Mitochondrion 10 (2010) 419–428

Contents lists available at ScienceDirect

Mitochondrion

journal homepage: www.elsevier .com/locate /mito

Review

Mitochondria, spermatogenesis and male infertility

Singh Rajender a,*, Pandey Rahul b, Abbas Ali Mahdi c

a Division of Endocrinology, Central Drug Research Institute, Lucknow, UP, Indiab Translational Health Science Unit, Research Cell, Chhatrapati Sahuji Maharaj Medical University, Lucknow, UP, Indiac Department of Biochemistry, Chhatrapati Sahuji Maharaj Medical University, Lucknow, UP, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 15 February 2010Received in revised form 24 May 2010Accepted 28 May 2010Available online 1 June 2010

Keywords:MitochondriaMale infertilityMitochondrial mutationSpermatogenesis

1567-7249/$ - see front matter � 2010 Elsevier B.V. adoi:10.1016/j.mito.2010.05.015

* Corresponding author. Address: Endocrinology DiInstitute, Lucknow 226 001, India. Tel.: +91 522 2613

E-mail address: rajender_singh@cdri.res.in (S. Raje

Mitochondria and its role in male reproduction has remained an enigma since long. Similarly, etiologyof male infertility in a large percentage of individuals, mainly primary infertility, has evaded concreteconclusions. Oxidative metabolism, energy production and free radical generation are the principal bio-logical reactions occurring inside mitochondria. In addition to the above, mitochondria participates inan important process of apoptosis. Mitochondrial causes of infertility have triggered interest because ofits presence in the tail of sperm and immense need of energy for sperm motility. Several studies onmitochondria have strongly suggested its role in fertility, some of which support mitochondrial rolepresenting numerous hypotheses, whereas others deny its very existence as a causative factor. Wehave, in the present review of existing literature, covered the role of mitochondria right from spermato-genesis to male infertility.

� 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction

Approximately half of the infertility cases are related to men (St.John et al., 2000). There are several potential causes of male infer-tility but a large number of cases are idiopathic, reflecting poorunderstanding of the basic process of spermatogenesis and theassociated regulatory mechanisms. Of late, attention has shiftedto genetic causes and as the arena of modern technological toolsincreased it became increasingly clear that genes play a significantrole in the etiology of male infertility (Shoffner et al., 1989; Ozawa,1995; St. John et al., 2000). The genetic causes of male infertilityare varied from chromosomal abnormalities to single genedisorders (Shoffner and Wallace, 1992; Michikawa et al., 1999;Pesole et al., 1999; Clayton, 2000). Several studies at genome levelhave suggested that disturbances in organization of the geneticmaterial in sperm are inversely related with its fertility potential(Folgero et al., 1993; Holyoake et al., 1999; St. John et al., 2000;Holyoake et al., 2001). Similarly, many studies have revealed thatDNA strand breaks are relatively higher in infertile patients (withboth abnormal and normal semen parameters) in comparison tofertile subjects (Folgero et al., 1993; Kao et al., 1995, 1998;Ankel-Simons and Cummins, 1996). The utility of sperm DNA dam-age as a diagnostic and prognostic marker in human fertility clinicshas been extensively studied, suggesting promising correlationwith infertility (Evenson and Wixon, 2006).

nd Mitochondria Research Society

vision, Central Drug Research894.nder).

Reduced sperm motility is one of the major causes of male infer-tility just next to reduced count (Gavella and Lipovac, 1992;Cummins et al., 1994; Kao et al., 1995, 1998; Ankel-Simons andCummins, 1996; Lestienne et al., 1997; Ruiz-Pesini et al., 2000; St.John et al., 2000; Spiropoulos et al., 2002a). Sperm travel throughmucus filled cervix to reach up to the site of fertilization by active fla-gellar movements, the energy for which is provided by the mito-chondria of the sperm (Folgero et al., 1993; St. John et al., 2000).Though sperm can survive by glycolytic energy, it is largely depen-dent on oxidative metabolism for its normal physiology and func-tion (Spiropoulos et al., 2002a). Roughly, 72–80 mitochondria arepresent in the mid-piece of mature mammalian spermatozoon(Ankel-Simons and Cummins, 1996; Cummins, 1998; St. Johnet al., 2000). The fact that a miniature and highly differentiated cellsuch as spermatozoa carries mitochondria which ultimately doesnot even become part of the zygote, indicates its importance in fer-tility. Sperm loses most of its cytoplasm during differentiation,which further indicates strong biological reason for retention ofmitochondria (Cummins, 1998; Spiropoulos et al., 2002a).

Looking at its importance in fertility, it has been proposed thatgenetic defects in mitochondria may compromise sperm physiol-ogy in general and motility in particular (Folgero et al., 1993;Spiropoulos et al., 2002a). Although mitochondrial DNA has beenextensively studied in the last few decades, there have been onlyfew studies investigating the role of mitochondrial DNA muta-tions/polymorphisms in male infertility (May-Panloup et al.,2003; Thangaraj et al., 2003). The role of mitochondria is stilldebatable since there is no consensus in the results of these stud-ies. We have taken up a review of literature to understand the role

. All rights reserved.

420 S. Rajender et al. / Mitochondrion 10 (2010) 419–428

of mitochondria in spermatogenesis, male fertility and associationbetween the genetic defects and male infertility/poor sperm motil-ity. We searched ‘Pubmed’, ‘Sciencedirect’ and ‘Google Scholar’using keywords; mitochondria, mitochondrial mutations, mito-chondria and male fertility, mitochondria and male infertility,and screened about 70 articles.

2. Basics of mitochondria

Mitochondrion is a complex sub-cellular organ present in thecytoplasm of all animal and plant cells. Mitochondria are doublemembrane structure consisting of outer and inner membranes sep-arated by an inter-membrane space. The inner membrane formsnumerous folds inside mitochondrial matrix known as ‘crystae’and is unusually high in protein content. It is the principle site of oxi-dative phosphorylation and its structure reflects its functional orga-nization. Electrons produced from hydrogen atoms are successivelytransferred from one cytochrome to the next, oxidising the formerduring the process. The proton gradient thus generated is ultimatelyutilised to produce ATP with ultimate union of proton, electron andmolecular oxygen resulting in H2O formation as a by-product.

Mitochondrial matrix contains its own genetic system. Humanmitochondrial DNA is a single double stranded molecule of16569bp (Shoffner and Wallace, 1992; Clayton, 2000; St. Johnet al., 2000). It encodes 2 rRNAs, 22 tRNAs and 13 of 67 polypep-tides forming mitochondrial complex. Mitochondria encoded poly-peptides consist of seven subunits of NADH CoQ reductase(complex I), cytochrome b (complex III), subunits I, II, and III ofcytochrome C oxidase (complex IV), and subunits 6 and 8 of theH+ ATPase (Anderson et al., 1981, 1982). Each of the complexesof the electron transport chain (ETC), except complex II, has pro-teins encoded by the mitochondrial genome, whereas the remain-der of the subunits of the ETC are encoded by the nucleus(Anderson et al., 1981; Cummins, 1994; Cummins, 1998; St. Johnet al., 2000). Unlike nuclear DNA, the mitochondrial genes havenone or only a few non-coding bases between them and thus lackintrons. Nevertheless, there is 1 non-coding sequence, the displace-ment loop (D loop), which is vital to mtDNA replication (Andersonet al., 1981; Clayton, 2000).

3. Mitochondrial disorders in general

The complex system of oxidation and reduction reactions in themitochondria makes it highly prone to oxidative stress and so isthe case with mitochondrial DNA. Mitochondrial DNA is 10–100times more prone to oxidative insult due to, first, its location inproximity to the ETC and second, lack of DNA repair mechanism.Although most of the knowledge about the structure and basicmechanisms of mtDNA replication and transcription was gener-ated about two decades ago (Clayton, 1982; Attardi, 1985), dissec-tion of the machinery involved in these processes, their regulationand coordination with nuclear gene expression has progressed rel-atively slow and much has still to be learned. Mitochondrial disor-ders follow both Mendelian and non-Mendelian patterns ofinheritance (Clayton, 1982; Attardi, 1985; Zeviani et al., 1989;Spiropoulos et al., 2002b). Mendelian inheritance is observed dueto complex interplay between nuclear and mitochondrial genome(Zeviani et al., 1989; Suomalainen et al., 1995). Nuclear changescan affect mitochondrial function and segregate in Mendelian fash-ion. Disorders due to changes in the mitochondrial genome segre-gate in non-Mendelian fashion because of heteroplasmy andvariation in mitochondrial copy number. Maternal inheritance isa characteristic of the mitochondrial disorders due to changes inthe mitochondrial genome while paternal inheritance may be evi-dent in mitochondrial disorders due to nuclear changes. Since sev-eral proteins and enzymes required for normal mitochondrial

function, such as DNA polymerase gamma are encoded by the nu-cleus, errors in these establish paternal inheritance (Holyoakeet al., 2001; Williams, 2002). Oocyte carries higher number ofmitochondria in comparison to sperm and since only oocytecontributes it to the next generation, maternal mitochondriadictate the terms (Shoffner and Wallace, 1992; Ankel-Simons andCummins, 1996; St. John et al., 2000). To understand the inheri-tance patterns, it would be interesting to elucidate if, duringthe course of evolution, there was selective distribution of mito-chondrial genes between the nucleus and mitochondria. To elabo-rate further on this issue is beyond the scope of this review.

