Epigenetics in fish gametes and early embryo

    Epigenetics in fish gametes and early embryo Catherine Labb´e, Vanesa Robles, Maria Paz Herraez PII: DOI: Reference:

S0044-8486(16)30375-1 doi: 10.1016/j.aquaculture.2016.07.026 AQUA 632246

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Aquaculture

Received date: Revised date: Accepted date:

9 December 2015 14 April 2016 21 July 2016

Please cite this article as: Labb´e, Catherine, Robles, Vanesa, Herraez, Maria Paz, Epigenetics in fish gametes and early embryo, Aquaculture (2016), doi: 10.1016/j.aquaculture.2016.07.026

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ACCEPTED MANUSCRIPT Epigenetics in fish gametes and early embryo

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Catherine Labbé1, Vanesa Robles2, Maria Paz Herraez3

INRA, Fish Physiology and Genomics, Rennes, France [email protected]

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IEO - Spanish Institute of Oceanograpy, Planta de Cultivos el Bocal, Monte Santander, Spain

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[email protected]

Department of Molecular Biology, University of León, León, Spain [email protected]

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Abstract

The spermatozoa and oocyte are very specialized cells which possess the unique ability to fuse and to

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produce an embryo. This embryo will develop into a mature organism capable of producing gametes again. This colloquial statement comprehends however the most complex mechanisms in biology

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that is cell differentiation and cell reprogramming. Epigenetic modifications are major actors in this process, both during gametogenesis and embryo development. The epigenetic concept is resulting

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from the growing scientific understanding of embryology and genetics, and it is made operational by generic molecular mechanisms, namely DNA methylation, histone tails modifications, and non coding RNAs. Epigenetic modifications associated with gamete differentiation and early embryo

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development are quite well described in mammals, but increasing research is developed in fish today, either in model or in aquaculture species. Because fish are living organisms thoroughly exposed to temperature and to water quality, environment is likely to influence the epigenetic pattern of the gametes, embryo, or adult organisms. Reproductive biotechnologies are also at risk to modify gametes and embryo epigenetics. This review is aimed towards giving the basic knowledge on the molecular actors of epigenetics, highlighting the developing knowledge in mammals which can be useful to fish research and proposing the possible area where epigenetics can enrich research on fish gametes and reproductive technologies.

Keywords Development; gametogenesis; epigenetic; reproductive biotechnology; sperm; oocyte; embryo. Bullet point statements

ACCEPTED MANUSCRIPT - Epigenetic encompasses gene expression regulation and is especially active during gametogenesis, development, and differentiation. - Gametes and embryo manipulation are at risk to influence the fish phenotype by way of epigenetic

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alteration.

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- Specific epigenetic data exist in fish which are to be treated distinctly from mammals because of

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fish close interaction with the environment (including toxicology and temperature).

Relevance/impact of the paper to the general field of Commercial Aquaculture

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Low applied impact today, and high scientific impact

ACCEPTED MANUSCRIPT Introduction

Although epigenetics has been extensively studied over the last twenty years in many research areas

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including cancer research and developmental disease, the thorough involvement of epigenetics in

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fish early development was more recently established. This review will present the most recent knowledge on fish gamete epigenetics, namely DNA methylation pattern and histone modifications,

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to underline the powerful model that is fish gamete and fish development for the general scientific community: a gametogenetic process completely exposed to temperature effect, an embryonic genome activation taking place after up to 10 mitotic divisions. The first section will introduce the

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molecular basis of epigenetics and the history of the scientific knowledge which led the research community to develop the concept of epigenetic. We will then review the increasing knowledge available in fish gametes, especially sperm, and the possible alteration of the epigenetic profiles. The

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description of the molecular events before the embryonic genome activation will additionally been included, with special emphasis on the setting up of proper epigenetics marks, with most of the knowledge being developed in zebrafish. How the constraint the gametes and parents are submitted

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to such as cryopreservation, parental history including xenobiotic exposure or nutrition is at risk of

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influencing the progeny through epigenetic phenomenon will be carefully addressed. The objective of this review is to provide some understanding of the importance of the epigenetic profile in fish

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gametes, to face very important question in fish gamete research. Above all, this review will point out the most critical questions raised by these topics which should be addressed in the future by the

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fish research community.

The epigenetic concept

The modern concept of epigenetic encompasses the question of cellular memory upon mitosis, environmental influence on the gene expression, together with the structural and molecular mechanism stabilizing gene expression pattern when the initial triggering event is no longer present. Epigenetic is following several centuries of an active quest towards understanding the basics of development and heredity1. It included the deciphering of cellular differentiation and embryonic development, and the discovering of the structural and molecular factors involved in gene expression and silencing. As this introduction will show, epigenetic should not be considered as a “new” area of science. Epigenetic is networking all the existing knowledge. It is nevertheless providing exciting insight on why we are not only the sum of our genes. 1

For this sub-chapter, we took as a thread the remarkable course of Edith Heard at the Collège de France (Heard, 2012-2013, in English). Several original papers of the oldest citations were found at http://www.esp.org/whatsnew/index.html

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1.1.

