Abstract: Sperm DNA fragmentation is known to compromise male fertility. The data entail that sperm DNA damage can be efficiently treated with oral antioxidant administration during a relatively short time period. It is an understanding that the 2 types of DNA appear to be distinct and independently packaged molecules; however, research has demonstrated the symbiotic nature of these structures in contributing to male infertility. Data must continue to be gathered to establish strong correlations between traditional semen analysis parameters and sperm DNA integrity; this information remains controversial and useless in clinical practice until new techniques for the diagnosis and treatment of sperm DNA can be established. More modern technology must be employed to correlate such information into useful clinical knowledge. This study is the compilation of reports pertaining to research on sperm DNA biochemistry and molecular biology to establish an opinion that sperm DNA integrity is both extremely fragile and exceedingly important for male fertility.
INTRODUCTION
Sperm DNA fragmentation is known to compromise male fertility. The data entail that sperm DNA damage can be efficiently treated with oral antioxidant administration during a relatively short time period (Greco et al., 2005). An improvement of basic sperm parameters by oral treatment with antioxidants has been reported in a number of studies (Agarwal et al., 2004), but DNA damage has been addressed in only a few of them (Geva et al., 1998; Suleiman et al., 1996; Kodama et al., 1997; Comhaire et al., 2000; Keskes-Ammar et al., 2003). Only two of these studies were controlled and randomized (Suleiman et al., 1996; Keskes-Ammar et al., 2003) and none of them used a direct assay for the detection of DNA strand breaks. Infertility is a growing problem among couples trying to conceive; in the past the female partner was singled out as the primary reason for being unable to bear a child. Research now reveals that male infertility may contribute in up to two thirds of all couples who seek treatment for infertility. For many years a conventional semen analysis (concentration, motility and morphology) was seen as sufficient to diagnose male infertility; however, scientific examination must now take into account 2 different kinds of DNA that have been proven to contribute to this diagnosis. Nuclear DNA (nDNA), contained in the head of the sperm, is responsible for packaging all of the paternal genetic information that will be needed for the fertilized egg. nDNA can be damaged or compromised via 4 interrelated courses: defective chromatin packaging, apoptosis, oxidative stress and genetic lesions. Mitochondrial DNA (mtDNA) is located in the midpiece of the sperm; when coupled with the tail, it is responsible for mobilizing the sperm toward the egg for fertilization. Scientists are only beginning to comprehend the relationship and interaction between these distinct DNA molecules and how they both contribute to male infertility. As the worldwide community continues to expand, an emerging subpopulation of couples has begun experiencing a common problem in making their contribution to the populace. These couples are experiencing a major health crisis, commonly referred to as infertility. Infertility is classically defined as a state in which a couple desiring a child is unable to conceive following 12 months of unprotected intercourse (Seshagiri, 2001). It affects approximately 15% of couples who seek clinical treatment to conceive a child and recent studies show that the number of infertile couples in the general population is growing (Feng, 2003). In infertile couples, responsibility for the lack of conception is generally divided into thirds, with one third due to male factors, one third due to female factors and the final third due to overlapping factors from both partners.
Application of nutraceuticals in sperm production: Nutraceuticals have been advocated as a way of potentiating sperm production and quality in the subfertile male. In a recent study, the administration of folic acid and zinc sulfate to subfertile males was shown to result in a significant improvement in sperm concentration compared to placebo. Treatment lasted 25 weeks and the daily dose of folic acid and zinc were 5 and 66 mg, respectively. Although the beneficial effect on fertility remains to be established, this finding opens new avenues of future fertility research and treatment (Wong et al., 2002). Arginine (Pryor et al., 1978), vitamin B12 (Sinclair, 2000), methylcobalamin (Moriyama et al., 1987) and ginseng (Salvati et al., 1996) have been used in the treatment of male infertility. However, most of these compounds have marginal effects on sperm production and quality and have not been tested for safety and efficacy in randomized placebo-controlled studies. In fact, ginseng has been shown to have estrogenic activity (Duda et al., 1996) and produce adverse reactions (Hammond and Whitworth, 1981; Dega et al., 1996). Because