4. Mitochondria and spermatogenesis

Spermatogenesis in human is expressed quantitatively in termsof daily sperm production which is the number of spermatozoaproduced per day. Low efficiency of spermatogenesis in human isdue to; first, the long duration of spermatogenesis which is approx-imately 74 days (Heller and Clermont, 1964) and second, a lowernumber of germ cells per unit volume of parenchyma (density)in comparison to non-human mammalian species (Johnson,1995). Several studies have indicated important role of mitochon-dria in spermatogenesis and fertility; however, most of them awaitconclusive evidence stating the exact role mitochondria play. Wenarrate the functions mitochondria play or the changes in mito-chondria that ensue during spermatogenesis (Fig. 1).

4.1. Mitochondrial function may be related to the meiotic process

Mitochondrial DNA mutations have been reported in mitochon-drial respiration defects giving rise to meiotic arrest and abnormal-ities of sperm morphology but to date no conclusive study hasbeen done to prove the same in human (Ankel-Simons andCummins, 1996; Cummins, 1998; St. John et al., 2000; Spiropouloset al., 2002a). If it is at all related, the threshold for phenotypicexpression of such defects relevant to male infertility remains un-known. The situation becomes perplexing since the amount of mu-tated and pathogenic heteroplasmic DNA is generally considered tobe low in actively dividing cells and high in non-dividing or ar-rested cells (Clayton, 1982; Shoffner and Wallace, 1992; Wiesneret al., 1992; Ankel-Simons and Cummins, 1996; Lightowlers et al.,1997; Cummins, 1998; Michikawa et al., 1999; Spiropoulos et al.,2002a, 2002b). It is, therefore, possible that factors other thanmitotic activity may affect function of the spermatozoa(Spiropoulos et al., 2002a, 2002b).

4.2. Mitochondrial depletion during spermatogenesis

Mitochondrial DNA copy number in human sperm is severaltimes less than oocyte. Oxidative phosphorylation has been sug-gested to be an important determinant of sperm motility; however,the functional significance of the low copy number in sperm re-mains unclear. A study on mouse reported that male could tolerateat least a threefold reduction in mtDNA copy number in theirsperm without impaired fertility (Wai et al., 2010). It also showedthat there is a critical post-implantation developmental thresholdof 40–50,000 copies of mtDNA in the mature oocyte. These obser-vations suggest that high mtDNA copy number in the mature oo-cyte is a genetic design for distribution of mitochondria to thecells of early post-implantation embryo before mitochondrial bio-genesis and mtDNA replication resumes, whereas down-regulationof mtDNA copy number is important for normal sperm function(Wai et al., 2010).

The down-regulation of key regulators of mitochondrial DNAduring spermatogenesis results in a decrease in mtDNA copy num-ber in sperm (Larsson et al., 1997). It has been hypothesised that a

S. Rajender et al. / Mitochondrion 10 (2010) 419–428 421

critical mtDNA threshold is essential for sperm function (Wai et al.,2010). It is not clear how fewer mitochondrial genomes would beadvantageous to developing spermatozoa, but it appears thatdown-regulation of both POLG and TFAM has been selected duringevolution of male germ-line, causing a marked reduction in mtDNAcopy number during spermatogenesis (John et al., 2010). Oligozoo-spermic and asthenozoospermic men have sperm containing sig-nificantly elevated levels of mtDNA (Tremellen, 2008), promptingthe hypothesis that reduced copy number in sperm decreases thelikelihood of ROS-mediated damage to mtDNA, thereby effectivelyavoiding potentially deleterious effects on sperm function. How-ever, till date no evidence exists to support the ROS hypothesis.

Majority of inherited human mtDNA diseases occur at a fre-quency of 1 in 5000 (Elliott et al., 2008) and are caused by muta-tions which are not effectively eliminated by c-DNA polymerase.The elimination mechanism of mtDNA mutations has yet to beidentified. It is hypothesised that the selective elimination of se-verely deleterious mutations is most efficient at low level of mito-chondrial content since clear differences in organellar fitnesswould manifest more quickly (Wai et al., 2010). If selection werebased on an indicator of mitochondrial function (e.g. membranepotential), which could be compromised by severe mtDNA muta-tions, increase in the number of cells during spermatogenesiswould provide a continuous opportunity to ensure that the devel-oping sperm were populated by the fittest mitochondria.

4.3. Molecular and physical changes in mitochondria duringspermatogenesis

Mitochondria comprise approximately 15–22% of the total cel-lular volume delivering 90% of the energy required. Mitochondriacan change their morphology, location in the cell and fuse to largerunits or separate (Bereiter-Hahn and Voth, 1994) depending on thecell type and the respective functional status. However, the chainof morphological change events in mitochondria of developingspermatogonial has no explained functional relevance as yet.Ultrastructural analysis revealed round-shaped, ellipsoid, cylindri-cal and filament like structures of mitochondria (Meinhardt et al.,1999).

Mitochondria are potentially immortal as they exist in daughtercells which complete their divided pool of proteins by synthesizingand by the import of nuclear encoded polypeptides from thecytoplasm. Import and maturation of these polypeptides is facili-

Fig. 1. Established/proposed roles of mitochon

tated by a variety of evolutionary conserved chaperones whichshow high similarity to the respective proteins. In basal compart-ment of the seminiferous tubules, spermatogonia have free accessto all nutrients in the inter-tubular space originating from the cir-culation and the lymph vessels. The germ cells that pass into lumi-nal compartment rely on lactate and pyruvate breakdown suppliedby the Sertoli cells. This reflects an indirect yet essential influenceon energy metabolism during spermatogenesis (Grootegoed et al.,1984) which is accompanied by obvious morphological changesand a sequential expression of different mitochondrial proteinssuch as hsp60, Lon protease, sulphydryl oxidase and cytochrome ct.

Heat shock proteins are the result of a large variety of stimulicharacterized by a cellular response in the form of an increasedsynthesis of a small group of specific proteins. They are one of mostconserved proteins and are found at various sites in the cell(Meinhardt et al., 1999). Mitochondrial hsp60 has a function in cor-rect folding of the native structure of imported mitochondrial pro-teins followed by a stepwise process of ATP-dependent release(Meinhardt et al., 1999). Studies have shown that mt-hsp60expression correlates well with mitotic activity of spermatogonia(Meinhardt et al., 1999). Its expression also correlates well withtype and activity of mitochondria (Meinhardt et al., 1999). It in-creases with cristae type of mitochondria and decreases with con-densed and intermediate type of mitochondria (Werner et al.,1997). One study on human testicular biopsies showed that mt-hsp60 was localised to spermatogonia, early primary spermato-cytes and Sertoli cells of normal unaffected tubules (Meinhardtet al., 1999). In general, the number of mt-hsp60 positive sperma-togonia decreases with the loss of spermatogenic function, as withmaturation arrest of spermatogenesis at the level of primary sper-matocytes (Werner et al., 1997). However, it will be too early toconclude that mt-hsp60 indicates perfect spermatogenic function.

Normal matrix proteins are degraded by ATP-dependent prote-ases after reaching their average life span of 3.5–5 days (Sonezakiet al., 1995; Meinhardt et al., 1999). Lon protease is a protein in-volved in catabolic activity (Sonezaki et al., 1995). Recently, itwas shown that Lon protease is a new and specific marker for anintermediate type of mitochondria which are just starting to con-dense into the form characteristic of mid-meiotic cells (Meinhardtet al., 1999). However, why Lon is expressed only during such ashort time frame of mitochondrial differentiation is still to be ex-plored. Sulphydryl oxidase, involved in oxidation of sulphydrylcompounds as cysteine, glutathione, is localised in a stage specific

dria in spermatogenesis and male fertility.

422 S. Rajender et al. / Mitochondrion 10 (2010) 419–428

manner during spermatogenesis (Bergmann et al., 1990); however,its distribution is less stage specific. Very little is known about otherfactors that influence mitochondrial differentiation during sper-matogenesis. Certain proteinaceous substances termed as paracrinemitochondrial maturation factors, for example activin, are releasedby the Sertoli cells (Spiteri-Grech and Nieschlag, 1993). These par-acrine factors are thought to be involved in mitochondrial differen-tiation; however, their exact role is still not worked out.