From epigenenesis to developmental biology

More than 300 years BC., Aristotle was holding from his observation that life starts from parts which

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are at the origin of the next parts (or organs) not yet existing at a given stage: “in all the productions

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of nature or of art, what already exists potentially is brought into being only by what exists actually”. This theory was later on called epigenesis (gradual emergence of form). It was opposed in the XVII

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and XVIII centuries to the preformation theory holding that the generation of offspring occurs as a result of an unfolding and growth of preformed parts contained in the sperm, or in the ovum (the Homunculus, or little man, of Paracelsus in 1572). Over the last three centuries, most important

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research provided a rather clear view of the embryonic development and the role of both sperm and eggs in the initiation of a differentiated individual, leading to the progressive receding of the preformation theory. Among the milestones, the German embryologist C.F. Wolff (Theoria

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Generationis 1759) proposed that the originally undifferentiated material in the egg develop through a series of steps termed morphogenesis, and von Baer (1792-1876) was the first to accurately describe the mammalian development from a fertilized egg. From then on, understanding the forces

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and mechanism leading to cellular differentiation and organ formation became the sacred grail of

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embryologists, and it sparked the development of experimental embryology. Among early achievements, Driesch (1867-1941) demonstrated in 1892 that any cell isolated from an early

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embryo could develop into a full embryo, because each cell still possess all the determinants, while Spemann (1869-1941, Nobel Prize for medicine in 1935) and his student showed that the induction and organization of cellular differentiation was directed in an organism by the neighboring cells or

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tissues (Spemann and Mangold, 1924). This major knowledge was still not answering however which fundamental mechanisms were involved.

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From heredity to gene regulation

The above described studies on early embryo development and differentiation (morphogenesis or epigenesis) was contemporaneous of the development of genetics and heredity laws. In 1902, Boveri (1862-1915) and Sutton (1877-1916) could suggest that the hereditary “factors” of Mendel (18221884) were borne by the chromosomes described earlier by Flemming (1843-1905), a view shared by the embryologist Hertwig (1850-1937). Sperm and eggs were accepted as being single cells containing the heritage of the species (Weismann 1834-1914; Wilson, 1900), and the cell nucleus was proposed to be the vehicle of inheritance. Morgan (1866-1945) later on provided the basic proofs that the genetic factors were physically located on the chromosomes, and confirmed that exchange of parts between chromosomes during gamete formation was triggering progeny diversity (Nobel Prize in Physiology or Medicine in 1933). Intense work on the DNA molecule was prompted by the

ACCEPTED MANUSCRIPT discovery that heredity was lying in DNA and not in proteins (Avery, MacLeod, McCarty, 1944). This led to the discovery of the double helix structure of DNA (Watson and Crick, 1953a, b, Nobel prize in Physiology or Medicine with Wilkins in 1962) and to the deciphering of the genetic code and its role

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in protein synthesis (Holley, Khorana and Nirenberg, Nobel Prize in Physiology or Medicine in 1968).

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The next major breakthrough was the demonstration of gene regulation in the lac operon model of Jacob and Monod (Nobel Prize in Physiology or Medicine in 1965 with Lwoff). In their review, Jacob

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and Monod (1961) proposed that in prokaryotes, some genetic determinants other than structural genes were acting as regulator and operator of the later, and that “the genome contains not only a series of blue-prints, but a co-ordinated program of protein synthesis and the means of controlling its

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execution” (Jacob and Monod, 1961). This work extended to eucaryotes was one first demonstration that genes are under the controls of some regulators.

Epigenetics, an old but evolving concept

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1.3.

In the 1960ies, connecting the new knowledge on gene regulation with a better understanding of cellular differentiation became an obvious task for the scientific community. This was formally

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emphasized in the theoretical model of Britten and Davidson (1969) where a network of different

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regulators were proposed to combine and interact to control genes expression in a specific sequence during cellular differentiation (Britten and Davidson, 1969).

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Although the word epigenetic was little in use, this model was echoing the short reflection published by the embryologist Waddington in 1942 “The epigenotype” in which the term epigenetics was first coined. In his visionary thinking at a time when genes were still a theoretical concept, Waddington

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was emphasizing the need to identify the “causal mechanisms at work” in the relation between genetics and phenotypes, and he called for the rapprochement of genetics with experimental embryology (Waddington, 1942). The name “epigenetics” was proposed to describe the study of the epigenotype (how genetics is involved in organ development). In his metaphoric drawing, the famous “epigenetic landscape” (Waddington, 1957), Waddington illustrated cell differentiation as a ball rolling down a hilly landscape. The choice of the path down to the last valley is influenced by the environment and by the actions and interactions of factors within the cell. Following one valley or another downhill is determining the fate of the cell, and the equilibrium reached in the bottom illustrates the stability of the differentiated state. This drawing is still widely used to illustrate the epigenetic concept, as well as the cellular differentiation process. We know today from the pioneer work on nuclear transplantation (Briggs and King, 1952; Gurdon, 1962) and from Yamanaka (Takahashi and Yamanaka, 2006) on mature cell reprogramming that mature cells can be reprogrammed into pluripotent cells (Nobel Prize in Physiology or Medicine, 2012 for Gurdon and Yamanaka), in other words, that the cell can climb back