ROS overproduction has been associated with defective sperm function (Aitken et al., 1989), infertile patients have been treated with antioxidant compounds including, ascorbic acid (Fraga et al., 1991), vitamin E (Kessopoulou et al., 1995; Mishra et al., 2009), selenium (Scott et al., 1998), gluthatione (Lenzi et al., 1998), vitamins, selenium, gluthatione, ubiquinol and carnitine (Moncada et al., 1992; Lenzi et al., 1993; Costa et al., 1994; Sikka et al., 1995; Vitali et al., 1995; Hawkes and Turek, 2001; Vicari and Calogero, 2001; Lenzi et al., 2003; Sharma et al., 2009; Mishra et al., 2009). However, the effect of this treatment on sperm quality is still controversial (Ford and Whittington, 1998; Geva et al., 1998; Lenzi et al., 1998; Tarin et al., 1998; Comhaire et al., 1999). In a randomized, placebo-controlled, double-blind study, oral high doses of vitamins C and E to infertile males did not show any statistically significant improvement in semen parameters (Rolf et al., 1999). However, in this study, patient recruitment was solely based on having a sperm concentration below 50 million mL-1. One of the main reasons studies looking at the efficacy of antioxidant therapy in the treatment of male infertility have not yet been conclusive may be due to inadequate patient selection. Not all infertile males have an increase in oxidative stress in their testis and semen. Therefore, in principle, only those men who have a quantifiable increase in oxidative stress should benefit from antioxidant therapy. Perhaps the best marker to identify these males would be Reactive Oxygen Species (ROS) levels in semen (Agarwal and Saleh, 2002). Another important aspect of antioxidant therapy is whether the antioxidant(s) and dose used in vivo are appropriate. Previous studies have indicated that the combination of vitamins E and C at high doses in vitro results in DNA fragmentation (Donnelly et al., 1999).
Types of sperm DNA and fertilization: The cornerstone of the evaluation of the man remains semen analysis. Although, it gives some quantitative and qualitative information about the sperm sample, recent insight into the molecular biology and genetics of the sperm cell have demonstrated that morphology and motility alone are not the only grounds upon which sperm should be evaluated. Commonly overlooked is the fact that sperm carry 2 different kinds of DNA. The nDNA, commonly called the genome, is located in the head of the sperm. The second DNA type is called the mtDNA and is responsible for delivering the sperm to the egg by providing ATP for cellular acceleration. Both types of DNA work toward the common goal of fertilization, but each is susceptible to a myriad of factors that could derail the fertilization process. Imperfections in both types of DNA contribute equally to the problem at hand. This article hopes to elucidate male factor infertility as contributed by both kinds of DNA. Origins of nDNA Damage nDNA in somatic cell nuclei is packaged into structures called nucleosomes. These structures consist of a protein core formed by an octamer of histones with 2 loops of wrapped DNA. The nucleosomes are then further coiled into regular helixes called solenoids, which increase the volume of the chromatin (Agarwal and Said, 2003). Sperm nuclei, however, need to be packaged much differently and more compactly to assure proper delivery of the nDNA. There are believed to be 4 levels of organization for packaging spermatozoon nDNA (Sakkas et al., 1999; Agarwal and Said, 2003). One level consists of anchoring the chromosomes to the nuclear annulus. In another, DNA loop domains are created as the DNA attaches itself to the newly added nuclear matrix. The arrangement of these loop domains ensures that the DNA can be delivered to the egg in a form that is both physically and chemically accessible to the growing embryo. Chromosome repositioning and organization within the matrix of the sperm head is another level. Condensation of nDNA into tiny, supercoiled dough-nuts called toroids by replacing the nuclear histones with structures called protamines completes the levels of chromosomal organization. Human sperm contain 2 types of protamines that are about half the size of typical histones; throughout evolution, they have increased the number of positively charged residues, allowing formation of a highly condensed complex with the negatively charged paternal genomic DNA. Also, the addition of cysteine residues allows the formation of disulfide bonds between adjacent protamine molecules, thereby strongly stabilizing the nucleo-protamine complex (Oliva, 2006). Prior to this re arrangement, recombination is essential for spermatogenesis to occur (Carrel et al., 2007); as seen in studies using animal knockout models, decreased recombination is associated with diminished spermatogenesis. Many factors (both endogenous and exogenous) can influence this, contributing to male infertility (Mishra et al., 2003; Nanda et al., 2010a). Scientists agree on 4 distinct