Review Article
Open Access
Sperm DNA Links from Sperm to Ovum to Implant
Genetic Changeability: an Overview
Sanjay Mishra1*, Amit Kumar Mani Tiwari1, Ram B. Singh2 and Abbas A. Mahdi3
1Department of Biotechnology, IFTM University, Delhi Road (NH 24), Moradabad 244102, Uttar Pradesh, India
2Halberg Hospital and Research Center, Civil Lines, Moradabad 244001, Uttar Pradesh, India
3Department of Biochemistry, King George’s Medical University, Lucknow-226 003, Uttar Pradesh, India
2Halberg Hospital and Research Center, Civil Lines, Moradabad 244001, Uttar Pradesh, India
3Department of Biochemistry, King George’s Medical University, Lucknow-226 003, Uttar Pradesh, India
*Corresponding author: Sanjay Mishra, Professor, Department of Biotechnology, IFTM University, Delhi Road (NH 24), Moradabad 244 102, UP, India.E-mail:
@
Received: March 18, 2018; Accepted: April 03, 2018; Published: April 10, 2018
Citation: Sanjay M, Tiwari AKM, Singh RB, Abbas AM (2018) Sperm DNA Links from Sperm to Ovum to Implant Genetic Changeability: an Overview. SOJ Gynecol Obstet Womens Health 4(1): 1-10. DOI: http://dx.doi.org/10.15226/2381-2915/4/1/00132
AbstractTop
Sperm DNA disintegration is known to cooperate male fertility. The
data bring about that sperm DNA cleavage can be competently treated
with orally administered antioxidant during a relatively short moment
period. It is an accepting thought that the two types of DNA become
visible to be distinctive and autonomously packaged molecules;
though, investigation has established the symbiotic nature of these
structures in contributing to male infertility. Data should continue to be
gathered to ascertain strong correlation between conventional semen
examination parameters and sperm DNA integrity; this information
remains contentious and inadequate in clinical practice until certain
novel techniques for the diagnosis and treatment of sperm DNA can
be established. Rather more modern technology should be employed
to associate such information into practical clinical awareness. This
overview compiles certain specific reports pertaining to studies
on sperm DNA physiology, biochemistry and molecular biology to
provide further new insights into establishing hypothesis that sperm
DNA integrity is both enormously fragile and remarkably significant
for male fertility.
Keywords: Apoptosis; Male infertility; Sperm DNA
Keywords: Apoptosis; Male infertility; Sperm DNA
Introduction
Sperm DNA disintegration is known to cooperate male
fertility. The data bring about that sperm DNA cleavage can
be competently treated with orally administered antioxidant
during a relatively short moment period. [1,2]. According to
these reports, the rise of DNA fragmentation index (DFI) level
can not only reduce the sperm motility but also affect the
clinical outcomes of in vitro fertilization (IVF) and intracyto
plasmic sperm injection (ICSI). While the outcomes of IVF and
ICSI are statistically similar when DFI level is less than 15%, the
curative effect of ICSI is better when DFI value exceeds 15%. An
upgrading of basic sperm parameters by oral administration with
antioxidants has been documented in a number of investigations,
but DNA break has been reported in only a few of them [3,4-6].
Infertility is a rising 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. Investigation now results
that male infertility may add in up to two thirds of all couples
who look for treatment to overcome infertility. For several years
a conventional semen analysis (concentration, motility, and
morphology) has been noticed as insufficient to diagnose male
infertility; however, scientific examination must now take into
account two different kinds of DNA that have been proven to be
worthwhile to this significant diagnosis. Nuclear DNA (nDNA),
enclosed in the head of the sperm, is accountable for packaging
all of the paternal genetic information, which will be needed
for the fertilized egg. nDNA can be damaged or compromised
through 4 interrelated courses: apoptosis, defective chromatin
packaging, oxidative stress, and genetic lesions. Mitochondrial
DNA (mtDNA) is cited in the mid piece of the sperm; as coupled
with the tail, it is liable for mobilizing the sperm toward the ovum
for fertilization. Scientists in relevance are only opening to figure
out the correlation between these distinct DNA molecules and
how they both contribute to male infertility. As the worldwide
community continues to spread out, an emerging subpopulation
of couples has started experiencing a general problem in
enabling their contribution to the population. 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 [7.8]. 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 increasing [9,10]. In infertile couples, liability for
the deficiency of conception is usually 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 the partners.
