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REVIEW
Potential implications of sperm DNA methylation functional
properties in aquaculture management
Songpei Zhang
1
| Yu Cheng
1
| Pavlína Vˇ
echtová
2
| Sergii Boryshpolets
1
|
Nururshopa Eskander Shazada
1
| Sayyed Mohammad Hadi Alavi
1,3
| Jacky Cosson
1
|
Otomar Linhart
1
1
University of South Bohemia in Ceske
Budejovice, Faculty of Fisheries and
Protection of Waters, South Bohemian
Research Center of Aquaculture and
Biodiversity of Hydrocenoses, Research
Institute of Fish Culture and Hydrobiology,
Vodnany, Czech Republic
2
University of South Bohemia in Ceske
Budejovice, Faculty of Science, Institute of
Chemistry and Biochemistry, Ceske
Budejovice, Czech Republic
3
School of Biology, College of Science,
University of Tehran, Tehran, Iran
Correspondence
Yu Cheng and Otomar Linhart, University of
South Bohemia in Ceske Budejovice, Faculty
of Fisheries and Protection of Waters, South
Bohemian Research Center of Aquaculture and
Biodiversity of Hydrocenoses, Research
Institute of Fish Culture and Hydrobiology,
Zatisi 728/II, 38925 Vodnany, Czech Republic.
Email: ycheng@frov.jcu.cz and linhart@frov.
jcu.cz
Funding information
Grantová Agentura ˇ
Ceské republiky,
Grant/Award Number: 20-01251S; Jihoˇ
ceská
Univerzita v ˇ
Ceských Budˇ
ejovicích,
Grant/Award Numbers: 097/2019/Z,
037/2020/Z; Národní Agentura pro
Zemˇ
edˇ
elský Výzkum, Grant/Award Number:
QK21010141; Ministry of Education, Youth
and Sports of the Czech Republic LRI
CENAKVA, Grant/Award Numbers:
LM2018099, CZ.02.1.01./0.0/0.0/16_025/
0007370; China Scholarship Council,
Grant/Award Numbers: 201908160003,
202108160002
Abstract
The biology of fish sperm, an important topic in the research of reproduction and
fisheries from basic science to evolutionary and applied aspects, has implications for
aquaculture management, fish breeding and biological conservation. The quality of
spermatozoa plays a vital role in fish fertility, and directly affects the health and per-
formance of offspring. Environmental factors that affect determinants of spermato-
zoa quality usually reflected in the DNA methylation pattern of the spermatozoa
epigenome, which may change offspring's performance. The aim of the present study
was to conduct a review on existing data about DNA methylation in fish spermatozoa
as a biological tool for identifying the quality of offspring based on their phenotypes
and performances. Furthermore, this study provides valuable knowledge from funda-
mental to applied sciences dealing with enhancement of breeding selection, fish
reproduction and environmental adaptation. The review also describes the individual
parts related to DNA methylation in fish, including overview of the methods which
can be used to study DNA methylation, DNA methylation dynamics and epigenetic
inheritance; identification of DNA methylation changes in sperm function in response
to internal and external environment constraints, and potential relationships between
DNA methylation and physiological regulation of spermatozoa quality determinants.
Overall, the present study revealed that our knowledge about intergenerational
inheritance of the performance and adaptability of fish through sperm DNA methyla-
tion is very limited, and no general conclusion could be approached from literature
mostly due to non-standardized experimental protocol or analytical tools.
KEYWORDS
environment factors, fish, imprinting, spermatozoa quality, transgenerational inheritance
1|INTRODUCTION
Along with the rapid development and alterations of aquaculture,
there are more opportunities and challenges in breeding selection,
livestock management and farming optimization. Fertilization success,
embryo quality and performance of the offspring are highly depen-
dent on the integrity of the gametes. Gametes of sexually reproducing
organisms carry hereditary materials for the development of the next
generation. The epigenetic elements including DNA/RNA methylation,
histone modification, non-coding RNAs and chromatin structure make
Received: 5 May 2022 Revised: 1 August 2022 Accepted: 21 August 2022
DOI: 10.1111/raq.12735
Rev Aquac. 2022;1–21. wileyonlinelibrary.com/journal/raq © 2022 John Wiley & Sons Australia, Ltd. 1
up the epigenome.
1
Epigenetics investigates the heritable phenotypic
traits in the offspring without changes in DNA sequencing, and pro-
vides new insights into the role of genome methylation as an impor-
tant mechanism that mediates an imprint of parent-of-origin effect in
combination with environmental stimuli.
2–5
As the comprehensively
studied epigenetic indictors so far, DNA methylation is catalysed by
the enzymes of DNA methyl transferases (DNMTs) and ten-eleven
translocations (TETs), which medicated on cytosine base including
CpG, CHG and CHH context (where H is either A, C or T). The enrich-
ment/depletion of involved ncRNA, histone modifications and DNA
methylation occurs at developmental loci, where they are noted in a
conspicuous way in genes associated with meiosis and spermatogene-
sis.
6,7
Labbé et al.,
8
Granada et al.
9
and Han et al.
10
greatly summa-
rized that DNA methylation get involved in gene expression and
maintenance of gene activity patterns by regulating the transcription
factor binding and the chromatin structure during gametogenesis and
embryogenesis in non-mammalian vertebrates including fish
(Figure 1a,b). Yet this does not seem to be sufficient for interpreting
alterations in response to environmental stimuli.
Extending knowledge about the importance of DNA methylation
for the organism's development has raised concerns about the effect
of environmental factors on gametes during gametogenesis and
spawning. Seemingly non-hereditary environmental factors and life-
style of an individual are reflected in the DNA methylation pattern of
the sperm genome that has shown to result in phenotypic changes in
the next generations.
11–16
The evident correlation of these phenom-
ena opened new questions regarding the impact of assisted reproduc-
tive techniques (ART) with particular consideration to the possible
negative impact of ARTs in human reproduction, and also on the
potential incidence of developmental and phenotypic abnormalities in
ART-conceived children.
17–19
Similar concerns have been also equally
recognized in other model organisms such as reproduction of live-
stock, poultry or fish.
8,20,21
In fish with external fertilization, variations in the properties of
the aquatic environment, where the sperm and oocyte meet upon fer-
tilization, not only influence gamete activation and fertilization suc-
cess, but also may cause another degree of variations to their
epigenome that eventually alter the phenotype of the resultant off-
spring.
22,23
These considerations in combination with maternal and
paternal effects were addressed as factors that modulate qualitative
parameters of the offspring in a natural fish population.
24
Additionally,
some studies have reported variation between artificially bred and
FIGURE 1 (a) DNA methylation involved in meiotic division during spermatogenesis and during zygotic genome activation modified from Han
et al.
10
(b) Comparative alterations of DNA methylation in spermatozoa prior to fertilization and in zygote during embryonic development in
zebrafish (Danio rerio), medaka (Oryzias latipes) and mouse obtained from Jiang et al.,
42
Potok et al.,
43
Wang et al.
44
and Wang et al.
45
2ZHANG ET AL.
natural populations, which affects genome methylation and thus influ-
ences the phenotypic features of the offspring.
25–31
Investigating the
above-mentioned phenomena is of crucial importance for fish repro-
duction and selection, where both high quality and quantity of off-
spring are the main indicator of successful breeding.
In aquaculture, fertilization, embryo development and progeny
performance are the candidate measures in broodstock management
that are most likely to be affected by the environmental conditions in
which sperm–oocyte communication takes place and fertilization
occurs.