methods, although there may be others, by which nDNA can be compromised or damaged: defective sperm chromatin packaging, apoptosis, oxidative stress and genetic lesions (Agarwal and Said, 2003; Sharma et al., 2004; Lewis and Aitken, 2005; Shafik et al., 2006). The effects of these damaging methods are often found to be interrelated. Defective Chromatin packaging refers to the highly complex and specific structure into which nDNA is folded to properly deliver the genetic information to the egg. Although, defects can arise at any of the 4 levels of packaging, the most common problems arise during DNA loop domain formation and histone-protamine replacement. nDNA loop domains can be difficult to arrange without inducing endogenous nicks to the nDNA (Sakkas et al., 1999). It is thought that these nicks exist naturally and serve to relieve torsional stress. The presence of these nicks is greatest during transition from round to elongated spermatids in the testis and occurs before complete protamination within the sperm. Topoisomerase II is the enzyme that creates an ligates the nicks within nDNA during this process (Sakkas et al., 1999; Henkel et al., 2004). Any defect in the enzyme itself will negatively affect the packaging of the genetic information and will contribute to male infertility. The enzyme may leave the nDNA fragmented with single- or double-stranded breaks; this may indicate an early apoptotic process in somatic cells and incomplete sperm maturation in the case of spermatozoa. Topoisomerase inhibitors have been proven to increase the levels of internal nDNA breaks by preventing their repair and increasing their susceptibility to damage (Lewis and Aitken, 2005). Also involved in sperm chromatin packaging is the replacement of histones with protamines. Protamines are major DNA-binding proteins essential for chromatin condensation (Vogt, 2004). During epididymal transport, histones are replaced by transition proteins, only to be replaced by protamines (Oliva, 2006); both intermolecular and intramolecular disulfide cross-linking among the cysteine-rich protamines compresses the DNA into one sixth the volume occupied by somatic cell nDNA (Lewis and Aitken, 2005; Zini and Libman, 2006). This high rate of cross-linking affords the sperm nDNA a measure of protection against exogenous assault and compensates for an impaired DNA-repair capacity. Human spermatozoa retain approximately 15% histones in their structure, leading to a less compact chromatin arrangement than in other mammals (Erenpreiss et al., 2006), perhaps to allow access for oocyte repair mechanisms. Human sperm contain 2 different types of protamines, which are believed to be present in equal amounts in fertile men: P1 and P2 (Carrell et al., 2007). Experiments have shown that the ratio of P1 to P2 is critical to male fertility (Oliva, 2006; Vogt, 2004; Erenpreiss et al., 2006; Carrell et al., 2007), more specifically to the sperm’s fertilization ability (Aoki et al., 2006). In addition, recent testing has demonstrated that P2 precursors (pre-P2) are vital in maintaining the delicate P1:P2 ratio. Translation of pre-P2 mRNA appears to cause abnormal head morphogenesis, reduced sperm motility and male infertility (Tseden et al., 2007). Also, a low pre- P2: P2 ratio suggests a link between deficient protamine processing and decreased nDNA integrity (Torregrosa et al., 2006). Apoptosis is the controlled disassembly of a cell from within; it is believed to have 2 roles during normal spermatogenesis (Seli et al., 2004; Shrama et al., 2004; Spano et al., 2005). The first role is to limit the germ cell population to numbers that can be supported by the surrounding Sertoli cells. The second is for the depletion of abnormal spermatozoa. As seen in the prior section, abnormal spermatozoa can be produced via defective sperm chromatin packaging, among other ways. In somatic cells, cells that enter into an apoptotic pathway usually have several classical indicators, such as phosphatidylserine (PS) relocation, Fas expression, nDNA strand breaks and caspase activity. PS relocation is perhaps the earliest indicator of apoptosis; normally located on the inner leaflet of the plasma membrane, PS migrates to the outer membrane once the apoptotic signal has been given (Henkel et al., 2004). To help control this signal, both pro- and anti-apoptotic proteins are present in the testis; they are members of the Bcl-2 family of proteins and provide a signaling pathway that is imperative to maintaining male germ cell homeostasis (Spano et al., 2005). Bcl-2 and Bcl-xL are both prosurvival proteins, while Bax is a pro-apoptotic protein. Disturbing the balance of these proteins from the Bcl-2 family has been demonstrated in mice to contribute to male infertility by disrupting normal apoptosis levels. Fas expression is another indicator that the apoptosis signal has been given. Fas is the type I cell surface protein, belonging to the tumor necrosis/nerve growth factor receptor family (Henkel et al., 2004); it