4.4. Gene expression in mitochondria during spermatogenesis

There is no mechanism known to co-ordinate mtDNA replica-tion with phases of cell cycle. Therefore, a particular mtDNA mol-ecule may be replicated several times or not at all during the cellcycle (Rantanen and Larsson, 2000). No qualitative or quantitativechanges occur in mitochondrial RNA transcripts (mtRNA) duringspermatogenesis (Alcivar et al., 1989). This is surprising due tosimultaneous down-regulation of mtDNA copy number (Hechtand Liem, 1984). The constant values of mtRNA could be due to in-creased stability of mitochondrial RNA transcripts in male germcells. Available data show that translation (into respiratory chainpeptides) continues to be active in the mitochondria of spermato-zoa (Hecht and Liem, 1984). Many mammalian genes expressingtestis- or sperm-specific isoforms have been identified (Ericksonet al., 1990; Siva et al., 2010). Some of the examples are: mitochon-drial transcription factor A (Tfam) (Larsson et al., 1996, 1997), heatshock protein 60 (HSP60) and HSP70 (Erickson et al., 1990;Meinhardt et al., 1995), oc-tubulin, lactate dehydrogenase (LDH-X),phosphoglycerate kinase 2 (pgk-2) (Erickson et al., 1990), prot-amine 1 (Prml) and Prml (Kleene et al., 1984), transition proteins1 and 2 (Erickson et al., 1990), diazepam binding inhibitor (Kolmeret al., 1997), and the Hox-1.4 homeobox gene (Erickson et al.,1990). Some of these are directly involved in the spermatozoonformation, for examples a-tubulin for cytoskeletal reorganization,and protamines and transition proteins for packaging the chromo-somes (Kolmer et al., 1997). These genes are transcribed post-mei-otically; however their expression is regulated at the translationallevel (Braun et al., 1989). The functions of other genes expressingtestis specific isoforms, including some oncogenes (c-int-1, c-abl,c-pim-1 and c-mos) and homeobox genes, are less vague (Ericksonet al., 1990).

Mitochondrial transcription factor A (mtTFA or Tfam) is a keyregulator of mtDNA copy number in mammals. This gene encodesa transcriptional activator, Tfam which is imported to mitochon-dria for regulating transcription of mtDNA. Tfam also has an impor-tant role in formation of the primers necessary for initiation ofmtDNA replication (Clayton, 2000). Expression of germ cell-spe-cific Tfam transcript isoforms occurs during spermatogenesis inmice and humans (Larsson et al., 1997). These Tfam transcript iso-forms have a structure which could prevent protein translation andtheir expression correlates with down-regulation of the mitochon-drial Tfam protein values. It is hypothesised that the down-regula-tion of mitochondrial Tfam protein levels down-regulates mtDNAcopy number during mammalian spermatogenesis (Larsson et al.,1997). Importance of Tfam can be realised from fact that homozy-gous Tfam knockout mice die between embryonic day 8.5 and 10.5(Larsson et al., 1996). The down-regulation of mitochondrial Tfamprotein concentrations in round and elongating spermatids coin-cides with the reduction of mtDNA copy number. Therefore, Tfamcould also be involved in regulating the amount of mtDNA duringspermatogenesis (Larsson et al., 1996, 1997).

4.5. Mitochondria in sperm quality assurance

Degeneration of germ cells affects the quantitative aspects ofspermatogenesis (Huckins and Oakberg, 1978; Johnson et al.,

1983; Johnson, 1985). However, the mechanisms of degenerationwith etiology and approaches for prevention have remained un-clear. Germ cell degeneration is associated with three critical stepsin spermatogenesis, namely spermatogonial mitosis, meiosis, and/or spermiogenesis. A significant loss (36–45%) by degeneration(Barr et al., 1971; Johnson et al., 1983) occurs during meiotic divi-sion. The reasons for germ cells degeneration are unclear. It is pro-posed that the degeneration of germ cells may be a mechanism foreliminating cells with genetic abnormalities (Oakberg, 1956;Clermont, 1962). However, non-random loss of spermatogoniaand relative constancy in the magnitude of degeneration is incon-sistent with simple selection for elimination of chromosomalabnormalities (Huckins and Oakberg, 1978). It has been proposedthat mitochondria play role in this degeneration process, therebyassuring that good quality meiotic products enter the process ofspermatogenesis to yield good quality mature sperm.

4.6. Mitochondrial proteins in sperm maturation/capacitation

Deposition of spermatozoa in the female reproductive tractstarts a multi-dimensional process of capacitation before fertiliza-tion. Sperm capacitation consists of events including hyperactiva-tion, acrosome reaction and tyrosine phosphorylation (Shivajiet al., 2009). Protein kinase A (PKA) activity has been proposed tobe essential for sperm tyrosine phosphorylation, hyperactivation,and acrosome reaction (Lefièvre et al., 2002). Similarly other ki-nases implicated in capacitation are protein kinase C (PKC), extra-cellular signal regulated kinase (ERK1), mitogen activated proteinkinase (MAPK), phosphoinositide 3-kinase (PI3 K) and protein ki-nase B (PKB/Akt) (Naor and Breitbart, 1997; Luconi et al., 2001). Re-cent studies have focussed on the role of tyrosine phosphorylationin sperm capacitation (Shivaji et al., 2009). Functional analysis ofsome of the tyrosine phosphoproteins may predict similar func-tions as observed for the proteins in somatic cells. However, situa-tion is complex for those proteins which are localised in themitochondria of somatic cells and have a known function, but inspermatozoa they are non-canonical in their localization. Examplesof such proteins include dihydrolipoamide dehydrogenase (DLD),pyruvate dehydrogenase A2 (PDHA2) and glycerol-3-phosphatedehydrogenase 2 (GPD2) (Mitra and Shivaji, 2005; Kumar et al.,2006; Kota et al., 2009). This leads to a new problem of determin-ing the functional relevance of such proteins. Another importantaspect is ascertainment of identical localisation of these mitochon-drial proteins between human and animal spermatozoa before cor-roboration between animal and human findings is established.

Tyrosine phosphorylation of numerous mitochondrial proteinsis known to occur in sperm (Shivaji et al., 2009). Paradoxically,all mitochondrial proteins are not localised to mitochondria, there-fore generating an array of compounding factors that may affectidentification of function of such proteins. The enzymes of theETC such as phospholipid hydroperoxide glutathione peroxidise(PHGPx), voltage-dependent anion channel (VDAC) are localisedto mitochondrion (i.e. the acrosome, head and/or principal piece)while enzymes like dihydrolipoamide dehydrogenase (DLD),PDHA2 {pyruvate dehydrogenase (lipoamide) alpha 2} and Glyc-erol-3-phosphate dehydrogenase 2 (GPD2) are extra-mitochon-drial in localization (Shivaji et al., 2009). Very few proteins areomnipresent in the sperm (Mitra and Shivaji, 2005). The mitochon-drial ETC in mammalian spermatozoa has been identified to under-go capacitation dependent tyrosine phosphorylation (Ficarro et al.,2003; Arcelay et al., 2008; Kota et al., 2009).

PHGPx, a major capsular protein embedding the helix of mito-chondria, has been implicated in sperm maturation and mitochon-drial function (Mitra and Shivaji, 2005). However, exact event oftyrosine phosphorylation of PHGPx in human sperm capacitationhas not been described. Similarly, there are no human findings

S. Rajender et al. / Mitochondrion 10 (2010) 419–428 423

for VDAC undergoing tyrosine phosphorylation during capacitationin spermatozoa. The down-regulation of DLD activity, a post-pyru-vate metabolic enzyme blocking acrosome reaction completely andhyperactivation partially in hamster spermatozoa appears to beinteresting finding (Mitra and Shivaji, 2004). The findings needstrength from human studies before final conclusions could bedrawn. Another issue is demonstration of DLD localization in mam-malian spermatozoa, where it is at extra-mitochondrial sites suchas acrosome and principal piece of the sperm flagellum (Shivajiet al., 2009). This invites mechanism of action affected by a numberof extra-mitochondrial factors. Similar are the findings withPDHA2, a constituent of pyruvate dehydrogenase A (PDHA). Giventhe localization of PDHA and the evidence that its activity posi-tively correlates with hyperactivation, it appears that it exhibitscapacitation-associated protein tyrosine phosphorylation. Studieshave also implicated ROS, cAMP and calcium with sperm capacita-tion, namely hyperactivation and acrosome reaction, possiblyinvolving PDHA somewhere downstream the sequence (Kumaret al., 2008). Correlation studies between GPD2 enzymatic activitywith hyperactivation and acrosome reaction has established itspossible involvement in capacitation (Shivaji et al., 2009).

Akinase anchoring protein (AKAP), a major structural protein inthe fibrous sheath of spermatozoa has been identified as a capaci-tation dependant phosphorylated protein (Jha and Shivaji, 2002;Shivaji et al., 2007). Role of chaperones influencing sperm motility,capacitation and fertilization has been extensively studied(Asquith et al., 2005). Conformational changes resulting from tyro-sine phosphorylation of sperm-surface chaperones is required forsperm-zona pellucida interaction (Asquith et al., 2005). Tyrosinephosphorylation of GSTs during capacitation has been reported insome recent studies (Ficarro et al., 2003; Arcelay et al., 2008; Kotaet al., 2009), hypothesizing their role in oxidative homeostasis cru-cial for sperm capacitation.

Similar to the changes at gene expression level, changes at pro-tein level are important to understand the complex process ofspermatogenesis before we seek role of these genes/proteins inmale infertility. Epigenetic changes also cannot be ruled out asthey are order of the day. As pointed out recently by Shivajiet al., (2009), functional identification and relevance of the proteinsin sperm capacitation is a necessity for better understanding therelationship between sperm protein tyrosine phosphorylation,hyperactivation and acrosome reaction and lastly in elucidatingmolecular mechanisms of male infertility.