ACCEPTED MANUSCRIPT the Waddington landscape. The fact itself that the differentiated cell need some extensive reprogramming before reaching back pluripotency raise once again the question of the molecular mechanisms at play. The complexity of the reprogramming induction, be it driven by external

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transcription factors or by exposure to epigenetic drugs and/or oocyte factors is to be emphasized,

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and reprogramming is still far from being fully mastered presently: a cell cannot ‘climb back’ alone. This cloistering of epigenetic to cellular differentiation opened up to a broader definition after

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methylation of the DNA cytosine (see 3.1.) was proposed (Holliday and Pugh, 1975; Riggs, 1975), and then proved to be one mechanism involved in maintenance and transmission of gene silencing. Two additional definitions of epigenetics took place near Waddington’s “1) The study of the changes in

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gene expression which occur in organisms with differentiated cells, and the mitotic inheritance of given patterns of gene expression, and 2) Nuclear inheritance which is not based on changes in DNA sequence” (Holliday, 1987, 1994). Very often, only this last item is reported, which cripple

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epigenetics of its two other most important components. More recently, Bird (2007) and Goldberg et al. (2007) proposed to refine again the definition of epigenetics to fit with the accumulated knowledge. Epigenetic events could be described as “ the structural adaptation of chromosomal

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regions so as to register, signal or perpetuate altered activity states” (Bird, 2007). In his definition,

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Bird portrays epigenetic marks as responsive to a change of state imposed by other events, not proactive. It opens up the possibility that identical combination of genes will have different

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developmental consequences. More recently, Bonasio et al. (2010) gathered these views and proposed the concept of trans and cis epigenetic signals, to categorize more clearly the mechanisms

1.4.

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allowing the self sustaining of the transcriptional response when the originating stimulus is gone.

Epigenetics or the reconciliation with Lamarck’s theory of evolution?

Nowadays, it is well accepted that environment affect the functioning of living organism, and that epigenetic mechanism are involved in the transmission of environmental cues to the cellular and molecular network (Feil and Fraga, 2012). It is therefore often proposed to parallel the environmental trigger of Lamarck’s theory to the environmental influence on epigenetic changes in the cells. Indeed, in his theory of evolution, the French naturalist Lamarck (1744-1829; Philosophie Zoologique, 1809) theorized that animals adapt to the constraint of the environment by changing during their lifetime. The use or disuse of one organ in response to environmental stimuli would be at the origin of animal diversification and evolution, as a fast adaptive response to environment since those acquired traits would be inheritable. The modern genetic was more prone to put forward the Darwin (1809-1882) theory (revisited and interpreted to the light of understanding heredity via the genes) in which natural selection of the most fitted was the driving force of evolution. Revisiting Lamarck’s theory to the light of epigenetic inheritance is however still controversial, as it would

ACCEPTED MANUSCRIPT require that acquired traits be stably transmitted over multiple generation by way of stabilized modifications in the gametes. Transgenerational transmission will be developed later in this paper.

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2. The molecular basis of epigeneticEpigenetic relies on molecular and structural mediators,

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discovered even before the term epigenetic was back in use (Holliday and Pugh, 1975). These include DNA methylation, posttranslational modification of histones, chromatin organization, and non coding

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RNAs. The different marks influence gene transcription by a combination of actions such as steric exclusion of transcription factors, recruitment of binding proteins with specific function in the transcription machinery (either inhibitory or activating), changes in DNA-protein affinity resulting in

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increase or decrease in chromatin compaction, and thus in DNA availability to the transcription

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regulatory network.

2.1. DNA methylation

This most widely studied epigenetic mark consists in the covalent binding of a methyl group to a DNA

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base, referred to as DNA methylation. In vertebrates, this modification takes place on the cytosine

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base, and exclusively when this cytosine is followed by a guanine (Figure 1A), leading to refer to the methylation status of the CpG dinucleotide. It was demonstrated that cytosine DNA methylation in specific genomic loci is associated with silent chromatin, and it is admitted that DNA methylation

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stands for a stable silencing status of genes expression. In mammals, some genomic areas statistically enriched in CpG are referred to as CpG islands (more often hypomethylated, Deaton and Bird, 2011). The concept of CpG islands is much related to the statistical algorythm used to identify them (Han

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and Zhao, 2008). These authors demonstrated that 5 different fish species (zebrafish, tetraodon, stickleback, fugu and medaka) could have very divergent CpG numbers and density. In mammals, DNA methylation is involved in silencing one of the two X chromosome in the female (X inactivation), in silencing one allele from a given parent to allow dosage of gene expression (parental imprinting), and in silencing of transposable elements. The same relation between DNA methylation and gene expression silencing is widely reported in fish (Lee et al., 2015 for the latest), although parental imprinting and X inactivation are still to be demonstrated (McGowan and Martin, 1997; Ma et al., 2011). At the opposite of methylation and silencing, the lack of methylation in promoter region is indicative of a permissive state of transcription. It is not always associated with active transcription as the regulatory network has to be turned on to trigger transcription. It should be kept in mind that in human, 70 to 80 % of the CpG sites are constitutively methylated.