32,33
Several studies have demonstrated that the spermatozoa
competitive capacity significantly influences fertilization success and
offspring fitness.
34–36
These studies suggest that a multitude of inter-
nal and external factors, such as environmental pollutants, diet, cryo-
preservation, temperature, salinity and oxidative stress. Capable of
altering the DNA methylation pattern of sperm was trans-
generationally inherited into the next generation.
Extensive studies of human epigenetics have demonstrated
ARTs-induced alterations of the DNA methylation pattern.
17,18,37
Analogous effects can also be expected in artificial reproduction in
the fish hatchery. Although concerns regarding the DNA methylation
stability in spermatozoa and offspring during artificial breeding have
already been addressed in fish,
38–41
exact mechanisms driving the
changes in genome methylation and their further manifestation in the
offspring's phenotype remain poorly understood. The aim of the pre-
sent review was to summarize existing data about DNA methylation
in fish spermatozoa, and to investigate the impacts of phenotypic
changes on the quality of offspring. This study provides valuable infor-
mation for undertaking research dealing with broodstock management
in aquaculture with particular consideration to breeding selection and
production.
2|OVERVIEW OF METHODS USED TO
STUDY DNA METHYLATION
Awareness of epigenetic-inherited biological features such as pheno-
type and adaptability has been increasing in living organisms over the
past decades. It follows the mechanisms that form and influence the
epigenome and its dynamics, as well as their effects on the associated
cellular processes. Accordingly, the repertoire of methods for charac-
terization and quantification of DNA methylation have been
increasing.
Up to now a plethora of different techniques studying DNA
methylation has been developed. These techniques are essentially cat-
egorized according to the way the 5-methylcytosines (5mCs) are dis-
tinguished from unmethylated Cs and then by the methods used for
methylation readout. An overview of these methods along with their
principles and applications have been extensively discussed.
46–50
Compared with currently mainstream methods of next generation
sequencing, Whole Genome Bisulphite Sequencing (WGBS) is the
most comprehensive and unbiased survey of all available methods. It
is based on single base resolution that can identify much higher CpG
density of genome revealing differentially methylated regions
precisely.
47,51
The short reads, however, have limitations in mapping
to repeated sequences, haplotyping and special sites.
48
Therefore,
whole genome reference sequence better compensates for its
drawbacks.
The selection of an appropriate method for studying DNA meth-
ylation, however, depends largely on the biological question of the
respective study.
47
Above all, the extent and resolution of DNA meth-
ylation to be analysed have to be taken into consideration. Other
important aspects include sensitivity, specificity and reproducibility of
the method, methodology complexity and costs or availability of
instrumentation (see Table 1).
Simultaneously, the application of new methods, such as third
generation sequencing and single-cell sequencing may provide more
strategies and insights for the methylation study.
52–55
However, none
of the existing methods provide an ideal combination of these param-
eters and the selection of appropriate assay to meet the research plan
usually involves a compromise. However, using the designated meth-
odology and careful data interpretation, many investigators in the field
of fish biology were able to successfully harness a wide range of tech-
niques for studying DNA methylation. A list of most popular tech-
niques currently used for methylation analysis in fish along with
references for studies using them was summarized in Table 1.
The focus of studies dealing with fish epigenetics is largely biased
by the major driver in fish aquaculture. The most attractive research
concentrates on the changes in fish breeding management and aqua-
culture production. Epigenetic changes are known as mediators of
environmental adaptations. Thus, the majority of fish aquaculture
studies aimed at describing DNA methylation changes induced by
aquaculture conditions and the effects of artificial breeding on the
epigenome of fish gametes in hatchery. Altered DNA methylation may
very well be inherited by the developing embryo and possibly affect
embryonic development, and eventually influence the offspring's fit-
ness. A summary of existed studies aimed at describing epigenetic
inheritance in model or economically important fish species along with
the methods used for DNA methylation analysis (detection and/or
quantification) was shown in Table 2.
3|DNA METHYLATION DYNAMICS AND
EPIGENETIC INHERITANCE IN FISH
3.1 |Characterization of DNA methylation in fish
With phenotypic plasticity, fish are among ideal biological models in
developmental epigenetic studies as they are directly and fully
exposed to the aquatic environment.
8,80
The fish phenotype can be
influenced by multiple environmental and biological factors, including
diet, toxicants, temperature, salinity, parental health status and popu-
lation density,
81,82
where the underlying mechanism results in differ-
ent forms of DNA methylation patterns. Compared with higher
vertebrates (birds and mammals), the global DNA methylation level is
much higher in fish.
83
The differences between fish and higher verte-
brates may be related to body temperature, duplication of highly
ZHANG ET AL.3
TABLE 1 An overview of DNA methylation methods used in the most recent research in fish
Method Resolution Scale Advantages Limitation References
BS, BA-seq,
oxBS-seq
mCpG Individual genes; amplicons; whole-genome
and locus-specific
Cost-effective; single nucleotide resolution;
Maintaining the 5mC stabilization
Low-scale; relies on error prone PCR and
efficacy of bisulphite conversion
[56–63]
RRBS mCpG mCpG rich fraction of genome Large-scale; cost-effective; small amount of
input material; single nucleotide
resolution
Covers only 1% of genome; relies on error
prone PCR and efficacy of bisulphite
conversion; requires existence of
reference genome
[25,64–66]
MeDIP-seq mCpG and mCpH per
100 bp
mCpG-rich fraction of genome Cost-effective; does not introduce errors to
target sequence
Low-resolution (150 bp); does not predict
absolute methylation level
[56,67]
WGBS mCpG Genome-wide Large scale; covers entire genome; single
nucleotide resolution
Costly and laborious; relies on error prone
PCR and efficacy of bisulphite
conversion; requires existence of
reference genome
[41,44,59,68]
MRE-seq mCpG within specific
sequence motifs
Genome-wide Cost-effective; does not introduce errors to
target sequence
Biased for specific nucleotide sequences [67]
LUMA mCpG in CCGG context Genome-wide Large-scale; no reference genome required;
single-nucleotide resolution
Costly and laborious; low-resolution; only
mCpGs within specific sequence motifs
[69]
ELISA Global mCpG level Genome-wide Large-scale; no reference genome required;
does not introduce errors to target
sequence
Low-resolution; does not predict absolute
methylation level
[29,44,70,71]
Abbreviations: BA, bisulphite amplicon sequencing; BS, bisulphite sequencing; ELISA, enzyme-linked immunosorbent assay; LUMA, luminometric methylation assay; MeDIP-Seq, methylated DNA
immunoprecipitation sequencing; MRE-seq, methyl-sensitive restriction enzyme digestion followed by sequencing; oxBS-Seq, oxidative bisulphite sequencing; RRBS, reduced representation bisulphite
sequencing; WGBS, whole genome bisulphite sequencing.
4ZHANG ET AL.
TABLE 2 Studies describing the effect of various environmental cues during artificial fish breeding on the DNA methylation profile in fish
gametes and progeny
Fish species Methodology Tissue Main results References
Danio rerio WGBS Spermatozoa, eggs and
embryo
DNA methylation pattern of the early embryo is
inherited from spermatozoa and not the oocyte.
[42]
Danio rerio BS Germ cells Cryopreservation increased the DNA methylation
level in promoters of vasa and cxcr4b genes,
causing significant downregulation of their
expression.