5. Free radicals, mitochondria and male fertility

In mammalian germ cells, ROS production has been shown to bean essential physiological event for maturation, capacitation andacrosomal reaction of spermatozoa, binding with zona pellucidaand oocyte fusion (de Lamirande and Gagnon, 1993). Excessivegeneration of ROS has been found to be associated with idiopathicmale infertility (Alvarez and Storey, 1995; Said et al., 2005), spermapoptosis (Gandini et al., 2000), impaired pre-implantation anddevelopment of the embryo (Aitken et al., 1989), and increased rateof early pregnancy loss (Ruiz-Pesini et al., 1998). Two ROS generat-ing systems have been proposed in sperm: nicotinamide-adeninedinucleotide phosphate oxidase, located in the plasma membrane(Cummins et al., 1994), and nicotinamide-adenine dinucleotideoxidase-dependent oxidoreductase (diaphorase), located in themid-piece of spermatozoa and integrated in the mitochondrialrespiratory system (Said et al., 2005).

Potential targets of ROS include all cellular components includ-ing DNA and unsaturated fatty acids. The sperm plasma membranehas very high concentrations of polyunsaturated fatty acids(approximately 22:6) (Alvarez and Storey, 1995), has no mem-brane repairing capacity, making it a soft target for ROS-mediated

damage. High physiological activity in sperm results in ROS gener-ation, which may impair sperm function through lipid peroxide in-duced changes in membrane fluidity and integrity, loss of themembrane-bound adenosine triphosphatase and adenosine tri-phosphate depletion (Aitken et al., 1989). At this level, it becomesdifficult to answer which mutations and injuries are tolerated orremoved by apoptosis, i.e., whether nuclear induced or inherentin mitochondria and how they are related (Moraes et al., 1991;Larsson et al., 1996). Nevertheless, it is clear that mitochondriaare liable to pay the penalty for harbouring the principle ROS gen-erating machinery.

6. Mitochondrial SNPs are often functionally inert

Although several SNPs have been reported in mitochondrialgenome (Shoffner and Wallace, 1992; Cummins, 1998; Kaoet al., 1998; Pesole et al., 1999; Spiropoulos et al., 2002a) theyare not functionally debilitating. This can be explained by the factthat most of substitutions are seen at third nucleotide of the co-don and thus are neutral, and those causing amino acids changehave little effect on mitochondrial function (Cummins, 1998; St.John et al., 2000; Spiropoulos et al., 2002a). Mutated codonsmay affect translation machinery efficiency probably due toselection of tRNAs affecting the level of protein products, how-ever, functional studies on the subject are scarce. High copynumber of mitochondria dilutes the effect of mutations in so-matic cells, but cells like sperm contain limited number of mito-chondria and a small change in translation ability may havegreater impact. The lack of repair machinery in mitochondriamay result in accumulation of new mutations/polymorphisms,which may give rise to functionally relevant haplotypes. Forexample, association between mitochondrial DNA haplotype andrespiratory chain dysfunction has been shown in asthenozoosper-mic patients (Ruiz-Pesini et al., 2000).

7. Mitochondrial mutations/deletions in Infertile men: role andimplications

Infertility affects 10–15% of the population, of which, approxi-mately 40% is due to male etiology consisting primarily of lowsperm count (oligozoospermia) and/or abnormal sperm motility(asthenozoospermia) (Ankel-Simons and Cummins, 1996; St. Johnet al., 2000; Spiropoulos et al., 2002a; Baker et al., 2005). As dis-cussed in detail above, there are studies supporting the role ofmitochondria in spermatogenesis and male fertility (St. Johnet al., 2000; Spiropoulos et al., 2002a). St John et al. have aptlyhypothesised that presence of mtDNA defects in sertoli cells andother support cells may cause energy production loss and lead todefective spermatogenesis (St. John et al., 2000). In another study,the authors (Folgero et al., 1993) showed that addition to the med-ia of complex II substrates, pyruvate and succinate, which by-passed the complex I mutation, resulted in a threefold increasein motility of defective sperm. Significant role of mitochondria insperm motility makes asthenozoospermic (low sperm motility)individuals the right candidates for mutation screening. Therefore,abnormal mitochondrial function has been hypothesised to associ-ate with asthenozoospermia/oligoasthenozoospermia (Cumminset al., 1994). However, only few mutations/deletions have been re-ported in mitochondrial genes/genome in infertile individuals(Fig. 2).

Mitochondrion was first correlated with male infertility in theearly 1990s when researchers (Folgero et al., 1993) reported re-duced sperm motility in the individuals having structural defectsin mitochondria (Table 1). Soon after, 4977bp deletion in mito-chondrial genome was correlated with spermatogenic failure(Cummins et al., 1994). Lestienne et al., (1997) reported multiple

Fig. 2. SNPs/mutations reported in mitochondrial genome in infertile maleindividuals. Substitutions are presented on the inner circumference of the circleand deletions are presented on the outer circle. The numbers indicate nucleotidepositions of the deletion boundaries with deletion size.

Table 1List of the published studies on mitochondrial genome variations in association withmale infertility.

Mutation Description Reference

3243 A > G Structural defects in mitochondriaassociated with reduced sperm motilityreported.

Folgeroet al.(1993)

4977bp deletion Spermatogenic failure in a 36-year-oldman might be caused by prematuretesticular ageing as a result ofmitochondrial 4977bp deletion.

Cumminset al.(1994)

MultiplemitochondrialDNArearrangements

The study reportedoligoasthenospermia associated withmultiple mitochondrial DNArearrangements.

Lestienneet al.(1997)

7345 and 7599bpdeletion

Associated with poor sperm motility. Kao et al.(1998)

8821 T > C in ATPase6gene

Reported in severeoilgoasthenteratozoospermics but notin oligozoospermic or normospermic.This mutation changed the amino acidserine to proline at residue 99 of themitochondrial ATPase6 in a regionwhich is highly conserved in othervertebrates.

Holyoakeet al.(2001)

11994 C > T mutationin ND4 gene

Cause of low sperm motility in Indiansub-continent.

Selvi Raniet al.(2006)

11994 C > T mutationin ND4 gene

Not a cause of low sperm motility inPortugal.

Pereiraet al.(2008)

424 S. Rajender et al. / Mitochondrion 10 (2010) 419–428

mitochondrial DNA rearrangements in oligoasthenozoospermicindividuals. Following this, multiple mitochondrial DNA rearrange-ments were reported in association with decreased sperm motilityand male fertility (Kao et al., 1998). The progress thereafter hasbeen slow and only one mutation in association with male infertil-ity has been reported in the last decade (Selvi Rani et al., 2006;Pereira et al., 2008). C19944T mutation in the ND4 gene of mito-chondria was reported by to be a single major cause of asthenozoo-spermia in India (Selvi Rani et al., 2006); however, another studyon Portuguese men denied the existence of this mutation in the lat-ter population (Pereira et al., 2008). It is more apprehensive tothink about a single major mitochondrial cause of male infertilitydue to the reasons that mitochondria display heteroplasmy and athreshold level of mutation is required for pathogenesis. As men-tioned earlier, it is more practical to think about a cumulative ef-fect of several mutations such that associations are sought at thelevel of mitochondrial haplotypes.

Substitutions of single nucleotides have been reported only inthree genes, namely ND4, ATPase 6 and 16S rRNA (Fig. 2 and Ta-ble 1). Mitochondrial DNA mutations in the ATP generating geneshave been recently reviewed to infer that motility is a very impor-tant factor for fertility and changes in mitochondrial genes mayimpair sperm motility (Shamsi et al., 2008). Apart from single basesubstitutions, large deletions in mitochondrial genome have beenreported in infertile individuals. The deletion events have been rec-ognized in the latter half of the genome between ATPaes 6 and Cyt bgenes. No single base deletion or insertion has been reported ininfertility. However, one intriguing issue is very less frequency ofall the reported mutations. Most of these mutations/deletions havebeen reported in single individuals or at the most two members ofa family. It is possible that the mutations/deletions in mitochon-dria are selectively rejected by rejecting the cells with very highload of these mutations; however, there is no clue to this phenom-enon. If not with all the mutations, such a phenomenon may betrue for mutations which could have serious consequences. Thesubstitutions we genotype for haplotype analysis may be thosehaving less serious consequences and are tolerated, ultimatelybeing used as markers in tracing origin and evolution of popula-tions. Another reason for low frequency of mutations could be rel-atively lesser numbers of studies completing sequencing of thewhole mitochondrial genome in the infertile individuals.

To elucidate functional importance of mtDNA changes, exten-sive studies through the use of cybrid cells like rho-0 need to beundertaken. Such cells are deficient in mitochondrial DNA butcan still synthesize mitochondrial proteins due to the transcriptionof nuclear genes that code for proteins associated with the mito-chondrion and those of the ETC (King and Attardi, 1996, 1988).However, to date no such cell model could be developed for humanspermatozoon mitochondrial study. In one study, transfection ofsperm mitochondria into a rho-0 cell line was attempted withthe recovery of viable cybrids post-selection less than 1% (Manfrediet al., 1997). Probably complex disulphide bonding in the spermmid-piece decreased the ability to isolate a pure populationof sperm mitochondria and reduced uptake by the rho-0 cells(Sutovsky et al., 1996). It, therefore, appears prudent that a fullanalysis of the role of mtDNA deletions and their ability to affectmitochondrial function and sperm viability needs to be under-taken. Functional studies should preferably be taken on the muta-tions/substitutions found in high frequency rather than very lowand similar frequency in cases and controls. Apart from mitochon-drial self genes, nuclear genes encoding mitochondrial proteins areequally good candidates for analysis in infertile individuals. Forexample, positive correlation has been shown between spermquality defects, male infertility and polymorphic variants in theCAG microsatellite repeats in mitochondrial DNA polymerase cgene (Rovio et al., 2001).