is induced by the binding of Fas ligand to the Fas receptor on the plasma outer membrane. Sertoli cells are known to express Fas ligand, demonstrating the fact that apoptosis is a commonly used mechanism to control the germ cell population at a level that can be supported by the Sertoli cells (Erenpreiss et al., 2006). Ligation of Fas ligand to the Fas receptor triggers activation of cytosolic aspartate-specific proteases, or simply caspases. Once caspase activation has taken place, a signal is transduced to synthesize caspase-activated deoxyribonuclease, which leads to DNA degradation by forming single and double-stranded breaks within the nDNA (Agarwal and Said, 2003). In infertile men, ejaculated spermatozoa often possess partially degraded nDNA, usually considered to be indicative of the apoptosis pathway; this escaping of the apoptosis signal is referred to as abortive apoptosis (Sakkas et al., 2002; Lewis and Aitken, 2005; Erenpreiss et al., 2006). The apoptotic pathway is an all or nothing response, meaning that once the signal has been given there is no reversing the process. Abnormalities in this pathway are often attributed to 1 of 2 possibilities: infertile men may not produce enough sperm to trigger Sertoli cell activation to produce Fas, or there may be a problem in activating the Fas mediated apoptosis signal (Agarwal and Said, 2003). It is believed that if the apoptotic cascade is initiated at the round spermatid phase when transcription is still active, this may be the origin of the nDNA breaks commonly seen in abortive apoptosis in ejaculated spermatozoa. However, nDNA breaks are known to be common during condensation of the genome. It is currently unclear whether these breaks are caused by an aborted apoptotic pathway or simply by incomplete chromatin packaging. Also, not all caspase activity has been shown to be indicative of the apoptotic signal. Recent work has demonstrated that there appears to be some caspase activity in human germ cells that is not associated with apoptosis and may indeed serve a viable function (Martinez et al., 2002). Another well-known inducer of the apoptotic pathway is telomere shortening. Telomeres are capping structures at chromosome ends that protect against rearrangements, preventing ends from being recognized as nDNA breaks (Shafik et al., 2006). They are usually composed of tandem TTAGGG sequence repeats that are bound to a complex array of proteins. Telomerase is a specialized reverse transcriptase that contains a catalytic subunit that synthesizes new telomeric repeats. In the absence of telomerase, telomeric sequences are lost after each round of replication, eventually creating a shifted sequence that could be recognized as an nDNA double-stranded break; this would then be recognized by a genomic surveillance mechanism that appears in the elongating spermatid (Shafik et al., 2006). This recognition is another way to induce an apoptotic response, possibly contributing to the abortive apoptosis theory. Abortive apoptosis is a theory that still requires much scientific evidence to be considered valid. Because of naturally occurring processes within the spermatozoa that mimic somatic cell apoptosis, many believe that this theory requires additional evidence. Oxidative stress upon spermatozoa is induced by an increase in the amount of Reactive Oxygen Species (ROS) that are present in the fluids filling the male genital tract (Nanda et al., 2010b). Sperm are particularly susceptible to oxidative stress due to the high content of unsaturated fatty acids in their membranes, as well as their limited stores of antioxidant enzymes (Baker and Aitken, 2005). Their increased susceptibility is enhanced by defective chromatin packaging, causing further damage to the genome; individuals with varicoceles are particularly susceptible to this type of damage (Saleh et al., 2003). The ROS are created by metabolizing ground-state oxygen into the superoxide anion and H2O2 (Vernet et al., 2001). They play an important physiologic role, modulating gene and protein activities vital to sperm proliferation, differentiation and function (Erenpreiss et al., 2006). The ROS also promote tyrosine phosphorylation to support sperm capacitation. Fertile men control ROS generation through seminal antioxidants; the pathogenic effects of ROS are apparent only when they are produced in excess of the antioxidant capabilities. It is known that the main source of excess ROS generation in semen is leukocytes; genital tract infections are considered to be the most common cause. However, secondary contributors are known to play an important role as well when an infection is not present. The origin of these secondary contributors has yet to be pinpointed in human sperm, but there are many sources under investigation. Three possible sources of excess ROS generation are from within the human sperm itself. The first is through leakage of electrons from the mitochondrial transport chain (Vernet et al., 2001; Balercia et al., 