[72]
Colossoma macropomum MSRE digestion Spermatozoa The viability of progeny was affected by DNA
methylation alterations caused by spermatozoa
cryopreservation.
[38]
Oryzias melastigma MSRE digestion Spermatozoa Hypoxia might pose a dramatic and long-lasting
threat to the sustainability of fish populations.
[73]
Danio rerio MeDIP-Seq Spermatozoa The environmental exposure to mercury promotes
the risk of disease development via epigenetic
transgenerational inheritance.
[74]
Oncorhynchus mykiss RRBS Spermatozoa The hatchery conditions induce DNA methylation
changes in the genome of steelhead in comparison
to wild populations.
[26]
Morone saxatilis MBD-Seq Spermatozoa WDR3/UTP12 and GPCR families are associated
with fertilization.
[75]
Anguilla anguilla LUMA Spermatozoa The cryopreservation using DMSO induces
hypomethylation in sperm while methanol-based
cryopreservation does not influence the sperm
DNA methylation in comparison to fresh sperm.
[76]
Oncorhynchus mykiss RRBS Liver, spermatozoa Family effects and temporal dynamics represent a
high degree of variation in methylation changes of
hatchery-reared fish in comparison to wild
population.
[27]
Carassius auratus, Danio
rerio
LUMA Spermatozoa The global DNA methylation pattern in
cryopreserved sperm show different degree of
stability upon treatment with different
cryoprotectants in two cyprinid species.
[40]
Gobiocypris rarus Dot-blot with anti-
5mC
Germ cells The global DNA methylation levels decreased
significantly in spermatozoa upon chronic
exposure of males to bisphenol A.
[77]
Cyprinus carpio WGBS Spermatozoa The DNA methylation levels in spermatozoa changes
dynamically during short-time storage.
[41]
Salmo salar WGBS Spermatozoa, liver Transgenerational plasticity mediated by
intergenerational inheritance of DNA methylation
acquired late in life for Atlantic Salmon.
[78]
Oreochromis niloticus WGBS Gonad Global DNA methylation level changes in high
temperature-induced sex-undifferentiation of
female gonads.
[61]
Lates calcarifer BA-seq Gonad The region-specific differences in length-at-sex
variation accompanied by differences in DNA.
Methylation coincided with alteration in water
temperature and salinity.
[62]
Ictalurus punctatus WGBS Embryo E
2
-induced sex reversal was a downstream process
independent of the sex determination process
regulated by DNA methylation.
[79]
Abbreviations: BA-seq, bisulphite amplicon sequencing; BS, bisulphite sequencing; LUMA, luminometric methylation assay; MBD-Seq, methyl-CpG binding
domain sequencing; MeDIP-Seq, methylated DNA immunoprecipitation sequencing; MSRE digestion, MspI and HpaII restriction enzymes digestion; RRBS,
reduced representation bisulphite sequencing; WGBS, whole genome bisulphite sequencing.
ZHANG ET AL.5
methylated regions, CpG islands and T bands.
83
Generally, the con-
texts of DNA methylation have been identified as GC (CpG), CHH,
and CHG in mammals genomes, where high level of DNA methylation
was especially found in GC. In fish, some similar results have been
reported, which complement the basic knowledge of methylation
levels in different fish genomes. For instance, higher DNA methylated
sites (%) were noted at CpG sites in zebrafish (80; Danio rerio),
84
Hybrid tilapia (70),
85
tiger pufferfish (65; Takifugu rubripes),
86
coral
reef fish (61–62; Acanthochromis polyacanthus),
87
ricefield eel (76–78;
Monopterus albus),
88
common carp (79; Cyprinus carpio),
41
grass carp
(75–81; Ctenopharyngodon idella)
89
and channel catfish (75–78;
Ictalurus punctatus)
90
corresponding to the CHH and CHG (%) which
were 1.2 and 0.9 in zebrafish, 0.5 and 0.6 in tilapia, 0.3 and 0.9 in tiger
pufferfish, 1.0–2.0 in coral reef fish, 0.3–0.4 in ricefield eel, 3.2 and
3.0 in common carp, 1.0–1.2 in grass carp and 0.3–0.4 in channel cat-
fish. Although CpG methylation was widely reported, there is some
evidence shown that non-CpG methylation played an important role
biological process.
91–93
The epigenetic biomarkers (epimarkers) are established during the
early developmental stage due to the prevalence of mitosis.
94,95
With
a sensitive response and phenotypic plasticity, fish have been used to
identify DNA methylation markers in medical diagnosis in human,
96
and age assessment,
58,97
sex prediction
57
and the effects of the envi-
ronment in fish.
39,98–100
These findings are conducive to further
revealing the methylation mechanism and its application in aquacul-
tural breeding and management.
3.2 |Imprinting
Genomic imprinting is an essential mechanism for proper embryonic
development and germline plasticity mediated by epigenetic mecha-
nism in other animals and humans,
101–104
however the strength of
genomic imprinting for non-mammalian vertebrates including fish and
egg-laying birds has not been clarified to date. Sensitive information
can be carried by gametes whether it is passed into future generations
or not. Epigenetic programming is essential for post-fertilization suc-
cess and offspring healthy development in the mammal's gam-
etes.
105,106
Abnormal spermatozoa quality is associated with
alternative DNA methylation in imprinted regions of the genes that
play a vital role in reproduction. In mammals, it has been shown that
spermatozoa concentration, motility and morphology are associated
with DNA methylation levels.
3,107
However, there is much debate on
whether alternations in DNA methylation of spermatozoa affect
embryos and offspring. This controversy exists not only in mammals
but also in fish.
108–110
Based on current knowledge, a better compre-
hensive and in-depth understanding of investigation and description
of DNA methylation in fish is required to be further elucidated.
Basically, the DNA sequence and epigenetic structure are copied
to the daughter cells. Although a controversy on the imprinted-
epigenetic features in offspring has been raised,
111
demethylation, de
novo methylation and DNA methylation reprogramming during cell
cycle progression are assumed to be the factors causing epigenetic
changes. Another scenario, compared to the unmethylated loci in
sperm, is an essential germline gene (Piwil1) which is methylated in
oocytes, and transferred to embryo.
112
Therefore, an asymmetry
beyond imprinted genes has been indicated. This makes the DNA
methylation inheritance more versatile and complicated. Except of
genes for inheritance, the methylated non-coding regions also can be
inherited. For example, the Snrpn promoter containing the endoge-
nous imprinted DMR region was found to be inherited by mouse
embryonic stem cells.
113
In addition, the nutrition status alters the
epigenome of the sperm that affects metabolic phenotypes of the off-
spring through several generations, which strongly implies that the
spermatozoa carry information of DNA methylation.
114,115
Further-
more, in mice, the offspring's health was found to be affected by the
changes in DNA methylation.
116
Hereby, the modification of DNA
methylation appear to be passed down not just to the next genera-
tion, but even across generations.
Similarly, establishment and maintenance of parental imprints in
fish is an unresolved question, which was raised by McGowan and
Martin
108
from an evolutionary viewpoint. From this perspective,
maintaining the imprinting in the embryo and offspring of fish is not
stable and definite.
108
The genome of medaka (Oryzias latipes) sperm
was hypermethylated prior to fertilization, but the methylation marks
were gradually erased after fertilization until the 16-cell stage.
44
The
developmental differentiation in DNA methylation patterns in fish
cause difficulties to understand the DNA methylation inheritance
mechanism.
