mPT pore becomes irreversibly open under a high conductance state with the accumulated Ca²⁺ in the mitochondria exceeding a certain threshold-concentration, and these irreversible opening results in the collapse of transmembrane potential and the release of cytochrome-c, which initiates apoptosis (Ichas et al., 1997).

It has been suggested that mPT pore opening is a protective mechanism against mitochondrial Ca²⁺ overload (Crompton, 1999). Buchholz et al.(2007) reviewed the influences of aging-caused functional changes of mitochondria and ER on Ca²⁺ regulation. These changes contribute to Ca²⁺ overload and apoptosis. However, the mechanism of Ca²⁺ overload is not well understood. Additionally, the Ca²⁺ threshold for mPT pore decrease is accompanied by an irreversible increase of oxidative stress (Ott et al., 2007).

Mitochondria and meiosis in oocytes

It is possible that embryos with normal appearance may contain genetic lesions. These abnormalities may affect the viability of postimplantation embryos or result in abnormal fetuses. Munne et al.(1995) indicated that aged female has a much higher proportion of embryos with normal appearance but abnormal genes, especially chromosomal aneuploidies, compared with younger ones. As we know, motor proteins and protein kinases are necessary for spindle assembly and chromosome alignment (Eichenlaub-Ritter, 1998). Chromosomal aneuploidies often occur during the first meiotic division (Robertson, 1998) and aberrant activity of motor proteins and kinetochores-related kinases may contribute to these chromosomal aneuploidies in aged female (Yin et al., 1998). Polo-like kinases (Plks) are key cytokinesis-related protein kinases in oocytes during meiotic maturation for spindle formation and chromosome separation (Pahlavan et al., 2000), and their activities are correlated with protein phosphorylation, which depends on the endogenous substrates and ATP as phosphate donor (Golsteyn et al., 1994).

Mitochondrial functional integrity is essential for ATP provision. Yin et al.(1998) cultured mouse oocytes with diazepam (DZ), a drug that binds with the benzodiazepine receptors on the outer mitochondrial membrane. This binding may limit ATP synthesis, affect calcium regulation, and disrupt the distribution of mitochondria, which can lead to the retardation of mouse oocyte maturation and chromosome aneuploidies. The similar conditions between the maternal age-related aneuploidy and drug-exposure aneuploidy suggest that mitochondrial dysfunction affects spindle formation and the segregation of chromosomes during the maturation of aged oocyte (Eichenlaub-Ritter, 1998). Oxidative stress can also influence the oocyte spindle assembly (Tarín, 1996), because oxidative stress may trigger mPT pore opening and decrease ATP content in oocytes (Zhang et al., 2006). It is therefore not surprising that injection of cytoplasm may improve embryo quality and support the embryo development (Cohen et al., 1998).

IMPROVEMENT OF OOCYTE QUALITY BY MANIPULATION OF MITOCHONDRIA

Many treatments are available for improving embryo quality and increasing success rate of IVF targeting at mitochondria, including mitochondrial transfer and various drugs. Accumulation of mtDNA mutation with aging mainly contributes to mitochondrial dysfunction, and the growing number of abnormal mitochondria impairs oocyte fertilization and subsequent embryo development. Pinkert et al.(1997) primarily microinjected mitochondria isolated from Mus spretus liver samples into fertilized ovary, and made mitochondrial genome applied for experimental and therapeutic purposes. Yi et al.(2007) have provided direct scientific evidence on the improvement of embryo development after mitochondrial transfer. Microinjecting a certain level of mitochondria into oocytes may compensate for the insufficiency of active mitochondria and raise the morula formation rate. However, allo-mitochondrial transfer may lead to potential risks such as mitochondrial heteroplasmy or mitochondrial genetic diseases (Malter and Cohen, 2002). Self-mitochondrial transfer can prevent these risks effectively, and the transfer of self-granulosa cell mitochondria was also proved to enhance the pregnancy rate by improving embryo quality during preimplantation development (Kong et al., 2004). Further research on mitochondria has also concentrated on the design and development of drugs. The rationale of targeting drugs to mitochondria is based on possible mitochondrial impairments during cellular metabolism, such as oxidative damage, apoptotic inducement, or uncoupling agent (Armstrong, 2007), which can help to overcome or reduce disadvantageous factors to mitochondria.