2004; Sharma et al., 2009). This was proposed because of tests performed on rat spermatozoa indicating increased translocation of mitochondrial free radicals into the sperm genome. However, further investigation has demonstrated that mitochondrial blockers do not have the same effects on human spermatozoa (Erenpreiss et al., 2006). The second proposed source is through NADPH oxidase in sperm. This theoretic oxidase would serve to transfer electrons from NAD (P) Hto ground-state oxygen to create the superoxide anion. It is known that NAD (P) H in leukocytes helps to contribute to ROS production in rat spermatozoa, but it has yet to be demonstrated in humans (Vernet et al., 2001; Saleh et al., 2003; Baker and Aitken, 2005; Balercia et al., 2004; Nanda et al., 2010b). The third proposed intracellular source of ROS production is through the generation of Nitric Oxide (NO) (Sanchez-Pena, et al., 2004; Sharma et al., 2009). The NO is a free radical created from the oxidation of L-arginine by 3 isoforms of Nitric Oxide Synthase (NOS). The NOS activity has been shown to be associated with the acrosome reaction and capacitation of mouse sperm, thus influencing their fertilizing potential. In humans, decreased NO concentrations are known to increase sperm capacitation and zona pellucida binding. The exact mechanism of its influence has yet to be elucidated. Other proposed sources of ROS come from outside the sperm’s immediate environment, usually from outside of the host’s body. They include xenobiotic agents such as organophosphorous pesticides that disrupt the endocrine system. These agents possess estrogenic properties that are capable of inducing ROS production by male germ cells (Agarwal and Said, 2005; Baker and Aitken, 2005). Cigarette smoking is also known to increase ROS levels through increased leukocyte generation. Infertile smokers are known to harbor increased levels of spermatid oxidative stress compared with infertile nonsmokers. This increase is associated with increased seminal leukocytes (Love and Kenney, 1999). Finally, scrotal heat stress has been demonstrated in stallions to damage sperm chromatin structure, possibly by oxidative stressors (Sheynkin et al., 2005). Recently similar analyses were performed on humans regarding the use of laptop computers in respect to elevated scrotal temperature (Tesarik et al., 2002). These findings also recognized the elevated temperature of the scrotal environment as having a negative effect upon spermatogenesis, warranting further research. Genetic Lesions Genetic lesions are another possible means of attack through which nDNA can influence male infertility; these lesions create insults or gaps within the genome and may yield effects ranging from minimal to catastrophic. They can be divided into 3 classes based on the type of impact they present (Vogt, 2004). The first class consists of chromosomal aneuploidies and rearrangements in which batteries of genes on specific chromosomes have changes in expression dosages or changes in their normal genomic environments. The second class embodies submicroscopic deletions (microdeletions), in which deletions or rearrangements of multiple genes mapped in a molecular environment have changes in their expression patterns. The third class is made up of single gene defects in which expression of a single gene (or key element) is changed or lost, causing male infertility. These lesions can affect all of the human chromosomes, including any of the 300 genes estimated to be involved in male fertility. They can occur within introns as well as exons, making their impact difficult to predict. Paternal nDNA Effects Prior to analyzing the second type of DNA found in spermatozoa, it is important to establish that nDNA integrity, as it relates to embryo quality, is still an intense topic of discussion. Paternal effects upon the embryo have been classified as both early and late. Early paternal effects appear to be mediated by centrosome destruction or a deficiency in oocyteactivating factors within the spermatozoa, implicating faulty sperm chromatin packaging and nDNA damage (Tesarik et al., 2004). Early effects are observed before the major activation of embryonic genome expression, which begins at the 4-cell stage in humans. Late paternal effects may involve sperm aneuploidy, nDNA damage, or abnormal chromatin packaging, which can influence the orderly activation of paternal gene expression (Kao et al., 1998). It has been found that there is no correlation between sperm nDNA fragmentation and the early paternal effect; however, many Assisted Reproductive Technology (ART) clinics perform embryo transfers on the third day after embryo retrieval, prior to the time when late paternal effects can be fully observed. Because of this fact, blastocyst transfer may be preferable, at the risk of having fewer eggs to transfer. mtDNA The mtDNA of a sperm is completely located in the sperm midpiece; it exists as a circular, double-stranded DNA molecule composed of 16 569 base