Application of Nutraceuticals in Sperm Creation
Nutraceuticals have been recognized as a way of potentiating
sperm production and quality in the sub-fertile male. Recently,
the administration of folic acid and zinc sulfate to sub-fertile
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 mg and 66 mg,
respectively. Although the beneficial effect on fertility remnants
to be recognized, this finding opens new avenues of future
fertility research and treatment [11]. Arginine, vitamin B12,
methylcobalamin and ginseng have been used in the treatment
of male infertility [11,12]. Though, most of these compounds
have minor effects on sperm production and quality and have not
been tested for safety and effectiveness in randomized placebocontrolled
studies [13]. In fact, ginseng has been shown to have
estrogenic activity and produce adverse reactions [13]. Because
ROS overproduction has been associated with defective sperm
function , infertile patients have been treated with antioxidant
compounds including, ascorbic acid, vitamin E, selenium,
glutathione [14,15-19]. However, the effect of this treatment
on sperm quality is still controversial [4]. In a randomized,
placebo-controlled, double-blind investigation, oral high doses
of vitamins C and E to infertile males could not show any
statistically significant improvement in semen parameters [4].
Although, in this study, patient recruitment was solely based on
having a sperm concentration less than 50 million/ml, and, thus
studies looking at the effectiveness of antioxidant therapy in the
treatment of male infertility have not yet been decisive may be
due to insufficient patient selection. Not all infertile males have an
augment in oxidative stress in their testis and semen. Therefore,
principally, only those men who have an experimental increase
in oxidative stress should gain from antioxidant therapy. Possibly
the best marker to identify these males would be reactive oxygen
species (ROS) levels in semen [20-22]. 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 [6,22].
Major Types of Sperm DNA and Fertilization
The foundation of the evaluation of the human remnants
semen analysis. Though it gives some quantitative and qualitative
information about the sperm sample, latest insight into the
physiology, biochemistry and molecular biology of the sperm cell
have demonstrated that morphology and motility alone are not
the only basis upon which sperm should be analyzed [23]. Over
the last years, major improvements in the field of male infertility
diagnosis have been achieved. The diagnostic usefulness of sperm
DNA integrity and sperm vacuolization for predicting outcome in
infertile couples undergoing in vitro fertilization (IVF) and
intracytoplasmic sperm injection (ICSI) treatments [23]. A cohort
study from 152 infertile couples undergoing sperm DNA
fragmentation and high-magnification tests prior to an assisted
reproduction treatment was designed. The most predictive cutoff
for pregnancy has been observed to be 25.5% of DNA
fragmentation with a negative predictive value of 72.7% (P=0.02).
For the degree of vacuolization, the best predictor of pregnancy
was 73.5% of vacuolated sperm grades III+IV with a negative
predictive value of 39.4% (P=0.09), which was not statistically
significant [23]. Consequently, sperm DNA fragmentation greater
than 25.5% could be associated with higher probability of failure
IVF treatment. Regarding the results of the sperm analysis at high
magnification, they do not allow us to predict whether or not
patients will become pregnant [23]. Generally unnoticed 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
[8]. The second DNA type is called the mtDNA and is responsible
for delivering the sperm to the egg by providing ATP for cellular
acceleration [22]. 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 [22].