42–44
Compared to mammals, some imprinted genes do not become the
epigenetic markers genes in fish. In zebrafish, peg1/mest, an ortholog
of the mammalian imprinted gene PEG1/MEST, is expressed during
embryogenesis; however, mest is not the imprinted gene.
117
There-
fore, imprinted genes of vertebrates might be identified in fish, how-
ever, maintaining the imprinting mechanism is delicate and
subtle.
62,118
In another example, the hypermethylated region was
detected in the goldfish (Carassius auratus)igf2 gene, which is the
paternal expression gene in the imprinting centre 1, but the methyla-
tion pattern of this gene was not maintained in embryo.
109
From
these analogical studies, it seems that parental imprinting probably
does not stabilize in fish, or that a special unknown inheriting mecha-
nism may be present.
Some crucial studies have revealed that paternal methylomes are
stably inherited by the early embryo, and the heavily methylated
sperm DNA determines and affects the DNA methylome during
embryo development in fish.
42,43,59,119
Due to the relationship
between the embryo and sperm DNA methylation patterns,
researchers have provided substantial evidence in helping to under-
stand the effects of sperm methylation on offspring performance. In
goldfish, it has been observed that the specifically methylated ntl
gene, one of the developmentally decisive genes, regulates the gender
of the offspring depending on the methylation level.
120
In zebrafish,
altered contents of DNA methylation and parental expression of
dnmt1 and dnmt3bb.2 inhibit the fecundity and impair the develop-
ment of the offspring,
70
and sperm DNA methylome affect the early
embryos.
42
During the DNA reprogramming, the level of DNA
6ZHANG ET AL.
methylation was maintained continuously, although it was pluripotent
at the period of embryogenesis in medaka.
44
The methylation pattern
in spermatozoa was regarded as the premise for the inheritance of the
methylation pattern in the medium blastula stage (MBT) embryo.
121
Following the paternal model, some studies have shown that repro-
gramming of the methylome occurred at embryogenesis and pre-
sented a similar pattern to sperm after reaching the MBT.
43,122
Intriguingly, Qin et al.
123
pointed out that DNA methylation in gyno-
genetic offspring may produce regulated hybrids by reprogramming
and developmental patterns.
This change in the DNA methylation pattern, which is reflected
immediately in the second generation, is called transgenerational
inheritance. Although the generations are never exposed to the pri-
mary disrupting events, transgenerational epigenetic alterations are
detected, especially in hatchery breeding. The offspring with the epi-
mutation should be considered as transgenerational inheritance in
fish, especially because the first generation is directly derived from
germinal cells that were affected by the agent during the F
0
situation.
For the F
1
offspring, the differences in methylation is dependent on
the individual methylated site within a given gene.
57
Both paternal
and maternal external stress affects the DNA methylation pattern in
fish.
124
The phenomenon of epigenetic reprogramming (e.g., erasing and
resetting most epimarkers) has been reported in vertebrates with sex-
ual reproduction, in which the spermatozoa, oocytes or both gametes
undergo hypomethylation, demethylation, methylation and de novo
methylation during early gametogenesis and embryogenesis. As
Granada et al.
9
summarized that epigenetic mechanism played a cru-
cial role in several biological functions, where genomic imprinting gain
considerable attention. Although, these results suggest that methyl-
ated genes in spermatozoa could be inherited by generations in fish;
however, genomic imprinting has not been demonstrated in fish.
Meanwhile, genome duplication exists in some fish, such as zebrafish,
sticklebacks (Gasterosteus aculeatus) and medaka, suggesting inheri-
tance of alterations in methylation pattern that needs to be consid-
ered in future research. Additionally, it is worth to note that natural
gynogenic population and sex determination in fish could be changed
by external conditions.
4|IDENTIFICATION OF DNA
METHYLATION CHANGES IN FISH
SPERMATOZOA FUNCTIONS IN RESPONSE
TO ENVIRONMENT CONSTRAINTS
Epigenetics modification has been considered as a joining point
between biological properties and environment factors,
5
and thus the
concept of “Environmental Epigenetics”has been proposed in recent
years. By overall consent, environmental epigenetics characterize
environmental-related molecular mechanisms that affect the off-
spring's phenotype and evolution without alterations in DNA
sequencing.
125,126
Environmental factors, therefore, possess the
capacity to influence epigenetic programming, and then dominate the
phenotype of the exposed individual. In this context, spermatozoa, as
FIGURE 2 A representative schematic showing the effects of various environmental cues on DNA methylation potentially applied to artificial
fish reproduction.
ZHANG ET AL.7
a genetic information carrier, is also affected by epigenetic changes
caused by environmental factors.
With regard to health perspectives, researchers have been very
concerned about the connection between the environment and dis-
ease, which was greatly reviewed and summarized by Åsenius et al.
127
Inevitably, DNA methylation has been pushed into the spotlight. Ciga-
rette smoke, recreational drugs and organic pollutants are the sub-
stances with high risk and hazards for human.
107,128,129
These risk
factors were put into context with changes of DNA methylation in
some genes and methylated regions that have been detected in
human spermatozoa.
130–132
These alterations of methylation associ-
ated with risks factors could not only respond to the parameters of
sperm count and motility, but also affect offspring development.
133
Furthermore, it is not restricted to these factors. The health status of
paternity and offspring were affected by diet, paternal body mass
index, organism functions (ageing and cancer) and others (genetic and
psychiatric, etc.) via whole DNA methylation level and methylated site
changes.
115,134–137
Therefore, the effects of environmental factors as
well as DNA methylation have become a critical issue in human and
fish spermatozoa.
There are various external and internal stimuli that are capable of
altering DNA methylation in spermatozoa, resulting in phenotypic
changes in the resultant offspring. Identification and characterization
of external and internal factors that alter DNA methylation patterns
provide a comprehensive overview of epigenetics inheritance of bio-
logical performance to develop fish breeding in aquaculture manage-
ment (Figure 2).
4.1 |Internal factors
4.1.1 | Reactive oxygen species
Reactive oxygen species (ROS) currently accepted as one of the major
influencing factors of the fish spermatozoa integrity and activity is the
key indicator of a cell under oxidative stress. It has been shown that
ROS affects spermatozoa motility,
138,139
DNA integrity
140,141
and
mitochondrial functions.
142
It plays a vital role in regulating cell apo-
ptosis signal transduction pathways.
143
ROS is generated during
short-term storage and cryopreservation of spermatozoa.
142,144
How-
ever, ROS-induced alternations of sperm methylation level have not
been properly characterized in fish reproduction, and need to be eluci-
dated in future studies. In mammals, it has been widely accepted that
ROS-induced abnormal DNA methylation alterations causes abnor-
malities in cellular and organismal function. Abnormal uncontrolled
5HmC levels, one of the ROS products of cytosine, can induce active
aberrant methylation processes.
145
ROS can also affect DNA methyl-
transferase activity itself, thus decreasing DNA methylation.
146
Mean-
while, protein modifications involved in DNA methylation are
modulated by ROS in spermatozoa (e.g., histone methylation and acet-
ylation) correlated with spermatozoa functions.
147,148
ROS-reduced
spermatozoa motility was associated with decreased DNA integrity
and alterations in DNA methylation.
149
Rahman et al.
150
reported that
oxidative stress-reduced spermatozoa motility was transgeneration-
ally inherited to F
2
generations in male mice exposed to bisphenol A
associated with increases in spermatozoa DNA methylation. Oxidative
stress in the spermatozoa of mouse leads to sex-related glucose and
fat-related metabolic pathologies in offspring.