ACCEPTED MANUSCRIPT At each cell division, the DNA methylation pattern is transmitted to the daughter cells during DNA replication (cellular memory). Cytosine methylation of the newly formed strand is catalyzed by a DNA methyl transferase (DNMT), DNMT1, which will specifically recognize hemi-methylated DNA. During

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early development or germline formation, de novo methylation of unmethylated DNA is catalyzed by

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other DNMTs, the DNMT3A and 3B. It is noteworthy that in fish, up to 6 Dnmts were found to be orthologs of Dnmt3a and b (Takayama et al, 2014). How DNMT3s recognizes the sites which are to be

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methylated in a naked landscape is the matter of extensive research (Jeltsch and Jurkowska, 2014). In some biological process (zygote formation after fertilization, primordial germ cell differentiation (see in 2.1.), DNA methylation has to be erased, either by a passive phenomenon amplified at each new

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cell division (during which the newly synthetized strand is not remethylated), or by active demethylation. The active demethylation process has been the matter of much debate some years ago as no demethylase could be convincingly identified. It is now well accepted that demethylation is

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taking place after the methyl group is hydroxylated by dioxygenases from the TET family proteins (reviewed by (Kohli and Zhang, 2013). The hydroxymethyl cytosine is then removed by a complex cascade involving different cellular machineries including the base excision-repair system (Kohli and

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Zhang, 2013) so that a naked cytosine finally replaces the methyl cytosine. The same process was

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also reported in fish (Kamstra et al., 2015).

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2.2. Post translational modification of histones and chromatin organization Histones are one major proteic component of chromatin, the nucleoproteic complex where all DNArelated processes function. In other terms, chromatin regulates the accessibility of the genetic

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information. Chromatin itself is organized in two different structures: euchromatin is gene rich, less electron dense as an indication of its open structure, and is the site of active gene transcription. Heterochromatin is much denser, gene poor and is the site where all silenced genome lies (Grewal and Jia, 2007). Two sub-class of heterochromatin are also considered: constitutive heterochromatin remains tightly packed throughout the cell cycle and contains the terminally locked genomic loci (transposons, imprinted genes and inactive X in mammals). On the contrary, facultative heterochromatin corresponds to loci where the gene activity can be restored upon cell signaling. The basic unit of chromatin is the nucleosome, well known from the beads on a string structure (Figure 1B). In somatic cells, the nucleosome consist in 2 sets of 4 different histones, core histones H2A, H2B, H3, and H4 (resulting in a proteic octamere). Almost two loops of DNA are wrapped around the octamere (145-147 base pairs) (Luger et al., 1997). Another histone, the linker H1 histone acts as a knot at the end of the nucleosome. Core histones have a central globular domain with a high affinity for DNA. This domain is responsible for DNA wrapping and nucleosome architecture. The second histone domain is the N-terminal tail protruding from the nucleosome (Luger and Richmond,

ACCEPTED MANUSCRIPT 1998) (Figure 1B). This amino-acid tail is lysine and arginine rich, and is the site where numerous post-translational covalent modifications take place (Rothbart and Strahl, 2014). These modifications are also coined “histone code” (Strahl and Allis, 2000). The best studied modifications are acetylation

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and methylation (both discovered by Allfrey et al., 1964). These modifications change histone-DNA

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and histone-histone interactions and serve as docking site for protein effectors also called readers (Musselman et al, 2012). The reviews of Sadakierska-Chudy and Filip (2015) and Musselman et al

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(2012) are providing an extensive survey of the histone tail modifications and histone code readers, and the review of Rothbart and Strahl (2014) emphasizes the complexity of the agonistic and antagonistic interactions between these modifications. The present review will be restricted to the

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most generic modifications for a simplified picture from the above-mentioned reviews.

Histone tail acetylation is set up on lysine residues of all four core histones by enzymatic “writers”,

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the histone acetyl transferases (HATs) and removed by “erasers”, the histone deacetylases (HDACs). Acetylation neutralizes the lysine positive charge and increases the tail bulkiness. This mark on histones H3 and H4 is mainly associated with a transcriptionally active state. Histone tail mono-, di or

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trimethylation of lysine or arginine residues is found mainly on H3 and H4 tails. Histone methylases

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(HMTs) and demethylases (HDMs) have been identified as writers and erasers. Depending on the site, histone tail methylation is associated with permissive or repressive state of gene expression.