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 two loops of wrapped
DNA. The nucleosomes are then further coiled into regular helixes
called solenoids, which increase the volume of the chromatin
[22,24]. 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 [24,25]. 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 [22]. The arrangement of these loop
domains ascertains that the DNA can be delivered to the ovum 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, super coiled dough- nuts called toroids by
replacing the nuclear histones with structures called protamines
completes the levels of chromosomal organization. Human sperm
contain two major types of protamines, which are about half the
size of typical histones; throughout evolution, they have
augmented the number of positively charged residues, allowing
formation of a highly condensed complex with the negatively
charged paternal genomic DNA. Besides, the addition of cysteine
residues allows the formation of disulfide bonds between
adjacent protamines molecules, thereby strongly stabilizing the
nucleo-protamine complex [22, 26-28]. Prior to this re
arrangement, recombination is essential for spermatogenesis to
occur [22, 26-28]; as seen in studies with animal knockout
models, lowering recombination is associated with diminished
spermatogenesis. Several factors (both endogenous and
exogenous) can influence this, contributing to male infertility.
Scientists agree on 4 principal techniques, although there may be
others, by which nDNA can be compromised or damaged:
defective sperm chromatin packaging, apoptosis, oxidative stress,
and genetic lesions [21,22,24,29,30]. 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 ovum. Though defects can come up at
any of the 4 levels of packaging, the most general problems occur
during DNA loop domain formation and histone-protamine
replacement. nDNA loop domains can be difficult to organize
without inducing endogenous nicks to the nDNA [22,31]. It is
contemplation that these nicks survive naturally and provide to
relieve torsional stress. The presence of these nicks is maximum
during transition from round to elongated spermatids in testis
and occurs before complete protamination within the sperm.
Topoisomerase II is a specific enzyme, which creates instantly
and ligates the nicks within nDNA during this process [22,32].
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 augment the levels of internal nDNA breaks by preventing their
repair and increasing their susceptibility to damage [29]. Also
involved in sperm chromatin packaging is the replacement of
histones with protamines. Protamines are major DNA-binding
proteins essential for chromatin condensation [31,33]. During
epididymal transport, histones are replaced by transition
proteins, only to be replaced by protamines [26]; both
intermolecular and intra molecular disulfide cross-linking among
the cysteine-rich protamines compresses the DNA into one sixth
the volume occupied by somatic cell nDNA [22,34]. This high rate
of cross-linking affords the sperm nDNA a measure of defense
against exogenous assault and compensates for an impaired
DNA-repair capability. Human spermatozoa preserve
approximately 15% histones in their native structure, leading to a
less compact chromatin arrangement compared to other
mammals [35,36], perhaps to allow access for oocyte repair
mechanisms. Human spermatozoa contain 2 different types of
protamines that are thought to be present in equal amounts in
fertile men: P1 and P2 [27,36]. The ratio of P1 to P2 is critical to
male fertility [22,26,27,33,35], more particularly to the sperm’s
fertilization ability [36]. Besides, recent analysis has confirmed
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, decreased sperm motility, and
male infertility [37]. Infertility and problems of impaired
fruitfulness have been a concern through ages and is also a
noteworthy clinical problem in modern era, ultimately affecting
about 10% of couples globally. Of all infertility cases, around 40–
50% are due to “male factor” infertility and as many as 2% of all
men will show suboptimal sperm parameters. It may be one or a
combination of low sperm concentration, poor sperm motility, or
abnormal morphology. The rates of infertility in less industrialized
nations are noticeably higher and infectious diseases are
accountable for a greater proportion of infertility [37]. Moreover,
a low pre- P2: P2 ratio suggests a link between deficient
protamines processing and decreased nDNA integrity [37,38].
These reports are thought to The present literature will help in
knowing the trends of male factor infertility in developing nations
like India and to find out in future, various factors that may be
responsible for male infertility. Apoptosis is the controlled
disassembly of a cell from within [19]; it is believed to have 2
roles during normal spermatogenesis [21,22,39,40]. 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 phosphatidyl serine (PS) relocation, Fas
expression, nDNA strand breaks, and capsize 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
[32,41]. 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 [40,42].