151
Based on these stud-
ies, spermatozoa maintain epigenetic mutations and transfer epigenet-
ically ROS-induced molecular modifications to the offspring,
especially through DNA methylation. Therefore, the epigenetic risks
of ROS-induced changes of methylation in spermatozoa are worthy of
being valued and noticed. Prospectively, these studies started to
involve ROS and DNA methylation alterations during fish sperm stor-
age.
38,72,76,152
As reviewed by Sandoval-Vargas et al.,
153
identification
of better indicators and markers may be the key to understanding this
mechanism, which it seems is especially urgent in fish.
4.1.2 | ATP production
In mammals, cellular disruption of ATP biosynthesis is a good indicator
of abnormal mitochondrial functions, which may lead to motility loss
in spermatozoa.
154
Decreased ATP supplies in spermatozoa with low
motility and fertilizing capacity were associated with increased DNA
methylation in mice.
150
Similar to mammals, ATP is key determinant
of spermatozoa motility kinetics in fish.
155,156
Also, energy required to
maintain adequate ATP for spermatozoa motility in fish is generated
through mitochondrial respiration.
157
Rahi et al.
158
verified that most
of the sperm energy was derived from stored ATP which was synthe-
sized in a quiescent state but bioenergetically active state. The
expression of Cyt b and Co I genes is associated with ATP production
and spermatozoa motility in fish.
159
ATPase activity-associated genes
and ATP-binding genes modulate ATP synthesis and cell
morphology,
160,161
in which these genes may also be the key for regu-
lation of energy kinetics and maintenance of spermatozoa function.
Furthermore, the alteration of DNA methylation is associated with
down-regulating of the relevant genes expression.
161,162
However,
these limited studies are insufficient to reveal the fact that regulatory
mechanism of ATP production in spermatozoa and DNA methylation
needs to be further explored. These may help us to understand
whether a physiological relationship exists between spermatozoa ATP
biosynthesis/depletion and the DNA methylation pattern.
4.1.3 | Spermatozoa ageing
A decline in spermatozoa performance (motility and velocity) and fer-
tilizing ability due to ageing was shown to be associated with changes
of membrane permeability, DNA integrity and mitochondrial
damage.
163–167
In mammals, DNA methylation alterations in age-
dependent spermatozoa were associated with changes in gene meth-
ylation and expression in offspring.
168
The age-related differentially
methylated regions (DMRs) in rat spermatozoa were shown to be
enriched for embryonic development.
106
The multiple age-associated
DMRs in human spermatozoa were used to speculate chronological
8ZHANG ET AL.
age with high accuracy.
169
Recently, spermatozoa ageing has been an
important topic in aquaculture management, particularly as short-term
storage sperm are used for artificial reproduction. In this regard,
Cheng et al.
41
reported that elevated DNA methylation in common
carp spermatozoa was negatively correlated with the spermatozoa
traits during short-term storage in vitro. However, DNA methylation
needs to be elucidated during spermatozoa ageing with consideration
of other internal factors, including ROS and ATP stores.
4.2 |External factors
4.2.1 | Hatchery breeding
It has been suggested that hatchery breeding affects DNA methyla-
tion dynamics and progeny performance. To explore the effects of
DNA methylation differences on transgenerational offspring,
researchers have found that stable and inducible DNA methylation in
wild animal populations follows predictions from the evolution theory
of selection-based and detection-based on epigenetic information
channels.
170
Generally, imprinted genes are recognized as an impor-
tant basis of inheritance. In addition to differences in spermatozoa
DNA methylation levels between parents and following generations,
the DMRs were detected in these generations.
59,115
Similarly, DNA
methylation alterations in F
1
and F
2
offspring were affected by the F
0
with low-activity in adulthood.
171
The methylation pattern in sperma-
tozoa of F
4
, however, resulted in the nearly complete restoration of
the wildtype, even though the epi-mutations were transferred to
future generations.
59
Remodelling or reprogramming of aberrant DNA
methylation in the paternal line is the most critical contributor to the
normalization of recurrent DNA. In addition, some de novo process
and special pathways could be the role of the modulating DNA meth-
ylation pattern. These studies, at the very least, suggest that transge-
nerational epigenetic DNA methylation patterns are formed in
paternal line by defined environmental stimuli given by hatchery
breeding conditions.
The differences of fertilization ability, phenotype, adaptability and
fitness between wild and hatchery populations are worthy of more
attention. Artificial selection of fish phenotypes as ecological and opti-
mized strategies has been widely used in hatchery practices and spe-
cies conservation. However, the debate on the role of hatcheries in
enhancing and restoring wild stocks has been invoked.
172–174
Reduc-
ing fitness and maladaptation of hatchery fish when they are released
to restore wild stock populations, are a subject of a heated debate. As
we all know, domesticated salmon could threaten the existence of
wild populations through induced epigenetic modifications.
25,28
The
spermatozoa of captive-reared broodfish with defined epigenetic
modification may be transmitted to the next generation after inter-
breeding with the wild population.
28,78,175
It is a potentially disrupting
local adaptation. Intriguingly, the study reports that the early-life epi-
marks in the coho salmon (Oncorhynchus kisutch) living under rearing
conditions seems to persist in the adult, even when released into the
natural environment.
31,176
Overall, paternal epigenetic patterns affect
transgenerational epigenetic patterns of DNA methylation. In this
context, the optimized breeding strategies of natural-origin and hatch-
ery fish is especially important.
Intriguingly, studies so far have failed to identify signatures of the
molecular basis of phenotypic changes in farmed Atlantic salmon
(Salmo salar), which is based on genetic analysis.
177,178
Using restric-
tion site associated DNA sequencing (RAD-Seq), Gavery et al.
26,27
were unable to differentiate wild and hatchery rainbow trout (Oncor-
hynchus mykiss), however the specific methylated regions were identi-
fied by reduced representation bisulphite sequencing (RRBS).
Compared with RRBS, WGBS and methylated DNA immunoprecipita-
tion (MeDIP) might be better tools to detect the special differences in
DNA methylation patterns.
27,30,31
Taken together, it cannot be denied that recent studies have pro-
vided us with a better understanding of whether DNA methylation
can be inherited by future generations, and the effects of spermato-
zoa, and of paternal DNA methylation patterns on offspring health
and performance. For understanding and applying the genetic mecha-
nism of DNA methylation, these studies; however, are limited and
insufficient. Therefore, high-quality multiomics and multitopic associa-
tion studies for this subject are essential and necessary. Such findings
will be necessary and highly instrumental in applied research in fish
reproduction practices, domestication and selection of species.
4.2.2 | Toxicants
Generally, fish living in the aquatic environments are both directly and
indirectly affected by the stress from toxicants than non-aquatic spe-
cies. With one accord, parental exposure to xenobiotics, such as
bisphenol A (BPA), bis (2ethylhexyl) phthalate (DEHP), dibutyl phthal-
ate and other toxicants could result in various abnormalities in fish
offspring generated by paternal defects. In early studies, the effects of
environmental toxicants have focused on their interference in fish
reproduction.
179
However, fundamental information and in-depth
mechanism of chemicals affecting reproductive success seem to have
been overlooked.
Due to environmental exposure, aberrant DNA methylation
changes have been reported in many animals and plants.
180
These
could severely affect spermatozoa and male fertility. For example,
DEHP could significantly impair the reproduction of zebrafish.