pairs (St. John et al., 2000a). The genetic information encoded by the mtDNA consists of 2 ribosomal RNAs, 22 transfer RNAs and 13 polypeptides essential for mitochondrial respiration and oxidative phosphorylation associated with the Electron Transport Chain (ETC). The most important function of the sperm mitochondria is to manufacture ATP. The mitochondria itself is composed of 2 distinct membranes, an inner membrane and an outer membrane. The outer membrane is relatively permissive and allows the transit of large molecules through nonspecific porin channels; the inner membrane is much more discriminatory. The inner membrane is heavily invaginated and forms cristae; enzymes for the ETC are located on the inner membrane and the particular nature of inner membrane transport helps to maintain the mitochondrial membrane potential, which drives the ETC (O’Connell et al., 2002a). It is important to remember the differences between mtDNA and nDNA (Ruiz-Pesini et al., 1998). The mtDNA is not afforded the same protection or basic upkeep that nDNA is given. First, there is no protection from histones or DNA-binding proteins within mtDNA; also it lacks introns. Because of this, every mutation in mtDNA has the potential to damage the function of the cell. mtDNA also lacks a significant proofreading system and replicates much more rapidly than nDNA; this causes the mutation rate found in mtDNA to be 10 to 100 times more than that of nDNA. mtDNA Deletions Because of the increased rate of occurrence, mitochondrial deletions have been exclusively investigated; of particular interest has been the relationship to sperm motility and forward progression. Deletions in the mitochondrial genome would directly affect the sperm’s ability to synthesize ATP through the ETC. Direct correlations have been found involving mtDNA deletions and decreased sperm motility (O’Connell et al., 2003). There are 6 distinct respiratory chain complexes that are required for the ETC to function properly. Of them, all but complex II are encoded by the mitochondrial genome; complex II is encoded by the nuclear genome and imported to the inner membrane of the mitochondria (St. John et al., 2001). Dysfunctions in these complexes are considered direct indications of mtDNA deletions. Deletions have been found to fall into 2 categories: small and large scale. While some large-scale deletions appear to be found in fertile men and may be considered common, they are usually associated with spermatozoa with low motility (St. John et al., 2000a; O’Connell et al., 2002a, 2003; Thangaraj et al., 2003). Small-scale deletions, however, can be equally devastating. Deletions as small as 2 base pairs have been proven to insert a stop codon into the mtDNA sequence and truncate vital proteins to ETC function (Rodrigo et al., 2004). It is important to note that no single deletion has been found to be indicative of poor sperm quality (Thangaraj et al, 2003). mtDNA deletions have also been compared to the ages of individuals seeking infertility treatment. Some authors argue that mtDNA deletions accumulate with increased age (O’Connell et al., 2002a), while others have found that there is no significant correlation between the two (St. John et al., 2000b). Epididymal and testicular mtDNA deletions have also been compared, suggesting that testicular sperm may be superior to epididymal sperm for use in ART (O’Cornell et al., 2003), however, recent publications suggest the opposite (O’Cornell et al., 2003; Buffat et al., 2006). Lastly, comparisons have been drawn between the incidences of nDNA deletions in combination with mtDNA deletions. Although results have only come out of a single laboratory, strong correlations between the 2 types of deletions have been found (O’Connell et al., 2002b; Buffat et al., 2006). mtDNA Copy Number The number of mtDNA molecules in a single spermatozoon is known as its mtDNA copy number. mtDNA copy number is controlled by the down-regulation of nuclear-encoded mitochondrial transcription factor A (May-Panloup et al., 2003). Laboratories have been trying to pinpoint the specific mtDNA copy number that is considered normal for fertile men, but have had little success; reported copy numbers for normal, fertile men range from 3.8 in 100% density layers (Kao et al., 2004) to 74.1 6 2.0 in healthy men (Diez-Sanchez et al., 2003) to 717 6 394 in motile spermatozoa (Marchetti et al., 2002). These discrepancies are usually attributed to the method of analysis used or the crosshybridization of mitochondrial pseudogenes found in the nDNA. All reports, however, appear to correlate on 1 important fact: progressive cells possess fewer mtDNA copy numbers than do nonprogressive spermatozoa. Mitochondrial DNA and Apoptosis While nDNA rearrangements can be associated with any number of possible abnormalities causing male infertility; mtDNA injury is most often attributed to the apoptotic pathway. Mitochondrial membrane potential is a measurable factor that has been used to predict the spermatozoa’s