Bcl-2 and Bcl-xL are both pro survival proteins, while Bax is a proapoptotic
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 [32,43]; 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 [35,44]. Ligation of Fas ligand to
the Fas receptor triggers activation of cytosolic aspartate-specific
proteases, or simply capsizes. Once capsize 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 [24]. 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’’ [29,35,45-47]. 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 [24,48].
It is believed that if the apoptotic cascade is initiated at the round
spermatids 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 [48,49]. 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 [30,50].
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 spermatids [30]. 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
[51]. 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 [52,53]. 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 [53,54]. ROS are created by metabolizing ground-state
oxygen into the superoxide anion and H2O2 [55,56]. 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 [56-
59]. 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 [35]. The
second proposed source is through NADPH oxidase in sperm.
This theoretic oxidase would serve to transfer electrons from
NAD(P)H to 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 [51,52,54,57,60] The third proposed
intracellular source of ROS production is through the generation
of nitric oxide (NO) [19,61,62]. NO is a free radical created from
the oxidation of L-arginine by 3 iso forms of nitric oxide synthase
(NOS). 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 organ
ophosphorous pesticides that disrupt the endocrine system.
These agents possess estrogenic properties that are capable of
inducing ROS production by male germ cells [52,63,64]. Cigarette
smoking is also known to increase ROS levels through increased
leukocyte generation. Infertile smokers are known to harbor
increased levels of spermatids oxidative stress compared with
infertile nonsmokers. This increase is associated with increased
seminal leukocytes [65]. Finally, scrotal heat stress has been
demonstrated in stallions to damage sperm chromatin structure,
possibly by oxidative stressors [66]. Recently similar analyses
were performed on humans regarding the use of laptop computers
in respect to elevated scrotal temperature [67]. 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 [8,33]. 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 (micro deletions), 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 oocyte activating factors within the spermatozoa,
implicating faulty sperm chromatin packaging and nDNA damage
[68,69]. 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 [70].
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. The mtDNA of a sperm is completely
located in the sperm mid piece; it exists as a circular, doublestranded
DNA molecule composed of 16569 base pairs [71-73].
Severe asthenozoospermia is one of the leading causes of male
infertility as spermatozoa cannot reach the oocyte and/or
penetrate normally. Identifying structural causes of sperm
immotility was of great concern before the advent of
intracytoplasmic sperm injection (ICSI), because immotility was
the limiting factor in the treatment of these patients. In these
cases, in vitro methods are used to identify live spermatozoa or
stimulate sperm motility to avoid selection of non-viable cells.
With these advances, fertilization and pregnancy results have
improved dramatically. The identification of genetic phenotypes
in asthenozoospermia is important to adequately inform patients
of treatment outcomes and risks. The one sperm characteristic
that seriously affects fertility prognosis is teratozoospermia,
primarily sperm head and neck anomalies. Defects of chromatin
condensation and acrosomal hypoplasia are the two most
common abnormalities in severe teratozoospermia. The
introduction of microscopic methods to select spermatozoa and
the development of new ones to evaluate sperm quality before
ICSI will assure that ultra structural identification of sperm
pathologies will not only be of academic interest, but will also be
an essential tool to inform treatment choice [73]. 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 significant 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 that drives the ETC [74]. It is important to remember
the differences between mtDNA and nDNA [75]. mtDNA is not
afforded the same defense or basic upkeep that nDNA is given.