181
Laing et al.
69
found that the expression of DNA methyltransferase
1(dnmt1) and the global DNA methylation level were reduced in the
gonad of zebrafish exposed to 1 mg/L BPA. A comprehensive review
on the effect of 17α-ethinylestradiol (EE2) and BPA on reproduction
in aquatic wildlife species showed relationships between the sexual
and neural development and the DNA methylation pattern.
182
This
study highlights that, in addition to EE2 and BPA effects on gonadal
development and reproductive behaviour, if the fish are fertile, the
adverse effects are trans generationally transmitted to their offspring
through their spermatozoa epigenome. On the downside, however,
the potential effects of DNA methylation on spermatozoa and off-
spring performance have not been elucidated.
ZHANG ET AL.9
During early spermatogenesis in zebrafish, BPA-induced epige-
netic changes were inherited by F
1
embryos.
183
The absence of
changes in DNA methylation in specific genes strengthened the fact
that heart failure at the embryonic stage was aroused by the alter-
ations of the epigenetic pattern.
184
Exposure to BPA increased DNA
methylation gene (dnmt7) expression suggesting that regulation of
genomic DNA methylation level in Chinese rare minnow (Gobiocypris
rarus), might be due to maintenance of DNA methylation mechanism
following expoesure.
77
Meanwhile, the insecticide permethrin could
induce transgenerational behavioural aberrance via DNA methyla-
tion.
171
The overexpression of hand2,esr2b and kat6a of F
1
embryos
from BPA exposed males was reduced by EGCG treatment,
185
in
which equilibrium methylation level might be correlated with the
demethylation process. Therefore, these studies indicate that toxi-
cants exposure disrupts fish reproduction with altering methylation
levels and suggests that xenobiotics influencing on offspring perfor-
mance might be mediated by DNA methylation.
4.2.3 | Diets
The impacts of the maternal nutrition level on future generations are
well validated and established phenomena in mammals and fish.
Paternal malnutrition related to programming metabolic disease of the
offspring has been recognized.
186
The changes in promoter methyla-
tion status and gene expression were produced by the parental diets
with low protein.
114
Additionally, numerous DMRs were found in the
spermatozoa of F
1
treated with high-fat diet, which was perfectly in
line with the F
0
.
115
Dietary nutrition levels, therefore, determined the
performance of parent and progeny by fluctuations in DNA
methylation.
Due to lack of high-quality assays in fish, however, it is unclear
whether diet affects spermatozoa methylation, or which compounds
could regulate the offspring's adaptability to the environment by trig-
gering spermatozoa DNA methylation. Nutritional (metabolic)-related
epigenetic studies in reproductive biology are considered a relatively
new area of research aiming to explore the status of metabolism-
related gene and DNA methylation in the offspring.
124,187–189
How-
ever, nutritional-induced DNA methylation alterations in gametes of
broodstock as possible causes of transgeneration changes seem to
have been overlooked.
Low vitamin B diets induced higher lipids inclusion in the hepato-
cytes of F
1
zebrafish livers.
190
With crude oil diets, global DNA meth-
ylation decreased in cardiac tissue and the bradycardia was also
observed in zebrafish offspring. Compared with cardiac tissue, global
DNA methylation levels in gonads were not affected by dietary crude
oil.
124
This might be due to the selection of sampling procedure as the
whole gonadal tissue was used. Several DMC-associated genes were
identified in stickleback, which suggested that advantageous epige-
netic changes provided a possible adaptative environmental mecha-
nism to respond to changes in diet.
191
Balanced nutrition is essential for the survival and healthy growth
of wild and farmed fish. The capacities of modulating the oxidative
stress, adapting to the environment, and adjusting epigenetic pattern-
ing were affected by the nutrition level. Influence of broodstock nutri-
tion on spermatozoa DNA methylation level in fish, however, is still
unknown and paternal and transgenerational inheritance is required
to be elucidated.
4.2.4 | Cryopreservation
Cryopreservation of gametes provides an effective method for hatch-
ery reproduction to biologically protect endangered species and to
make a gene bank of genetic resources. However, whether the cryo-
preservation with cryoprotectants affect spermatozoa DNA methyla-
tion is still virtually unknown.
Riesco and Robles
72
reported cryopreservation-induced hyper-
methylation in the promoter regions of vasa gene that is important for
regulating the transcription, However the functions of cryoprotectant
agents (CPAs) appeared to be ignored. Yang et al.
192
compared the
cryopreserved spermatozoa with and without cryoprotectant in yel-
low catfish (Pelteobagrus fulvidraco), and observed down-regulation of
the transcriptome of spermatozoa cryopreserved with Me
2
SO. De
Mello et al.
38
found that lower methylation levels were detected in
spermatozoa cryopreserved with CAPs, which resulted in delays and
abnormalities during embryonic development. Similarly, the DNA
methylation level in goldfish spermatozoa was decreased during cryo-
preservation with dimethylsulfoxide (DMSO and 1,2-propanediol).
40
However, cryopreservation of zebrafish spermatozoa with methanol
induced only a slight increase in global DNA methylation.
40
These
studies suggest that cryopreservation causes changes in the sperma-
tozoa DNA methylation levels.
38,193
Abnormal methylation alterations
detected in cryopreserved semen may result from cryoprotectant(s),
and involve both hypomethylation and hypermethylation pro-
cesses.
194
However, the methanol protocol did not change the global
DNA methylation level in the European eel (Anguilla anguilla) sperma-
tozoa.
76
It is not clear, therefore, if the cryoprotectant molecule is the
categorical risk factor for DNA methylation alterations.
Undoubtedly, however, cryopreservation is a crucial tool, com-
monly applied in ARTs and germplasm banking. However, the cryo-
protectant induced a potentially harmful epigenetic changes during
the cryopreservation process. As there was a significant correlation
between DNA methylation and offspring performance,
38
and cryopro-
tectants and species,
195–197
the optimum and appropriate cryopreser-
vation methods must be used for different fish species, and an
appropriate cryoprotectant should be selected and established to
reduce the risk from DNA methylation alteration.
4.2.5 | Salinity
A recent study reported that DNA methylation plays a potential role
in facilitating adaptation to divergent salinities.
191
For the progeny,
the differentiated DNA methylation level was found in offspring fertil-
ized at several environmental salinity levels.
198
In addition, in
10 ZHANG ET AL.
calculating the changes of spermatozoa function over generations, it
was shown that the offspring of the round goby Neogobius melanosto-
mus underwent epigenetic acclimatization to the environment by tran-
sitioning to a new level of salinity.
199
In aquaculture, acclimatization
to environmental salinity is a common biological process for fish
domestication that may affect spermatozoa DNA methylation levels
resulting in phenotypic changes in the offspring.
4.2.6 | Temperature
Frequent studies clearly indicate temperature have significant
impacts on physiological performance correlated with modification
of DNA methylation in fish, including sex determination
and differentiation,
56,57
development,
200,201
gamete maturation and
reproduction capability
202–204
and immunity.
205,206
Sex control and
selection for growth performance are important elements for improv-
ing production and increasing profitability in aquaculture.
Worthwhile, the effects of temperature on sex change in fish
have already been an important and major topic in aquaculture.
207,208
It is known that the diversity and plasticity are the typical characteris-
tics in fish considering temperature-dependent sex determination and
differentiation.
209
Herein, intriguingly, paternal and temperature fac-
tors are independently or interactively involved in sex-determining
system,
208
where epigenetic modification has been reported to be
involved in the processes.