8. High mutant load in mitochondria is required for phenotypicexpression

Most mammalian cells contain hundreds of mitochondria and,each mitochondrion in turn, contains several (2–10) copies of

S. Rajender et al. / Mitochondrion 10 (2010) 419–428 425

mtDNA (Wiesner et al., 1992; Shuster et al., 1988). The presence ofidentical mtDNA copies in an individual/tissue sample is named ashomoplasmy, but mutations arise, are maintained and/or amplifiedto different levels and co-exist with wild-type mtDNA, giving riseto the condition of heteroplasmy (Lightowlers et al., 1997; Cum-mins, 1998). At cell division, mitochondria and their genomes arerandomly distributed to the daughter cells and hence, startingfrom a given heteroplasmic situation, different levels of hetero-plasmy and even homoplasmy can arise in different cell lineages.As a consequence of this, it is common to find a ‘threshold effect’in mtDNA-linked human diseases i.e. the mutation has to reach acertain percentage, usually higher than 60–80%, in order to mani-fest its pathological effects (Lightowlers et al., 1997; Zeviani andAntozzi, 1997). Another facet of the point under considerationraises questions about differential expression of same polymor-phisms in mitochondria (Folgero et al., 1993).

Presence of heteroplasmy should be seen as protective phe-nomenon conferred by nature on mitochondria. As discussedabove, mitochondria are 10–100 times more prone to oxidativeinsult. Why Mother Nature has thought not to protect mitochon-drial DNA from damage is confounding although mitochondrialdiseases have severe expressions. A group of researchers hassuggested that spermatozoa do not eliminate any rearrangementor mutation in their mitochondria thereby implying that mito-chondria may play a role in the differentiation of these cells(Lestienne et al., 1997). The authors point to the same inferencequoting findings of double localization (nuclear or mitochon-drial) of the mitochondrial transcription factor activator (mtTFA)in the testis (Dairaghi et al., 1995), a factor which triggersmtDNA transcription at the light strand promoter and initiationof replication (Lestienne et al., 1997). Role of mitochondria indifferentiation is not well proved and needs further research.Similarly, exact role of two mitochondrial transcription factorsin complex process of spermatogenesis is not elucidated. Wehypothesize that this may partly be due to multiple copy num-ber of mitochondrial genome and high mutant load (80–90%) forphenotypic expression and stringent screening process for thosewhich are expressed.

Another important aspect is the plasticity in mitochondrialfunction in sperm. Point mutations may or may not be pathogenicdepending upon the plasticity of sperm mitochondria and spermmotility, in particular (Huang et al., 1994). Spermatozoa are pro-duced in the testes under anoxic conditions of partial pressure of4–16 mm Hg (Setchell, 1978). Further, spermatozoa have 22–80mitochondria per cell, a relatively low number considering theirneed for motility (Anderson et al., 1981; Ankel-Simons andCummins, 1996; Cummins, 1998; St. John et al., 2000; Williams,2002). Sperm mitochondria tend to be responsive to glucose con-centration of the female reproductive tract (Storey, 1980), and de-pend on anaerobic utilization of glucose relegating importance ofrequirement of glucose in mitochondrial ATP generation for motil-ity. All these features indicate good plasticity in mitochondrialsperm function. After all, why it should not be so in a process asimportant as reproduction for a species?

9. Mechanism of mutation in mitochondria

Mitochondrial DNA mutations/deletions occur at a frequency ofless than 1% and increase with advancing age (Michikawa et al.,1999). mtDNA is especially prone to large scale deletions in the re-gions characterized by presence of flanking repeats though it is notuniversal (Ozawa, 1995; St. John et al., 2000). Jumping of polymer-ase enzyme at the mutation site is also hypothesised as a cause ofpersistence of mutations in H strand for 50 end repeats (Ozawa,1995; St. John et al., 2000). Following theories have been proposedfor origin of mitochondrial mutations:

1. Slip mismatching.2. Illegitimate elongation of the D-loop strand during replication.3. Free radical induced deletions and mutations.4. Clonal expansion of existing deletions in the stem cells.

According to the slip mismatch theory, illegitimate alignmentoccurs between direct repeats of H and L strand in mtDNA duringreplication, leading to excision of the DNA loop. This leads to for-mation of shortened template of newly formed L strand for subse-quent elongation (Ozawa, 1995).

According to the illegitimate elongation of the D-loop strandtheory, the process begins with elongation of the D-loop duringreplication. The elongation of the D-loop strand leads to exposureof the first repeats with extension of the H-strand. This causes dis-placement of the elongated D-loop strand, misaligning it with adownstream repeat on the L-strand. During the next round of rep-lication, in elongated D-loop strand, the region between the re-peats is leftover resulting in a smaller genome. However, theabove two theories do not account for deletions that occur wherethere are no repeats. (St. John et al., 2000) Further, they fail to ac-count for point mutations specifically.

Numerous studies have shown a clonal expansion of cells hav-ing specific deletions present early in life (Johnston et al., 1995;Oldfors et al., 1995; Tengan et al., 1997; Zhang et al., 1999) andthey are not associated with the ‘‘vicious cycle.’’ In one study,age and common deletion levels were positively correlated in con-trols (r = 0.80) and in patients with skeletal muscle disorders(r = 0.69), which showed that each group had same rate of accumu-lation of the age related common deletions (Tengan et al., 1997).This accumulation of deletions in the cells supports the conceptof the founder molecule theory (Marchington et al., 1997) andthe exponential increase in deletions with age giving speculationto the clonal expansion hypothesis: but there is no plausible expla-nation for this theory (St. John et al., 2000). The questions stillunanswered are; why there would be bias for replication of deletedmolecules, who actually controls such bias and what is the role ofnuclear DNA in such a process? With the identification of a muta-tion or deletion in the mitochondrial genome, it is important todetermine if the defect had an adverse effect on cellular function.

With increased evidence of the free radical induced injury, to-day free radical induced mtDNA deletions and mutation theoryseems more relevant (Venkatesh et al., 2009). This theory relieson the fact that mtDNA is up to 100 times more susceptible tooxidative insult than nuclear DNA due to lack of the histone pro-teins (Pesole et al., 1999; Zhang et al., 1999). Very basic mechanismof DNA repair exists in mitochondria (Bohr and Anson, 1995;Thyagarajan et al., 1996; Lunt and Hyman, 1997; Yakes and VanHouten, 1997; Beckman and Ames, 1999; Bohr and Dianov,1999). Disturbance in homeostasis between free radical generationsystem (e.g. cytochrome C generates OH� free radical) and scav-enging (e.g. SOD and glutathione peroxidise) leads to large scaledeletions at hot spot regions. Although a strong correlation be-tween free radical levels and mtDNA deletions has been made,the accuracy of techniques for measuring OH_ and its conversionto 8–OH–dG, for example, is controversial. Often such data are cor-relative and hence must be viewed with caution (St. John et al.,2000). According to one study, quantification of oxidative DNA ad-ducts is biased by artifactual oxidation to the extent that it is notclear whether mtDNA is more or less prone to oxidative damagein comparison to the nuclear DNA (Beckman and Ames, 1996).

The free radical theory is also supported by the fact that freeradical induced cell death occurs through mitochondria. A stronglink has been established between apoptosis and mitochondria,and specifically that mitochondria initiates the cascade of down-stream events (Wilson, 1998). This complex process is partiallyregulated by two members of the Bcl-2 family. Bcl-2, the anti-

426 S. Rajender et al. / Mitochondrion 10 (2010) 419–428

apoptotic factor, is involved in maintaining inner mitochondrialmembrane electrochemical gradient through regulation of normalflow of Ca2+ into and out of the mitochondria, whereas Bax, thepro-apoptotic member of the Bcl-2 family, induces the disruptionof inner mitochondrial membrane electrochemical gradient(Knudson et al., 1995; Xiang et al., 1996). For normal cellular func-tioning, heterodimerization of Bcl-2 and Bax is required so that abalance is established between cell proliferation and cell death(Kroemer, 1997). This is important part of screening process forcells that are affected by free radicals.

Testis appears to be finely regulated by Bax and Bcl-2 to the ex-tent that, in mice, overexpression of Bcl-2 (Selvi Rani et al., 2006)and deficiency of Bax (Furuchi et al., 1996) leads to spermatogen-esis that fails to complete to the mature spermatozoa stage. Apop-tosis has been proposed to be a regulator of the over proliferationof the clonal progeny. St. John et al., (2000) have interestinglyhypothesised that defective spermatozoa though were markedfor apoptosis by Fas receptors they escaped cell death throughup-regulation of anti-apoptotic Bcl-2 genes. However, what causesthis up-regulation is unknown. One might argue that defectivemitochondria might be the cause of imbalance but such cellsshould be destroyed, in principle. The situation is perplexing andneeds further research. Presence of high levels of mtDNA muta-tions in poor quality sperm samples, therefore, requires physiolog-ical and biochemical studies to determine if these deletions cantruly affect both survival and function of the sperm cell.