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Both marks (acetylation and methylation) have been identified in fish as well (Lindeman et al., 2011; Andersen et al., 2012; Andersen et al., 2013): euchromatin is characterized by the presence of the transcriptionally permissive modification H3K4me3, standing for trimethylation of the 4th lysine

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residue (from the N-terminal end) on histone H3. Three other major permissive modifications deal with histone acetylation: H3K9ac, H4K9ac, H3K27ac. Repression is signed mainly with some trimethylation, the most prominent being H3K9me3, but also H3K64me3 and H4k20me3. In facultative heterochromatin, H3K27me3 signs a transient silenced state, and this mark can be found with permissive ones such as H3K4me3. The main histone modifications are summarized table . It should be noted that some writers such as HMT can be found in multifunctional protein complexes. Among the best studied are the polycomb repressive complexes (PRCs) involved in H3K27 trimethylation and chromatin compaction, and the trithorax complexes (TRX) involved in H3K4 trimethylation and maintenance of gene expression (Noordermeer and Duboule, 2013).

It should be kept in mind that the histone and DNA modifications can influence each other in very complex sequences which are the subject of intense research. In the same line, interactions between readers and writers/erasers are paramount. Deregulation of these interactions is leading to alteration in tissue function and disease.

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2.3. Non coding RNAs Non coding RNAs (ncRNAs) are a huge molecular class encompassing infrastructural (or

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housekeeping) RNAs such as transfer or ribosomal RNAs, and regulatory RNAs (Kaikkonen et al, 2011;

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Morris and Mattick, 2014). The later include (micro) miRNAs, (piwi-interacting) piRNAs, (small interference) siRNAs and (long) lncRNAs displaying a wide range of activities at the transcriptional

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and post-transcriptional level. The vast functional array of those ncDNA and their involvement in differentiation and development makes difficult the sorting of exclusive epigenetic modulators (reviewed in Sadakierska-Chudy and Filip, 2015), but it is often proposed that the ncRNA contributes

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to link environmental signals and the epigenetic modulation of transcription. They also contribute to the cellular memory by driving the epigenetic modulators (writers/erasers, readers) at the right place in the genome after mitosis, and after fertilization. Piwi RNAS are also described as modulators of

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transposon silencing in the germline. However, the consideration of all these above mentioned noncoding RNAs as key players in epigenetics could be controversial when epigenetic definition is considered sensu stricto. By contrast, the best described epigenetic modulators are lncRNAS (Ponting

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et al., 2009; Mercer and Mattick, 2013), involved among others in the striking case of the entire X

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chromosome inactivation in female (in mammals). The Xist non coding RNA is expressed on the targeted X chromosome and mediates the silencing of the genome via recruiting the polycomb

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repressive protein complexes (PRC1 and 2) which then initiate trimethylation of H3K27 and compact chromatin, thereby establishing a silent chromatin state. Once chromatin is modified, Xist can be deleted without restoration of the permissive state of the X chromosome. More generally, lncRNAs

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guide many of the proteins that catalyze DNA methylation and posttranscriptional histone modifications.

3. Epigenetics of the germ cells As introduced above, the epigenome is extensively reprogramed during the two major processes in an organism’s life: gametogenesis and early embryo development (Hales et al., 2011; Jammes et al., 2011) (figure 2). Before gametogenesis, the primordial germ cells (PGCs) in the embryo undergo a complete erasure of the somatic marks acquired during the first steps of embryo development. Then, upon gametogenesis, new epigenetic marks are acquired in relation to gamete function, but also to ensure proper embryo development after fertilization. The same epigenetic resetting is observed after fertilization, during the first steps of embryo development. Gametes are far from being only gene and food bags, they carry sensitive information which is to be passed on or not to the offspring. Epigenetic programming in male and female gametes is crucial for post-fertilization success and for a

ACCEPTED MANUSCRIPT healthy offspring development in mammals (Bromfield et al., 2008; Boissonnas et al., 2013). As it will be developed later in this review, this statement is very likely true in fish as well. Epigenetic changes in germ cells have been documented only in mammals so far, mainly in human

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and mice, and bovine to some extent. In fish, it is the final product, spermatozoon and oocytes,

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which are well described today.

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3.1. Primordial germ cells

Early during embryo development PGCs specification occurs, the PGCs migrating towards the

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embryonic genital ridges. In mice, committed cells undergo a genome-wide methylation erasure, hence achieving the more intense hypomethylation in any cell type, known as the “epigenetic ground

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stage” (Seki et al 2005, Hajkova, 2011, Seisenberg et al 2012, 2013). Demethylation is probably driven by the expression of pluripotency genes, which are at this stage highly methylated in somatic cells, such as Nanog and Oct4 (Magnúsdóttir et al 2014). The PGC-specific demethylation resets all genes

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into an open configuration. In mammals, it allows that X inactivation in female is erased, and that

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parental imprinting is lost. From this “ground stage” remethylation of germinal cells begins in male embryos after sex determination, whereas in females it is initiated at later stages (see below). This exceptional germline epigenetic reprogramming is required to reestablish the imprints in the next

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generation according to the sex of the embryo, to help in acquiring the pluripotency of germ cells, and to erase aberrant epigenetic information preventing their inheritance to the offspring via

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germinal cells.