210
For instance, the big mount of differen-
tially methylated sites was noted on the sex chromosome 19 with the
bias toward of methylation level between females and males in three-
spine stickleback.
211
High density of differentially methylated CpG
sites were noted on the sex chromosome (chromosome 4) in channel
catfish.
90
Meanwhile, the temperature-dependent masculinization
with the differentially methylated genes in the gonads of Nile tilapia
(Oreochromis niloticus).
56
Similarly, changed DNA methylation on the
promoter of aromatase Cyp19a was considered as the response to
environmental temperature to determine the sex in European sea bass
(Dicentrarchus labrax).
210
Additionally, it has been suggested that the
capability to adapt to temperature changes by methylation could be
inherited by future generations.
8
Moreover, Ryu et al.
212
revealed the
possible association of DNA methylation with transgenerational adap-
tation to temperature change. Although these results provide more
and more evidence for possible methylation mechanism of genetic-
induced and/or temperature-induced phenotypic plasticity in sex
determination and offspring performance, however it is still not clear
how they mediate plasticity and acclimatization responses. This may
be the key point for a breakthrough in genetic breeding of farmed fish,
particularly in monosex production.
4.2.7 | Hypoxic stress
Hypoxic stress has already become a widespread environment prob-
lem raising the concerns in aquaculture. The major adverse effects of
hypoxia in fish include reduction in feeding and growth alternations of
sex hormone levels, delay in development, interfere with gametogene-
sis and may be death.
213–217
The possibly negative effects of hypoxia
are usually attributed directly to F
0
, so the transgenerational genetic
effects of hypoxia seem to be overlooked by current understanding.
Most important, the researcher has been aware of the potential risk in
male reproductive health.
218–220
Earlier, the studies reported that exposing monkeys to high alti-
tude with hypoxic stress significantly changed number of spermatozoa
and spermatozoa motility.
221
Subsequently, environment hypoxia
resulted transgenerational task was detected in rats.
222
Intriguingly,
breakthrough finding was reported in fish that hypoxia impairs male
reproduction in Oryzias melastigma by declining of spermatozoa qual-
ity and quantity, via the methylation regulation.
73,223
Probing the
mechanism of hypoxic stress on fish physiology through methylation
is being emphasized, including reproductive impairments,
73,223,224
embryogenesis,
225
immunity,
226
growth and development
227
and
metabolism.
228
Therein, the functions of hypoxia-responsive miRNAs
(miR-125-5p, miR-103b and miRNA-210-5p), genes (HIPKs, SYCP3,
STAT3 and HIF-1a), signalling pathway were investigated in both
model species and economically important species.
73,223,226–228
These
results revealed the adverse effects of hypoxic stress on paternal
reproduction impairments and offspring performance in mammals and
fish with phenotypic and epigenetic levels. Therefore, avoiding hyp-
oxic stress is a very important aspect of aquaculture management,
especially for gametogenesis during the spawning season.
5|POTENTIAL RELATIONSHIPS
BETWEEN DNA METHYLATION AND FISH
SPERMATOZOA QUALITY
In all animals, spermatozoa quality parameters (e.g., motility, velocity
and concentration) are determinants of male fertility, and are directly
associated with fertilization, hatching and malformation rates.
229
Envi-
ronmental factors affect spermatozoa quality during the period of tes-
ticular development and after discharge of sperm into the aquatic
environment at spawning.
28,129,133,181
Our current knowledge indi-
cates a relationship between spermatozoa quality parameters and
changes of the DNA methylation pattern.
3,107,230
For instance, declin-
ing spermatozoa velocity and the percentage of spermatozoa motility
are found over generations as the offspring epigenetics evolve in
response to a novel environment.
199
A significant positive correlation
between global DNA methylation levels and spermatozoa concentra-
tion and motility has been observed in human, and animal models
including fish.
3,20,21,231,232
Furthermore, a significant reduction in the
value of parameters, including sperm volume, number of spermatozoa
and number of motile spermatozoa and elevating DNA damage has
been observed in the human smoking population with stochastic DNA
methylation alterations.
107
Vitally, the offspring performance (such as
metabolic syndrome, and growth and sexual development) was corre-
lated with paternal exposure to environmental factors.
232,233
There-
fore, to better understand the association with spermatozoa quality,
DNA methylation patterns, and offspring performance, it is crucial to
ZHANG ET AL.11
consider the environmental conditions when the male produces sper-
matozoa during spermatogenesis, when spermatozoa acquire the
potential for motility in the sperm duct, and when the sperm is ejacu-
lated at spawning. In these regards, the aforementioned internal and
external environmental stimuli can affect both spermatozoa quality
and the DNA methylation pattern, which consequential effects on the
performance and health of offspring.
As a consequence, it can be anticipated that alterations of DNA
methylation patterns in fish spermatozoa will also be transmitted to
progeny. The evaluation of progeny fitness and performance is equally
important in order to cover the whole process of fish reproduction
and address all factors that could influence it via DNA
methylation.
171,183
These factors and their relationships in the field of fish reproduc-
tion, however, have not been sufficiently discussed to date. Existing
studies on changes in the spermatozoa DNA methylation pattern pro-
vide only limited insights for the interpretation of their significance
for fish biology. Below DNA methylation dynamics in different stages
of spermatozoa development and their effects on spermatozoa quality
parameters and progeny performance are discussed.
5.1 |Regulation of pathways involved in
spermatozoa motility signalling
Ion signalling is essentially linked with spermatozoa quality. Notably,
CatSper, a cation channel that regulates Ca
2+
signalling, plays a key
role in spermatozoa motility and fertilization in vertebrates including
fish.
234,235
Lin et al.
236
recently studied the structure of CatSper com-
plex in the mouse spermatozoa, and re-named it as a channel-
transporter ultracomplex the CatSpermasome. The interpretation of
CatSpermasome structure reveals a deep and mechanistic under-
standing of spermatozoa motility signalling it as a determinant of male
fertility. So far, differential-methylated regions in particular genes
associated with Ca
2+
signalling have been shown that include adeny-
late cyclase 5 (Adcy5), phospholipase C β1(Plcb1) and protein kinase C
β(Prkcb).
237
Watkins et al.
59
reported that spermatozoa of a low-
protein diet (LPD) fed mice displayed global hypomethylation
compared with a normal protein diet (NPD) fed to males. Further tran-
script analyses of the genes involved in Ca
2+
signalling revealed that
transcripts of Adcy,Plcb1 and Prkcb were decreased in the liver and
heart of LPD offspring.
116
These studies show that dietary-altered
spermatozoa DNA methylation patterns may affect spermatozoa
motility via affecting the expression of genes involved in Ca
2+
signal-
ling required for the initiation of spermatozoa motility.
In fish ionic regulation of spermatozoa motility signalling has been
extensively studied and reviewed.
156,238,239
The ions also play vital
roles in spermatozoa viability and DNA integrity.
240–242
Some studies
reveal an optimum Na
+
/K
+
and Ca
2+
/K
+
ratio in the seminal plasma
to maintain spermatozoa motility.
243,244
Among the ions, Ca
2+
regu-
lates spermatozoa axonemal beating by several types of channels such
as voltage-gated calcium channels and CatSper.
235,245
If the pH of the
sperm activating medium is similar to or higher than that of the
seminal plasma, the motility and fertilization ability of spermatozoa
can be enhanced.