First, there is no protection from histones or DNA-binding
proteins within mtDNA; moreover it lacks introns. Because of
this, every mutation in mtDNA has the potential to damage the
function of the cell. mtDNA also lacks an important proofreading
system and replicates much more rapidly than nDNA; this results
in the mutation rate created in mtDNA to be 10 to 100 times more
compared to that of nDNA. Deletions in the mitochondrial genome
would directly affect the sperm’s capability to synthesize ATP
through the ETC. Direct correlations have been noticed to involve
mtDNA deletions and decreased sperm motility [76,77]. There
are six different 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 [76,78]. 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 [74,76,79,80] Small-scale
deletions, on the other hand, can be equally disturbing. Deletions
as small as two base pairs have been proven to insert a stop codon
into the mtDNA sequence and shorten vital proteins to ETC
function [81,82]. It is important to note that no single deletion
has been found to be indicative of poor sperm quality [79]. mtDNA
deletions have also been compared to the ages of individuals
looking for infertility treatment. Some authors discuss that
mtDNA deletionsgather with increased age [74], while others
have registered that there is no noteworthy correlation between
the two [71,72]. Epididymal and testicular mtDNA deletions have
also been compared, signifying that testicular sperm may be
superior to epididymal sperm for use in ART [76], however,
recent publications suggest the opposite [76,83]. Finally,
comparisons have been drawn between the incidences of nDNA
deletions in combination with mtDNA deletions. Though results
have only come out of a single laboratory, strong correlations
between the two types of deletions have been observed [83-85].
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 [70,86-88]. These data are
usually attributed to the technique of analysis used or the crosshybridization
of mitochondrial pseudo genes found in the nDNA.
All reports, however, appear to correlate on an important fact:
progressive cells acquire fewer mtDNA copy numbers compared
to non-progressive spermatozoa [89]. There is an ongoing debate
over the cause and effect of apoptotic signaling in mitochondria
reflecting whether the sperm mitochondria respiratory system
contributes to the ROS environment, causing apoptosis, or
increased ROS environment results in mitochondrial respiratory
failure. The first hypothesis 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 [19,22,51].
Proposed free radical generation by mitochondrial involvement
has been explained by Ozawa’s hypothesis [22,51]. This
hypothesis outlines a ‘‘viscous cycle’’ while mtDNA deletions
cause the mitochondria to remove ATP from the sperm, inducing
an energy crisis within the spermatozoa; as the cellular demand
for ATP continues, the acceleration of electron leakage connected
with ROS generation is increased. This cycle is likely to continue,
having catastrophic results and eventually concluding with the
spermatozoa entering the apoptotic pathway [19]. The latter
hypothesis, signifying that the ROS environment leads to
mitochondrial respiratory failure, is supported by the thought
that the appearance of mtDNA damage can be seen before any
other indications of the apoptosis pathway [19,90]. The first signs
of stress induced by increased ROS levels are seen in the
distraction of the mitochondrial membrane potential [89,91].
Besides, 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 [87].
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 [78,87,91]; on the
other hand, the pathway by which paternal mitochondria are
eliminated is still debated. A few existing hypotheses involve
paternal mtDNA dilution within the fertilized egg or oxidative
damage to the entering paternal mtDNA as probable explanations
for maternal mtDNA dominance. The most reasonable theory
involves the ubiquitination of the paternal mtDNA [87,92].
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
thought to be ubiquitinated is prohibin [91-93], which is an
evolutionarily conserved, 30-kd integral protein of the inner
mitochondrial membrane, 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 remove the
mtDNA. This hypothesis is further supported by the fact when gel
electrophoresis is performed upon mature spermatozoa mtDNA,
there exists three different bands for the protein prohibin: 1 at
the predicted 30-kd location and 2 others in the range of 47 to 50
kD, probably suggesting the phosphorylation of the protein in
preparation for the attachment of the ubiquitin tag [93,94]. Latest
evidence, though, disputes the ubiquitin tag hypothesis in the
elimination of paternal mtDNA [71,72]. A protein known as t-tpis,
located in the testis and involved in spermatogenesis (complete
function still obscure), has been given special focus for the cause
of its involvement in a vital Tom complex within the mitochondria
of spermatozoa. Tom complexes are translocations of the
mitochondrial outer membrane. t-tpis is found to be expressed
solely in the mid-piece of spermatozoa, connecting it to probable
mitochondrial function. Further investigation has revealed that
t-tpis is a protein member of the Tom complex assembled using
Tom 22 and Tom 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
probable way of paternal mitochondrial recognition and
elimination. Contrary substantiation of exclusive maternal
mitochondrial inheritance comes from abnormal embryos, which
failed to eliminate paternal mtDNA; never-the-less, these embryos
frequently fail to develop past the blastocyst stage [95]. In the
rare event that paternal mtDNA is observed in adults [96-100],
recombination events are often attributed to this phenomenon.