One characteristic of fish compared to mammals is that a matrix of mRNAs and proteins located in the oocyte, called the germplasm, will distribute into a subset of embryonic cells very early during development (1-2 cell stage). It is the presence of the germplasm in these few embryonic cells which is responsible for their further specification as germ cells (reviewed by Raz, 2003). This occurs very early during embryo development, long before the embryonic genome activation is set up, and it is not known yet whether there is any need for an erasure/rewriting of the epigenetic marks as the one described during gametogenesis in mammals.

3.2. Spermatogenesis and the spermatozoon epigenetic profile

The spermatozoon is a unique cell in terms of nuclear organization and function, resulting from the highly regulated process that is spermatogenesis. After the nascent PGCs have migrated to the

ACCEPTED MANUSCRIPT genital ridges, PGCs become the gonocytes and show active proliferation while interacting with the Sertoli precursors. This mitotic period ends up at the spermatogonia stage, before meiosis is triggered, the later leading to the spermatocytes formation. At the end of meiosis, spermiogenesis is

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initiated, changing the haploid round shaped spermatids into spermatozoa. It is during

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spermiogenesis that the nucleus is packaged with new specific proteins (protamines, or modified histones, see the companion paper by Herraez et al, 2016, this volume), and that transcription is

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gradually repressed.

3.2.1. Spermatogenesis

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In mammals (with most knowledge coming from human and mice studies), all three epigenetic actors that are DNA methylation, histone modification and ncRNA activity are involved in spermatogenesis

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(Miller et al, 2010, Carrell and Hammoud, 2010; Yadav and Kotaja, 2014): at the beginning, the mitotic period in the genital ridge is characterized by an increase in the permissive marks H3/H4ac and H3K4me, and a low DNA methylation level. In gonocytes however, DNA methylation of

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transposable elements and of imprinted genes is being established. Then, at meiosis (after birth), the

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decrease in the permissive H3K4me3 histone in spermatocytes parallels the exit from the stem cell stage. This meiotic phase is also showing an increase in H3K27me3 and H3K9me3 marks, promoting progressive gene silencing. At the end of meiosis, the formed spermatids display a very repressed

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chromatin, with a high level of silencing modifications in the histones and a more methylated DNA. During spermiogenesis, the proteins replacement requires a new wave of epigenetic changes in the histones, to allow protamine establishment (Nair et al 2008). As stated above, data are still missing in

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fish about germline epigenetic changes during gametogenesis. What is known is that during spermiogenesis nevertheless, the nucleosome disassembly preceding nucleoprotamine incorporation requires histone hyperacetylation in trout (Christensen et al, 1984).

3.2.2. Spermatozoon

In mammals, the final product that is the highly packed spermatozoa is in fact not completely devoid of histones, and it has been shown that the genomic loci with remaining histones were involved in early embryo development (reviewed by Hales et al., 2011). This was initially stressed by Hammoud et al., 2009 and Brykczynska et al., 2010 who showed that these histone-bearing loci contained developmental key genes whose promoters were highly enriched in the permissive H3K4 me2 and me3 marks, although some were poised by H3K27me3. In these loci, hypomethylation was also matching developmental promoters of the self-renewal network of embryonic stem cell. This pattern

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the establishment of the proper DNA methylation status during spermatogenesis has been linked in

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humans to different infertility disorders (Rajender et al 2011, Heyn et al 2012). Most strikingly, it has been shown that in human, constitutive heterochromatin (silenced sequences) in sperm bore specific

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histone marks which were transmitted to the oocyte, allowing to establish back the constitutive heterochromatin in the paternal half of the embryo chromatin (van de Werken et al., 2014). This emphasizes further how the epigenetic pattern of the sperm is important for the proper gene

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expression and silencing in the embryo, and at the origin how epigenetic marks can explain male subfertility (Boissonnas et al, 2013).

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If there is a huge lack of information on epigenetics during gametogenesis in fish, it is different for the fish gametes. Zebrafish is being increasingly used to study epigenetics in gametes (Wu et al., 2011; Jiang et al., 2013; Potok et al., 2013), because most genomic tools are available in this model

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species. Zebrafish sperm epigenome has been deeply analyzed in those three papers that provided a

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genome wide perspective of the histone marks (Wu et al., 2011) and DNA methylation pattern (Wu et al., 2011; Jiang et al., 2013; Potok et al., 2013) using ChIP (immunoprecipitation of chromatin

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fragments bearing specific histone marks), MeDIP (methylated DNA immunoprecipitation), and methyl-Seq after DNA bisulfite treatment. There is no similar knowledge on the epigenome in any

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other teleost.