246
Furthermore, Na
+
and Cl
are predominant ions
of the seminal plasma to maintain osmolality.
156
Despite known ionic
regulation of spermatozoa motility signalling, the molecular identity of
the ion channels is very limited in fish spermatozoa. Fechner et al.
241
identified a gene encoding putative cyclic nucleotide-gated K
+
chan-
nel (CNGK) in zebrafish spermatozoa. Moreover, association between
spermatozoa DNA methylation levels and transcripts or expressions
of ion channels involved in spermatozoa motility signalling has not
been investigated. Kumar et al.
247
found that Na,K-ATPase alpha4
(Atp1a4) gene expression is regulated by methylation level changes in
ES cells. Therefore, it might be biologically rational to investigate
whether environmental-induced changes of spermatozoa DNA meth-
ylation patterns affect ionic regulation of spermatozoa motility, shown
by changes in expressions of ion channels; for instance, CatSper that
regulates Ca
2+
-dependent axonemal beating.
Collectively, our current knowledge shows that altered spermato-
zoa DNA methylation could result in reducing fertility or reproductive
efficacy in fish, and thus evaluation of DNA methylation might be a
valuable criterion to assess spermatozoa quality. Revealing more
unknown DNA methylation changes, however, will require more
advanced methods and analysis in candidate genes with deep
sequencing. Furthermore, even as these studies attempt to elucidate
the regulatory mechanisms underlying the relationship between DNA
methylation and spermatozoa quality, it is particularly important to
see whether DNA methylation in spermatozoa may affect phenotypes
of embryos and offspring.
5.2 |Regulation involved in other spermatozoa
function
Abnormal spermatozoa parameters are associated with alternative
DNA methylation in imprinted regions of the gene which play a vital
role in reproduction. In humans, the concentration of spermatozoa,
and morphology and motility of spermatozoa were associated with
the spermatozoa DNA methylation level.
3,107
The DNA methylation
level separated the samples identically with spermatozoa motility and
abnormal semen parameters in buffalo bull
248
and horse.
249
Targeted
analysis of MTHFR promoter DNA methylation showed that hypo-
methylation of MTHFR was associated with low motility spermato-
zoa.
230
Furthermore, Laqqan et al.
130,250
found that oligozoospermia
and subfertility were relative to some genes with DNA methylation
modification, such as PRRCA2,ANXA2,MAPK8IP3 and UBE2G2. Addi-
tionally, these genes and non-coding RNAs, such as gsk3a,sf1, miR-21
and miR-34c, are involved in spermatozoa activity and male
infertility.
251–253
From these studies, aberrantly methylation alter-
ations always resulted in poor-quality spermatozoa in mammals.
Limited studies have been carried out on fish spermatozoa. Some
studies found that the genes of bdnf,kita,amh,gsk3a and dmrt1 are
potential predictors of spermatozoa quality in fish.
252,254,255
As an
important regulator of gene expression, miRNAs should also not be
ignored. Several studies have indicated the role of miRNAs in
12 ZHANG ET AL.
differentiation and maturation of various cell types.
256,257
For exam-
ple, miR-34a and miR-200 modulate spermatozoa motility in zebra-
fish.
258,259
Notably, p53/miR-200s may control zebrafish embryo
size.
260
In a study detecting miRNA expression profile in human semi-
nal plasma associated with ageing, an altered expression of 67 miR-
NAs, especially miR146a, miR371 and miR122 was found.
261
Therefore, considering that signalling pathways such as mRNA and
miRNA affect spermatozoa quality and offspring performance, it is
particularly important to search for new and optimal biomarkers can-
didates and to study signalling pathways in depth.
6|CONCLUSION AND FUTURE
PERSPECTIVES
There are various internal and external environmental stimuli that
affect fish phenotypes and performance associated with alterations in
the DNA methylation pattern of spermatozoa. As similar environmen-
tal factors affect spermatozoa quality parameters, it has been sug-
gested that environmental factors-induced phenotypic changes could
be due to alterations in spermatozoa genome methylation. There is no
denying that some promising results have been achieved so far, how-
ever, studies in fish progeny performance and adaptability through
DNA methylation based on paternal intergenerational inheritance are
still insufficient, limited and not standardized. To better understand
epigenetic influence of environmental factors, further studies should
consider investigating: (1) a link between spermatozoa quality, DNA
methylation and fertility to answer whether low quality spermatozoa
with aberrant DNA methylation results in diminished fertility?
(2) Although DNA methylation seems to be inherited by the next gen-
erations, potential regulatory mechanisms that stabilize/destabilize
the transgenerational inheritance are largely unknown. (3) The existing
literature have focused in particular on within- and trans-generational
inheritance of environmental adaptation capability with consideration
to the effects of gametes, however the major focus has been put on
the role of oocytes, while the spermatozoa have been overlooked. In
fertilization, spermatozoa are genetic information carriers and transfer
half of the genome to the offspring. In this context, new technologies
and analytical methods will provide researchers with better tools to
elucidate the link between DNA methylation patterns of spermatozoa
and fish phenotype and performance facilitating aquaculture develop-
ment. The combinations of Multi omics with different methodological
approaches are important to make up for the one-sided and inade-
quate research of the single method used in most fish sperm studies.
It is highly important and urgent need to select and standardize suit-
able methods for different species and research purposes.
AUTHOR CONTRIBUTIONS
Songpei Zhang: Conceptualization; formal analysis; investigation; meth-
odology; writing –original draft; writing –review and editing. Yu Cheng:
Conceptualization; formal analysis; investigation; methodology; writing –
review and editing. Pavlína Vˇ
echtová: Conceptualization; writing –
review and editing. Sergii Boryshpolets: Writing –review and editing.
Nururshopa Eskander Shazada: Investigation; writing –review and edit-
ing. Sayyed Mohammad Hadi Alavi: Writing –review and editing. Jacky
Cosson: Writing –review and editing. Otomar Linhart: Conceptualiza-
tion; funding acquisition; project administration; supervision; writing –
review and editing.
ACKNOWLEDGEMENT
Songpei Zhang and Yu Cheng were supported by the Chinese Scholar-
ship Council.
FUNDING INFORMATION
Ministry of Education, Youth and Sports of the Czech Republic, LRI
CENAKVA, Project Numbers: LM2018099, CZ.02.1.01./0.0/0.0/
16_025/0007370; Grant Agency of the University of South Bohemia
in Ceske Budejovice, Grant Numbers: 097/2019/Z, 037/2020/Z;
Czech Science Foundation, Grant Numbers: 20-01251S; National
Agency for Agriculture Research, Czech Republic, Grant Numbers:
QK21010141; China Scholarship Council, Award Number:
202108160002, 201908160003.
CONFLICT OF INTEREST
The authors declare to have no conflict of interests, neither personal
relationship nor financial interest that could have appeared to influ-
ence this review.
DATA AVAILABILITY STATEMENT
All data was shown in the present review paper, and there is no addi-
tional data. All information can be found in the cited references.
ETHICS STATEMENT
There are no ethical concerns with the article.
ORCID
Songpei Zhang https://orcid.org/0000-0001-5982-9233
Otomar Linhart https://orcid.org/0000-0001-7123-3192
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How to cite this article: Zhang S, Cheng Y, Vˇ
echtová P, et al.
Potential implications of sperm DNA methylation functional
properties in aquaculture management. Rev Aquac. 2022;1‐21.
doi:10.1111/raq.12735
ZHANG ET AL.21