10. Conclusion

Mitochondria are unique organelles since they require the con-tribution from two physically separated genomes. Regulatorymechanisms participating in mitochondrial gene expression arestill poorly understood, and may unveil its participation in sper-matogenesis and male infertility. Though most of the studies fa-vour the proposal that mitochondria is important for spermfunction and fertilization, issues such as low frequency and func-tional importance of mutations, threshold level and inheritanceneed to be explored to uproot the causes. In the absence of muta-tions with severe consequences, haplotype analysis needs to be ta-ken such that maximum numbers of mutations/polymorphismsare analyzed in the patients. With recent knowledge in the controlof cell function through epigenetic control it becomes necessary tostudy mitochondrial function in both normal and infertile menwith a new approach.

Acknowledgements

Singh Rajender is thankful to the Ministry of Health and FamilyWelfare (MOH & FW), Govt. of India for financial support. PandeyRahul is thankful to the Department of Biotechnology, Govt. of In-dia for M. Phil. Fellowship. CDRI communication number for thisarticle is 7922.

References

Aitken, R.J., Clarkson, J.S., Fisel, S., 1989. Generation of reactive oxygen species, lipidperoxidation, and human sperm function. Biol. Reprod. 40, 183–197.

Alcivar, A.A., Hake, L.E., Millette, C.F., Trasler, J.M., Hecht, N.B., 1989. Mitochondrialgene expression in male germ cells of the mouse. Dev. Biol. 135, 263–271.

Alvarez, J.G., Storey, B.T., 1995. Differential incorporation of fatty acids into andperoxidative loss of fatty acids from phospholipids of human spermatozoa. Mol.Reprod. Dev. 42, 334–346.

Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H.L., Coulson, A.R., Drouin, J.,Eperon, I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J.H.,Staden, R., Young, I.G., 1981. Sequence and organisation of the humanmitochondrial genome. Nature 290, 457–465.

Anderson, S., Bankier, A.T., deBarrell, B.G., Bruijn, M.H.L., Coulson, A.R., Drouin, J.,Eperon, I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J.H.,Staden, R., Young, I.G., 1982. Comparison of the human and bovine

mitochondrial genomes. In: Slonimski, P., Borst, P., Attardi, G. (Eds.),Mitochondrial Genes. Cold Spring Harbour, New York, pp. 51–57.

Ankel-Simons, F., Cummins, J.M., 1996. Misconception about mitochondria andmammalian fertilization: implications for theories on human evolution. Proc.Natl. Acad. Sci. USA 93, 13859–13863.

Arcelay, E., Salicioni, A.M., Wertheimer, E., Visconti, P.E., 2008. Identification ofproteins undergoing tyrosine phosphorylation during mouse spermcapacitation. Int. J. Dev. Biol. 52, 463–472.

Asquith, K.L., Harman, A.J., McLaughlin, E.A., Nixon, B., Aitken, R.J., 2005.Localization and significance of molecular chaperones, heat shock protein 1,and tumor rejection antigen gp96 in the male reproductive tract and duringcapacitation and acrosome reaction. Biol. Reprod. 72, 328–337.

Attardi, G., 1985. Animal mitochondrial DNA: an extreme example of geneticeconomy. Int. Rev. Cytol. 93, 93–145.

Baker, M.A., Aitken, R.J., Baker, M.A., Aitken, R.J., 2005. Reactive oxygen species inspermatozoa: methods for monitoring and significance for the origins of geneticdisease and infertility. Reprod. Biol. Endocrinol. 3, 67.

Barr, A.B., Moore, D.J., Paulsen, C.A., 1971. Germinal cell loss during humanspermatogenesis. J. Reprod. Fertil. 25, 75–80.

Beckman, K.B., Ames, B.N., 1996. Detection and quantification of oxidative adductsof mitochondrial DNA. Methods Enzymol. 264, 442–453.

Beckman, K.B., Ames, B.N., 1999. Endogenous oxidative damage of mtDNA. Mutat.Res. 424, 51–58.

Bereiter-Hahn, J., Voth, M., 1994. Dynamics of mitochondria in living cells: shapechanges, dislocations, fusion and fission of mitochondria. Microsc. Res. Tech. 27,198–219.

Bergmann, M., Kumari, M., Aumüller, G., Hoffmann, K., Seitz, J., 1990. Distributionpattern of testicular sulphydryloxidase immuno-activity in the djungarianhamster (Phodopus sungorus) during photoperiodically induced involution andrecrudescence. Int. J. Androl. 13, 488–499.

Bohr, V.A., Anson, R.M., 1995. DNA-damage, mutation and fine-structure DNA repairin aging. Mutat. Res. 338, 25–34.

Bohr, V.A., Dianov, G.L., 1999. Oxidative DNA damage processing in nuclear andmitochondrial DNA. Biochimie 81, 155–160.

Braun, R.E., Behringer, R.R., Peschon, J.J., Brinster, R.L., Palmiter, R.D., 1989.Genetically haploid spermatids are phenotypically diploid. Nature 337, 373–376.

Clayton, D.A., 1982. Replication of animal mitochondrial DNA. Cell 28, 693–705.Clayton, D.A., 2000. Transcription and replication of mitochondrial DNA. Human

Reprod. 15 (Suppl. 2), 11–17.Clermont, Y., 1962. Quantitative analysis of spermatogenesis of the rat: a revised

model for the renewal of spermatogonia. Am. J. Anat. 111, 111–129.Cummins, J., 1998. Mitochondrial DNA in mammalian reproduction. Rev. Reprod. 3,

172–182.Cummins, J.M., Jequier, A.M., Kan, R., 1994. Molecular biology of human male

infertility: links with aging, mitochondrial genetics, and oxidative stress? Mol.Reprod. Dev. 37, 345–362.

Dairaghi, D.J., Shadel, G.S., Clayton, D.A., 1995. Human mitochondrial transcriptionfactor A and promotor spacing integrity are required for transcription initiation.Biochim. Biophys. Acta 1271, 127–134.

de Lamirande, E., Gagnon, C., 1993. Human sperm hyperactivation and capacitationas parts of an oxidative process. Free Radic. Biol. Med. 14, 255–265.

Elliott, H.R., Samuels, D.C., Eden, J.A., Relton, C.L., Chinnery, P.F., 2008. Pathogenicmitochondrial DNA mutations are common in the general population. Am. J.Human Genet. 83, 254–260.

Erickson, L.D., Rizza, S.A., Bergert, E.R., Charlesworth, M.C., McCormick, D.J., Ryan,R.J., 1990. Synthetic alpha-subunit peptides stimulate testosterone productionin vitro by rat Leydig cells. Endocrinology 126, 2555–2560.

Evenson, D.P., Wixon, R., 2006. Clinical aspects of sperm DNA fragmentationdetection and male infertility. Theriogenology 15, 979–991.

Ficarro, S., Chertihin, O., Westbrook, V.A., White, F., Jayes, F., Kalab, P., Marto, J.A.,Shabanowitz, J., Herr, J.C., Hunt, D.F., Visconti, P.E., 2003. Phosphoproteomeanalysis of capacitated human sperm. Evidence of tyrosine phosphorylation of akinase-anchoring protein 3 and valosin-containing protein/p97 duringcapacitation. J. Biol. Chem. 278, 11579–11589.

Folgero, T., Bertheussen, K., Lindal, S., Torbergsen, T., Oian, P., 1993. Mitochondrialdisease and reduced sperm motility. Human Reprod. 8, 1863–1868.

Furuchi, T., Masuko, K., Nishimune, Y., Obinata, M., Matsui, Y., 1996. Inhibition oftesticular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 inspermatogonia. Development 122, 1703–1709.

Gandini, L., Lombardo, F., Paoli, D., Caponecchia, L., Familiari, G., Verlengia, C.,Dondero, F., Lenzi, A., 2000. Study of apoptotic DNA fragmentation in humanspermatozoa. Human Reprod. 15, 830–839.

Gavella, M., Lipovac, V., 1992. NADH-dependent oxidoreductase (diaphorase)activity and isozyme pattern of sperm in infertile men. Arch. Androl. 28, 135–141.

Grootegoed, J.A., Jansen, R., van der Molen, H.J., 1984. Spermatogenic cells in thegerminal epithelium utilize alpha-ketoisocaproate and lactate, produced bySertoli cells from leucine and glucose. Ann. NY Acad. Sci. 438, 557–560.

Hecht, N.B., Liem, H., 1984. Mitochondrial DNA is synthesized during meiosis andspermiogenesis in the mouse. Exp. Cell Res. 154, 293–298.

Heller, C.G., Clermont, Y., 1964. Kinetics of the germinal epithelium in man. RecentProg. Horm. Res. 20, 545–575.