Nonetheless, it is generally more accurate to consider artificial
recombination (ie, errors in testing) before considering actual
recombination events to have occurred. Treatment of Sperm DNA
for better ART outcomes. Regrettably, 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 technique of treatment involves cessation of
all activities that are recognized to be harmful to the production
of healthy sperm; this includes smoking and exposure to probable
environmental estrogens, such as pesticides [15,19,22,24,51].
This is in general accompanied by oral antioxidant treatment at
least two months prior to ART treatment in an effort to minimize
oxidative stress [22,49]. One more suggested line of treatment is
the use of surgically retrieved testicular spermatozoa as an
alternative of epididymal sperm. The motive to use testicular
sperm is to minimize sperm with fragmented nDNA and acquire
specimens with better mtDNA for use with in vitro fertilization
(IVF) and intracytoplasmic sperm injection (ICSI) procedures
[22,49,101]. However, latest evidence suggests the exact opposite,
indicating epididymal sperm to be superior to testicular sperm
for ICSI outcome [81,83]. Never-the-less, a high-magnification
optical system can be employed to sort better spermatozoa for
ICSI. In this way, 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), permitting the
embryologist to select better sperm for ICSI fertilization
[22,96,100,101]. Other ways to upgrade sperm nDNA include
enhanced preparation techniques. This involves reducing the
centrifugal forces exerted on the sperm while concentrating it
and eliminating leukocytes as rapidly as possible from the sample.
Besides, the swim-up technique can be employed to avoid use of
the centrifuge. It is hypothesized that the supplementation 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 hours at 37 0C has
been recommended to improve motility, along with decreasing
the proportion of spermatozoa containing single-stranded nDNA
breaks [22,102]. 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
un reacted acrosomal status, without damaging the specimen
[22,51,101,103]. One principle of this assay lies in the expression
of the chaperone protein HspA2; in spite of its pivotal role in
meiosis, HspA2 levels have become physiological and biochemical
markers of sperm maturation [22,101,104]. Low levels of HspA2
expression are connected with diminished sperm maturity,
increased frequency of chromosomal aneuploidies, presence of
apoptotic processes, and fragmented nDNA. The second principle
involved considers remodeling of the cytoplasmic and membranespecific
physiological and biochemical markers, facilitating the
formation of sperm binding sites for the zona pellucida of oocytes
and for the binding sites of HA. Immature sperm, which fail to
undergo membrane remodeling are unable to bind to immobilized
HA and thus not likely to be selected in this assay [22,101,104].
Chromosomal disomies are said to be reduced between fourfold
and fivefold in HA-selected sperm compared with semen sperm
[22,51,101,105] reflecting that HA preferentially selects for
chromosomally normal sperm. Because of such fascinating
consequences, a kit for this specific assay has become
commercially available. The sperm-hyaluronic binding assay
(HBA) has been marketed for routine testing of sperm motility
and fertility [101,106,107]. Regrettably, HBA data have been
observed quite inadequate of expectations in predicting
successful fertilization rates in IVF, expressing rather less
significance compared to sperm morphology and limiting its
clinical predictive worth. Besides, comprehensive research is
required to upgrade the existing protocols so that to improve the
quality of spermatozoa likely to be sorted out for fulfilling the
purpose of majority of ART techniques to elevate outcomes.
Acknowledgements
The authors are grateful to Prof. A.K. Ghosh, Vice Chancellor,
IFTM University, Moradabad, U.P., India for providing the
necessary facilities and time-to-time encouragement.
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