risk for apoptosis (Donnelly et al., 2000). There is an ongoing debate over the cause and effect of apoptotic signaling in mitochondria. In other words, does the sperm mitochondria respiratory system contribute to the ROS environment, causing apoptosis, or does the increased ROS environment cause mitochondrial respiratory failure? The first theory is supported by the fact that the mitochondrial respiratory system is a substantial producer of intracellular free radicals that might be able to escape the mitochondria and influence the production of ROS (Sharma et al., 2009; Nanda et al., 2010b). Proposed free radical generation by mitochondrial involvement has been explained by Ozawa’s hypothesis (Nanda et al., 2010b). This hypothesis outlines a viscous cycle whereby mtDNA deletions cause the mitochondria to deprive the sperm of ATP, inducing an energy crisis within the spermatozoa; as the cellular demand for ATP continues, the acceleration of electron leakage associated with ROS generation is increased. This cycle will continue, having catastrophic results and eventually concluding with the spermatozoa entering the apoptotic pathway (Sharma et al., 2009). The latter theory, indicating that the ROS environment causes mitochondrial respiratory failure, is supported by the idea that the appearance of mtDNA damage can be seen before any other indications of the apoptosis pathway (Sutovsky et al., 2004). The first signs of stress induced by increased ROS levels are seen in the disruption of the mitochondrial membrane potential (Donnelly et al., 2000). In addition, structural evidence implies that the location of the mitochondria within the sperm midpiece leaves these structures in closest proximity to the stressors of increased ROS levels (May-Panloup et al., 2003). Because of mtDNA’s lack of DNA protection, this implies that these DNA molecules would be the first to be damaged. Although, both theories have substantial support, further research is necessary to distinguish the cause and effect pathway. Elimination of Paternal Mitochondria by the Egg It is well known that mitochondrial inheritance is of maternal origin (St. John et al., 2001); however, the pathway by which paternal mitochondria are eliminated is still debated. A few existing theories involve paternal mtDNA dilution within the fertilized egg or oxidative damage to the entering paternal mtDNA as possible explanations for maternal mtDNA dominance. The most plausible theory involves the ubiquitination of the paternal mtDNA (Sutovsky et al., 2000). Ubiquitination is a process through which a ubiquitin tag is attached to a protein molecule, imprinting it for destruction. The protein molecule within paternal mitochondria most commonly believed to be ubiquitinated is prohibin (Sutovsky et al., 2000; Hayashida et al., 2005), which is an evolutionarily conserved, 30-kd integral protein of the inner mitochondrial membrane that is expressed during spermatogenesis as well as after fertilization. Studies demonstrate that prohibin is ubiquitinated by the spermatozoa itself and is already destined for destruction before it even fertilizes the egg. Upon fertilization, prohibin would encounter the egg’s cytoplasmic destruction machinery, recognizing the ubiquitin tag and would eliminate the mtDNA. This theory is further supported by the fact that when gel electrophoresis is performed upon mature spermatozoa mtDNA, there exists 3 distinct bands for the protein prohibin: 1 at the predicted 30 kd location and 2 others in the range of 47 to 50 kd, possibly signifying the phosphorylation of the protein in preparation for the attachment of the ubiquitin tag (Hayashida et al., 2005). Recent evidence, however, disputes the ubiquitin tag hypothesis in the elimination of paternal mtDNA (St. John et al., 2000a). A protein known as t-tpis, located in the testis and involved in spermatogenesis (full function unknown), has been given special attention due to its involvement in a vital Tom complex within the mitochondria of spermatozoa. Tom complexes are translocators of the mitochondrial outer membrane. T-tpis is found to be expressed solely in the midpiece of spermatozoa, linking it to possible mitochondrial function. Further investigation has revealed that t-tpis is a protein member of the Tom complex assembled using Tom 22 and 40 complexes; they are known to be required for cell viability and are localized on the cytosolic side of the mitochondrial outer membrane. A potential ‘knob and key-hole model involving t-tpis expression has been proposed as a possible way of paternal mitochondrial recognition and elimination. Contrary evidence of exclusive maternal mitochondrial inheritance comes from abnormal embryos that failed to eliminate paternal mtDNA; however, these embryos frequently fail to develop past the blastocyst stage (Quintana-Murci et al., 2001). In the rare event that paternal mtDNA is observed in adults (Emiliani et al., 2001; Schwartz and Vissing, 2002; Bandelt et al., 2005; Tesarik et al., 