The main characteristics in zebrafish sperm is that DNA is highly methylated (91-95% of the analyzed CpGs, (Jiang et al., 2013; Potok et al., 2013)), more than in human. It was also stressed that hypomethylated loci are not randomly distributed, as they are found in promoters regions of genes related to developmental regulators, transcription factors and metabolism/biosynthesis. The top 250 hypomethylated genes are dominated by embryonic transcription factors (Wu et al., 2011). As observed in human, the hypomethylated regions correspond to genes expressed early during embryo development. In sperm, those permissive hypomethylated regions are not necessarily enriched in active histones marks. The highest enrichments in active histone marks (H3K4me3, H3K4me2, H4K16ac) are found in in genes related to meiosis and other spematogenic events, whereas those marks are only moderately enriched in developmental loci (Wu et al., 2011). Additionally, some loci important for developmental processes are even shown to be enriched in the transient repressive modifications (H3K27me3) even though they are hypomethylated. On a general scale, the marks in sperm seem to correlate with the gene expression timing in the embryo: genes expressed before or

ACCEPTED MANUSCRIPT at early mid blastula transition (MBT, when the embryonic genome is massively activated) are enriched with activating histone marks, while those which are expressed later during development, or which are undergoing hypermethylation in adult stages, are poised in a multivalent chromatin

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pattern, bearing together H3K4me3, H3K27me3 and DNA hypomethylation (Wu et al., 2011). The

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authors hypothesize that repressive marks are needed to avoid spurious expression of these genes in the germline, and activating marks prevent the DNA methylation of these loci, avoiding their early

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inactivation in the embryo. DNA hypomethylation would be required soon after fertilization in order to enable transcription of these essential transcription factors under paternal control (Wu et al

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2011).

3.3. Oogenesis and the oocyte epigenetic profile

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Gametogenesis in female germline has a different timing from that of male germline. Meiosis starts during embryo development but is stalled around birth: the primary oocyte will resume meiosis only after puberty: at each sexual cycle, oocyte growth will include active transcription to accumulate

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maternal mRNAs, then the primary oocyte will resume meiosis to form a secondary oocyte. Meiosis

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is then stalled again in secondary metaphase stage for a short period, up to fertilization. Because the gametogenetic material is difficult to obtain in quantities compatible with genome wide analysis,

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most histone modifications data are obtained from immunological studies: the acute transcriptional activity at the end of the primary oocyte growing phase is associated with increased histone acetylation, while meiosis resumption signals acetylation reduction (Duffié and Bourc'his, 2013). De

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novo DNA methylation begins only several days before ovulation, in growing oocytes during the germinal vesicle stage (GV) (Bourc'his and Proudhon, 2008; Tomizawa et al., 2012). Again, it is technically challenging to obtain mature oocytes in sufficient quantities without using superovulation (that could have epigenetic effects), and there is little information about the epigenetic profile of the mammalian female gamete (Hales et al 2011).

This in one advantage of most fish species, including cyprinids, over mammals: females are releasing several hundreds of mature oocytes, making their epigenetic study easier. Zebrafish oocytes have a less methylated nuclear DNA than sperm (75% of the CpGs), and the extremely abundant mitochondrial DNA is almost entirely hypomethylated (Potok et al 2013). The functional analysis performed by these authors on the oocyte methylome revealed that some clusters of genes display the same pattern in both sperm and oocyte: genes involved in early development and metabolism which are hypomethylated and genes related to later functions which are hypermethylated. On the contrary, other clusters show a differential methylation (14% of them according to Jiang et al (2013)):

ACCEPTED MANUSCRIPT hypermethylated in sperm and hypomethylated in oocytes (factors expressed in mid- to latedevelopment) or vice versa (genes involved in germline dazl, piwi and vasa, some developmental factors and many hox genes) (Potok et al 2013). Those hypermethylated developmental factors

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should therefore undergo demethylation of the maternal allele after fertilization before the onset of

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embryonic genome activation. To the extreme, Jiang et al (2013) suggested that the oocyte-specific methylation pattern is gradually discarded after fertilization, and that the paternal chromatin would

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“direct” the methylation pattern of the early embryo. This will be discussed in the next section.

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3.4. Parental imprinting in fish gametes

As introduced earlier in this review, one question remains to be addressed in fish, in relation with the establishment and maintenance of parental imprinting: do fish gametes bear specific silencing of

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some genes which would allow that only one allele is expressed in the offspring? This question was raised from an evolutionary point of view almost 2 decades ago (McGowan and Martin, 1997). In the past, several groups tested whether some genes known to be parentally imprinted in mammals

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would bear differential epigenetic marks in fish. For example, a region in the goldfish igf2 gene is

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hypermethylated in sperm, and hypomethylated in oocyte. However, the parent-specific methylation pattern is not maintained in the embryo (Xie et al, 2009). In the same line, the zebrafish ortholog of

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the PEG1/MEST imprinted gene in mammals showed bi-allelic expression in zebrafish larvae (Hahn et al, 2005), therefore refuting the presence of a parental-of-origin dosage. Paulsen et al (2005) also demonstrated that some imprinting control elements identified in mammals could not be identified