Holyoake, A.J., Sin, I.L., Benny, P.S., Sin, F.Y., 1999. Association of a novel humanmtDNA ATPase6 mutation with immature sperm cells. Andrologia 31, 339–345.

S. Rajender et al. / Mitochondrion 10 (2010) 419–428 427

Holyoake, A.J., McHugh, P., Wu, M., O’Carroll, S., Benny, P., Sin, I.L., Sin, F.Y., 2001.High incidence of single nucleotide substitutions in the mitochondrial genomeis associated with poor semen parameters in men. Int. J. Androl. 24, 175–182.

Huang, C.C., Chen, R.S., Chen, C.M., Wang, H.S., Lee, C.C., Pang, C.Y., Hsu, H.S., Lee,H.C., Wei, Y.H., 1994. MELAS syndrome with mitochondrial tRNA leu (UUR)gene mutation in a Chinese family. J. Neurol. Neurosurg. Psychiatry 57, 586–589.

Huckins, C., Oakberg, E.F., 1978. Morphological and quantitative analysis ofspermatogonia in mouse testes using whole mounted seminiferous tubules, I.The normal testes. Anat. Rec. 192, 519–528.

Jha, K.N., Shivaji, S., 2002. Protein serine and threonine phosphorylation,hyperactivation and acrosome reaction in in vitro capacitated hamsterspermatozoa. Mol. Reprod. Dev. 63, 119–130.

John, J.C., Facucho-Oliveira, J., Jiang, Y., Kelly, R., Salah, R., 2010. Mitochondrial DNAtransmission, replication and inheritance: a journey from the gamete throughthe embryo and into offspring and embryonic stem cells. Human Reprod., inpress.

Johnson, L., 1985. Increased daily sperm production in the breeding season ofstallions is explained by an elevated population of spermatogonia. Biol. Reprod.32, 1181–1190.

Johnson, L., 1995. Efficiency of spermatogenesis. Microsc. Res. Tech. 32, 385–422.Johnson, L., Petty, C.S., Neaves, W.B., 1983. Further quantification of human

spermatogenesis: germ cell loss during postprophase of meiosis and itsrelationship to daily sperm production. Biol. Reprod. 1, 207–215.

Johnston, W., Karpati, G., Carpenter, S., Arnold, D., Shoubridge, E.A., 1995. Late onsetmitochondrial myopathy. Ann. Neurol. 37, 16–23.

Kao, S., Chao, H.T., Wei, Y.H., 1995. Mitochondrial deoxyribonucliec acid 4977bpdeletion is associated with diminished fertility and motility of human sperm.Biol. Reprod. 52, 729–736.

Kao, S.H., Chao, H.T., Wei, Y.H., 1998. Multiple deletions of mitochondrial DNA areassociated with the decline of motility and fertility of human spermatozoa. Mol.Human Reprod. 4, 657–666.

King, M.P., Attardi, G., 1988. Injection of mitochondria into human cells leadsto a rapid replacement of the endogenous mitochondrial DNA. Cell 52,811–819.

King, M.P., Attardi, G., 1996. Isolation of human cell lines lacking mitochondrialDNA. Methods Enzymol. 264, 304–313.

Kleene, K.C., Distel, R.J., Hecht, N.B., 1984. Translational regulation anddeadenylation of a protamine mRNA during spermiogenesis in the mouse.Dev. Biol. 105, 71–79.

Knudson, C.M., Tung, K.S.K., Tourtellotte, W.G., Brown, G.A.J., Korsmeyer, S.J., 1995.Bax-deficient mice with lymphoid hyperplasia and male germ cell death.Science 270, 96–99.

Kolmer, M., Pelto-Huikko, M., Parvinen, M., Höög, C., Alho, H., 1997. Thetranscriptional and translational control of diazepam binding inhibitorexpression in rat male germ-line cells. DNA Cell Biol. 16, 59–72.

Kota, V., Dhople, V.M., Shivaji, S., 2009. Tyrosine phosphoproteome of hamsterspermatozoa: role of glycerol-3-phosphate dehydrogenase 2 in spermcapacitation. Proteomics 9, 1809–1826.

Kroemer, G., 1997. The proto-oncogene Bcl-2 and its role in regulating apoptosis.Nat. Med. 3, 614–620.

Kumar, V., Rangaraj, N., Shivaji, S., 2006. Activity of pyruvate dehydrogenase A(PDHA) in hamster spermatozoa correlates positively with hyperactivation andis associated with sperm capacitation. Biol. Reprod. 75, 767–777.

Kumar, V., Kota, V., Shivaji, S., 2008. Hamster sperm capacitation: role of pyruvatedehydrogenase A and dihydrolipoamide dehydrogenase. Biol. Reprod. 79, 190–199.

Larsson, N.G., Garman, J.D., Oldorfs, A., Barsh, G.S., Clayton, D.A., 1996. A singlemouse gene encodes the mitochondrial transcription factor A and a testis-specific nuclear HMG box protein. Nat. Genet. 13, 296–302.

Larsson, N.G., Oldfors, A., Garman, J.D., Barsh, G.S., Clayton, D.A., 1997. Down-regulation of mitochondrial transcription factor A during spermatogenesis inhumans. Human Mol. Genet. 6, 185–191.

Lefièvre, L., Jha, K.N., de Lamirande, E., Visconti, P.E., Gagnon, C., 2002. Activation ofprotein kinase A during human sperm capacitation and acrosome reaction. J.Androl. 23, 709–716.

Lestienne, P., Reynier, P., Chretien, M.F., Penisson-Besnier, I., Malthiery, Y., Rohmer,V., 1997. Oligoasthenospermia associated with multiple mitochondrial DNArearrangements. Mol. Human Reprod. 3, 811–814.

Lightowlers, R.N., Chinnery, P.F., Turnbull, D.M., Howell, N., 1997. Mammalianmitochondrial genetics, heredity, heteroplasmy and disease. Trends Genet. 13,450–455.

Luconi, M., Marra, F., Gandini, L., Filimberti, E., Lenzi, A., Forti, G., Baldi, E., 2001.Phosphatidylinositol 3-kinase inhibition enhances human sperm motility.Human Reprod. 16, 1931–1937.

Lunt, D.H., Hyman, B.C., 1997. Animal mitochondrial DNA recombination. Nature387, 247.

Manfredi, G., Thyagarajan, D., Papadopoulou, L.C., Pallotti, F., Schon, E.A., 1997. Thefate of human sperm-derived mtDNA in somatic cells. Am. J. Human Genet. 61,953–960.

Marchington, D.R., Hartshorne, G.M., Barlow, D., Poulton, J., 1997. Homopolymerictract heteroplasmy in mtDNA from tissues and single oocytes: support for agenetic bottleneck. Am. J. Human Genet. 60, 408–416.

May-Panloup, P., Chretien, M.F., Savagner, F., Vasseur, C., Jean, M., Malthiery, Y.,Reynier, P., 2003. Increased sperm mitochondrial DNA content in maleinfertility. Human Reprod. 18, 550–556.

Meinhardt, A., Parvinen, M., Bacher, M., Aumüller, G., Hakovirta, H., Yagi, A., Seitz,J., 1995. Expression of mitochondrial heat shock protein 60 in distinct celltypes and defined stages of rat seminiferous epithelium. Biol. Reprod. 52,798–807.

Meinhardt, A., Wilhelm, B., Seitz, J., 1999. Expression of mitochondrial markerproteins during spermatogenesis. Human Reprod. Update 5, 108–119.

Michikawa, Y., Mazzucchelli, F., Bresolin, N., Scarlato, G., Attardi, G., 1999. Agingdependent large accumulation of point mutations in the human mtDNA controlregion for replication. Science 286, 774–779.

Mitra, K., Shivaji, S., 2004. Novel tyrosine-phosphorylated post-pyruvate metabolicenzyme, dihydrolipoamide dehydrogenase, involved in capacitation of hamsterspermatozoa. Biol. Reprod. 70, 887–899.

Mitra, K., Shivaji, S., 2005. Proteins implicated in sperm capacitation. Indian J. Exp.Biol. 43, 1001–1015.

Moraes, C.T., Shanske, S., Trischler, H.J., Aprille, J.R., Andreetta, F., Bonilla, E., Schon,E.A., DiMauro, S., 1991. MtDNA depletion with variable tissue expression: anovel genetic abnormality in mitochondrial diseases. Am. J. Human Genet. 48,492–501.

Naor, Z., Breitbart, H., 1997. Protein kinase C and mammalian spermatozoaacrosome reaction. Trends Endocrinol. Metab. 8, 337–342.

Oakberg, E.F., 1956. A description of spermiogenesis in the mouse and its use inanalysis of the cycle of the seminiferous epithelium and germ cell renewal. Am.J. Anat. 99, 391–413.

Oldfors, A., Moslemi, A.R., Fyhr, I.M., Holme, E., Larsson, N.G., Lindberg, C., 1995.Mitochondrial DNA deletions in muscle fibers in inclusion body myositis. J.Neuropathol. Exp. Neurol. 54, 581–587.

Ozawa, T., 1995. Mechanism of somatic mitochondrial DNA mutations associatedwith age and diseases. Biochim. Biophys. Acta 1271, 177–189.