2006), recombination events are often attributed to this phenomenon. Nonetheless, it is generally more accurate to consider artificial recombination (i.e., errors in testing) before considering actual recombination events to have occurred. Treatment of sperm DNA for better ART outcomes unfortunately, there is no treatment for mtDNA deficiencies; instead, scientists have focused upon ways in which to isolate sperm with improved nDNA status, as well as selecting better sperm for ART use to generate better ART outcomes. The first method of treatment involves cessation of all activities that are known to be harmful to the production of healthy sperm; this includes smoking and exposure to possible environmental estrogens, such as pesticides (Agarwal and said, 2003; Sharma et al., 2009; Mishra et al., 2009; Nanda et al., 2010 b). This is generally accompanied by oral antioxidant treatment at least 2 months prior to ART treatment in an attempt to minimize oxidative stress (Martinez et al., 2002). Another suggested line of treatment is the use of surgically retrieved testicular spermatozoa instead of epididymal sperm. The reason to use testicular sperm is to minimize sperm with fragmented nDNA and obtain specimens with better mtDNA for use with in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) procedures (Martinez et al., 2002). However, recent evidence suggests the exact opposite, indicating epididymal sperm to be superior to testicular sperm for ICSI outcome (Rodrigo et al., 2004; Buffat et al., 2006). Also, a high-magnification optical system can be used to select better spermatozoa for ICSI. In this manner, spermatozoa can be selected by visualizing morphology under conditions not possible with normal laboratory equipment. Subtle morphologic abnormalities become visible under this high magnification (66006) that cannot be seen under normal high power objectives (4006), allowing the embryologist to select better sperm for ICSI fertilization (Emiliani et al., 2001; Tesarik and Mendoza, 2007). Other ways to improve sperm nDNA include enhanced preparation techniques. This involves lowering the centrifugal forces exerted on the sperm when concentrating it and removing leukocytes as quickly as possible from the sample. Also the swim-up technique can be used to avoid use of the centrifuge. It is postulated that the addition of sperm wash medium to raw semen prior to liquefaction may inhibit bacterial binding to the sperm surface as well as diminish nDNA damage caused by ROS. Oddly, in vitro culture of surgically retrieved testicular spermatozoa for 48 to 72 h at 37°C has been suggested to improve motility, along with decreasing the proportion of spermatozoa containing single-stranded nDNA breaks (Huszar et al., 2003). Very recently, a novel sperm selection assay has been proposed to select viable sperm free of chromosomal anomalies for use with ICSI. Sperm Hyaluronic Acid (HA) binding has demonstrated the ability to isolate mature, viable sperm with unreacted acrosomal status, without damaging the specimen (Jakab et al., 2005; Nanda et al., 2010a, b). One principle of this assay lies in the expression of the chaperone protein HspA2; in spite of its key role in meiosis, HspA2 levels have become indicative of sperm maturation (Huszar et al., 2006). Low levels of HspA2 expression are associated with diminished sperm maturity, increased frequency of chromosomal aneuploidies, presence of apoptotic processes and fragmented nDNA. The second principle involved takes into account remodeling of the cytoplasmic and membrane-specific biochemical markers, facilitating the formation of sperm binding sites for the zona pellucida of oocytes and for the binding sites of HA. Immature sperm that fail to undergo membrane remodeling are unable to bind to immobilized HA and will not be selected in this assay (Huszar et al., 2006). Chromosomal disomies are said to be reduced between fourfold and fivefold in HA-selected sperm compared with semen sperm (Ye et al., 2006) reflecting that HA preferentially selects for chromosomally normal sperm. Because of such promising results, a kit for this assay has become commercially available. The sperm Hyaluronan Binding Assay (HBA) has been marketed for routine testing of sperm motility and fertility (Nasr-Esfahani et al., 2008). Unfortunately, HBA results have fallen well short of expectations in predicting successful fertilization rates in IVF, demonstrating less significance than sperm morphology and limiting its clinical predictive value. Further research is needed to perfect techniques that will be employed to improve the quality of spermatozoa that are selected for use in all ART techniques to improve outcomes.
ACKNOWLEDGMENTS
The authors are grateful to Prof. R.M. Dubey (Managing Director) and Prof. A. Srivastav (Director), CET, IFTM Campus, Moradabad, U.P., India) for providing the necessary facilities and encouragement. The author, SN, is a Ph.D. scholar and registered at U.P. Technical University, Lucknow, India.