Pereira, L., Goncalves, J., Bandelt, H.J., 2008. Mutation C11994T in the mitochondrialND4 gene is not a cause of low sperm motility in Portugal. Fertil. Steril. 89, 738–741.

Pesole, G., Gissi, C., DeChirico, A., Saccone, C., 1999. Nucleotide substitution rate ofmammalian mitochondrial genomes. J. Mol. Evol. 48, 427–434.

Rantanen, A., Larsson, N.G., 2000. Regulation of mitochondrial DNA copy numberduring spermatogenesis. Human Reprod. 15, 86–91.

Rovio, A.T., Marchington, D.R., Donat, S., Schuppe, H.C., Abel, J., Fritsche, E., Elliott,D.J., Laippala, P., Ahola, A.L., McNay, D., Harrison, R.F., Hughes, B., Barrett, T.,Bailey, D.M., Mehmet, D., Jequier, A.M., Hargreave, T.B., Kao, S.H., Cummins, J.M.,Barton, D.E., Cooke, H.J., Wei, Y.H., Wichmann, L., Poulton, J., Jacobs, H.T., 2001.Mutations at the mitochondrial DNA polymerase (POLG) locus associated withmale infertility. Nat. Genet. 29, 261–262.

Ruiz-Pesini, E., Diez, C., Laperia, A.C., Pérez-Martos, A., Montoya, J., Alvarez, E.,Arenas, J., López-Pérez, M.J., 1998. Correlation of sperm motility withmitochondrial enzymatic activities. Clin. Chem. 44, 1616–1620.

Ruiz-Pesini, E., Lapena, A.C., Diez-Sanchez, C., Perez-Martos, A., Montoya, J., Alvarez,E., Diaz, M., Urries, A., Montoro, L., Lopez-Perez, M.J., Enriquez, J.A., 2000. HumanmtDNA haplogroups associated with high or reduced spermatozoa motility.Am. J. Human Genet. 67, 682–696.

Said, T.M., Agarwal, A., Sharma, R.K., Thomas Jr, A.J., Sikka, S.C., 2005. Impact ofsperm morphology on DNA damage caused by oxidative stress induced by beta-nicotinamide adenine dinucleotide phosphate. Fertil. Steril. 83, 95–103.

Selvi Rani, D., Vanniarajan, A., Gupta, N.J., Chakravarty, B., Singh, L., Thangaraj, K.,2006. A novel missense mutation C11994T in the mitochondrial ND4 gene as acause of low sperm motility in the Indian subcontinent. Fertil. Steril. 86, 1783–1785.

Setchell, B.P., 1978. The Mammalian Testi. Elek Books, London.Shamsi, M.B., Kumar, R., Bhatt, A., Bamezai, R.N., Kumar, R., Gupta, N.P., Das, T.K.,

Dada, R., 2008. Mitochondrial DNA Mutations in etiopathogenesis of maleinfertility. Indian J. Urol. 24, 150–154.

Shivaji, S., Kumar, V., Mitra, K., Jha, K.N., 2007. Mammalian sperm capacitation: roleof phosphotyrosine proteins. Soc. Reprod. Fertil. Suppl. 63, 295–312.

Shivaji, S., Kota, V., Siva, A.B., 2009. The role of mitochondrial proteins in spermcapacitation. J. Reprod. Immunol. 83, 14–18.

Shoffner, J.M., Wallace, D.C., 1992. Mitochondrial genetics: principles and practice.Am. J. Human Genet. 51, 1179–1186.

Shoffner, J.M., Lott, M.T., Voljavec, A.S., Soueidan, S.A., Costigan, D.A., Wallace, D.C.,1989. Spontaneous Kearns Sayre/chronic external opthalmoplegia plussyndrome associated with a mitochondrial deletion: a slip replication modeland metabolic therapy. Proc. Natl. Acad. Sci. USA 86, 7952–7956.

Shuster, R.C., Rubenstein, A.J., Wallace, D.C., 1988. Mitochondrial DNA in anucleatehuman blood cells. Biochem. Biophys. Res. Commun. 155, 1360–1365.

Siva, A.B., Kameshwari, D.B., Singh, V., Pavani, K., Sundaram, C.S., Rangaraj, N.,Deendayal, M., Shivaji, S., 2010. Proteomics-based study on asthenozoospermia:differential expression of proteasome alpha complex. Mol. Hum. Reprod. 16,452–462.

Sonezaki, S., Okita, K., Oba, T., Ishii, Y., Kondo, A., Kato, Y., 1995. Protein substratesand heat shock reduce the DNA-binding ability of Escherichia coli Lon protease.Appl. Microbiol. Biotechnol. 44, 484–488.

Spiropoulos, J., Turnbull, D.M., Chinnery, P.F., 2002a. Can mitochondrial DNAmutations cause sperm dysfunction? Mol. Human Reprod. 8, 719–721.

Spiropoulos, J., Chinnery, P.F., Turnbull, D.M., 2002b. Pathogenic mitochondrial DNAmutations and human reproduction. Human Fertil. 2, 133–137.

Spiteri-Grech, J., Nieschlag, E., 1993. Paracrine factors relevant to the regulation ofspermatogenesis–a review. J. Reprod. Fertil. 98, 1–14.

St. John, J.C., Sakkas, D., Barratt, C.L., 2000. A role for mitochondrial DNA and spermsurvival. J. Androl. 21, 189–199.

428 S. Rajender et al. / Mitochondrion 10 (2010) 419–428

Storey, B.T., 1980. Strategy of oxidative metabolism in bull spermatozoa. J. Exp.Zool. 212, 61–67.

Suomalainen, A., Kaukonen, J., Amati, P., Timonen, R., Haltia, M., Weissenbach, J.,Zeviani, M., Somer, H., Peltonen, L., 1995. An autosomal locus predisposing todeletions of mitochondrial DNA. Nat. Genet. 9, 146–151.

Sutovsky, P., Navara, C.S., Schatten, G., 1996. Fate of the sperm mitochondria, andthe incorporation, conversion, and disassembly of the sperm tail structuresduring bovine fertilization. Biol. Reprod. 55, 1195–1205.

Tengan, C.H., Gabbai, A.A., Shanske, S., Zeviani, M., Moraes, C.T., 1997. Oxidativephosphorylation dysfunction does not increase the rate of accumulation of age-related mtDNA deletions in skeletal muscle. Mutat. Res. 379, 1–11.

Thangaraj, K., Joshi, M.B., Reddy, A.G., Rasalkar, A.A., Singh, L., 2003. Spermmitochondrial mutations as a cause of low sperm motility. J. Androl. 24, 388–392.

Thyagarajan, B., Padua, R.A., Campbell, C., 1996. Mammalian mitochondria possesshomologous DNA recombination activity. J. Biol. Chem. 271, 27536–27543.

Tremellen, K., 2008. Oxidative stress and male infertility–a clinical perspective.Human Reprod. Update 14, 243–258.

Venkatesh, S., Deecaraman, M., Kumar, R., Shamsi, M.B., Dada, R., 2009. Role ofreactive oxygen species in the pathogenesis of mitochondrial DNA (mtDNA)mutations in male infertility. Indian J. Med. Res. 129, 127–137.

Wai, T., Ao, A., Zhang, X., Cyr, D., Dufort, D., Shoubridge, E.A., 2010. The roleof mitochondrial DNA copy number in mammalian fertility. Biol. Reprod.,in press.

Werner, A., Meinhardt, A., Seitz, J., Bergmann, M., 1997. Distribution of heat-shockprotein 60 immunoreactivity in testes of infertile men. Cell Tissue Res. 288,539–544.

Wiesner, R.J., Ruegg, J.C., Morano, I., 1992. Counting target molecules by exponentialpolymerase chain reaction, copy number of mitochondrial DNA in rat tissues.Biochem. Biophys. Acta 183, 553–559.

Williams, S.R., 2002. Another surprise from the mitochondrial genome. N. Engl. J.Med. 347, 609–611.

Wilson, M.R., 1998. Apoptosis: unmasking the executioner. Cell Death Differ. 5,646–652.

Xiang, J., Chao, D.T., Korsmeyer, S.J., 1996. Bax-induced cell death may not requireinterleukin 1 beta-converting enzyme-like proteases. Proc. Natl. Acad. Sci. USA93, 14559–14563.

Yakes, M.F., Van Houten, B., 1997. Mitochondrial DNA damage is more extensiveand persists longer than nuclear DNA damage in human cells followingoxidative stress. Proc. Natl. Acad. Sci. USA 94, 514–519.

Zeviani, M., Antozzi, C., 1997. Mitochondrial disorders. Mol. Human Reprod. 3, 133–148.

Zeviani, M., Servidei, S., Gellera, C., Bertini, E., DiMauro, S., DiDonato, S., 1989. Anautosomal dominant disorder with multiple deletions of mitochondrial DNAstarting at the D-loop region. Nature 339, 309–311.

Zhang, C., Lee, A., Liu, V.W., Pepe, S., Rosenfeldt, F., Nagley, P., 1999. MitochondrialDNA deletions in human cardiac tissue show a gross mosaic distribution.Biochem. Biophys. Res. Commun. 254, 152–157.