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in fugu or in zebrafish, and imprinted gene clusters in mammals were poorly conserved in fish (Dunzinger et al, 2007). Although this may lead to the conclusion that imprinting does no exist in fish, Ma et al (2011) showed that the goldfish ntl gene is differentially methylated between sperm and eggs, and that the paternal allele showed a transcription activity earlier than the maternal one in the embryo. Some hints of a possible imprinting mechanism were also provided in a study exploring a transgene expression in zebrafish (Martin and McGowan, 1995). From these divergent studies, it is still impossible to definitely state about the existence or not of parental imprinting in fish. It should be kept in mind that natural gynogetic population exist, or that sex determination in fish can change with environmental conditions, or that some fish underwent two additional whole genome duplication compared to mammals. These specificities may render imprinting mechanism more subtle and less generic than in mammals.

4. Changes in the epigenetic pattern during embryo development

ACCEPTED MANUSCRIPT The epigenome of the new individual is established soon after fertilization, during early embryo development. Global changes during this period affect DNA methylation, suffering from bulk and fast variations as well as from histones modification. As stated before, the methylation degree is high in

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the spermatozoa and lower in oocyte, with several differentially methylated CpG clusters among

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male and female gametes. In a short period of time after fertilization the embryo shall i) recover the epigenetic signature of embryonic cells, maintaining their pluripotency and differentiation abilities

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and ii) drive a specific epigenetic program in a subset of cells, the primordial germ cells (PGCs), to develop the germline. Mistakes that occur during this stage of development can have a later impact

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on life or even being inherited by future generations (McCarrey 2014).

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4.1. Epigenetic changes in mammalian early embryo

Most knowledge accumulated on the methylation changes in the parental chromatin after fertilization was obtained in mouse and human, leading to the well known canonical figure (Figure 2,

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Reik et al, 2001): in mice, fertilization is followed by a fast demethylation of the sperm pronuclei ,

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from which escape some genomic regions including the imprinted genes (Hales et al 2011). It is important to know that studies in other mammalian species failed to demonstrate such a thorough

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demethylation process in the paternal chromatin (Beaujean et al, 2004 ; Beaujean, 2014). Demethylation of the maternal genome is slower and thought to rely on a passive mechanism in a replication-dependent manner. In this process, the inactivation of DNMT1 is responsible for the inability to methylate the new DNA strand in the successive cell cycles and, at the morula stage, a low

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overall DNA methylation is reached (Hales et al 2011, Seisenberger et al 2012). Then, DNMT1 activity is restored and de novo methylation begins, reaching at gastrula similar levels to that of somatic cells. X chromosome inactivation in females also takes place at this late stage: long non-coding RNAs and DNA methylation will silence one of the two X chromosomes (Augui et al, 2011), either the paternal or maternal one in a random fashion within the embryonic cells. With regards to protein modification, sperm protamines are readily replaced by maternal histones soon after fertilization while the maternal chromatin completes meiosis. As reviewed by Hayes (2011) in the mouse, there is a clear difference in the modified histones composition between the paternal and maternal chromatin up to the 4 cells stage of the embryo. Later on, the pluripotent inner cell mass –ICM, at the origin of the embryo- and the trophectoderm cells –TE, at the origin of the extraembryonic tissues- will form in the blastocyst, bearing very different histone modification pattern and gene expression which can no longer be compared to fish embryo development.

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4.2. Epigenetic changes in fish early embryo

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Although it is often proposed that methylation changes during early development in fish follow the

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mouse pattern, with a global DNA demethylation after fertilization followed by the re-establishment

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of methylation upon embryo genome activation, divergent data requires cautiousness with this statement. Back in 1999, Martin et al demonstrated that the establishment of DNA methylation in the zebrafish embryo just before embryonic genome activation was necessary to allow normal development. Collas (1998) also demonstrated that the early embryos were displaying a DNA

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demethylation activity, followed by a DNA methylation activity. These two studies agreed on the existence of methylation changes, but they did not assess whether it was linked to a global change

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involving a ground zero methylation level in the embryo. At the same time, Macleod et al (1999) were showing that no genome wide changes in DNA methylation were taking place in zebrafish, although this assumption was relying on the sole study of the promoter region of 3 candidate genes,

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and on a global study with restriction enzymes whose sensitivity was questionable. Indeed, with the

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same method in the same species, Mhanni and McGowan (2004) were proposing later on that the 2 cell stage was hypomethylated and that methylation was increasing at stages previously described as

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stable by Macleod et al (1999). And with the same method, Walter et al (2002) were concluding in medaka that the global DNA methylation level was stable during development. Mackay et al (2007) proposed a preliminary immunolabeling of embryo methyl cytosine. There was a tendency to a lower

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labeling at early stages, although it cannot be excluded that the bigger embryonic cells at early stages are not as easily accessible to the antibody as later on. In the same type of experiment using immunofluorescence, we failed to see convincing global changes during development (Labbé et al, 2013, Figure 3). In conclusion we really believe that answering this question with the above mentioned tools will deserves a carefully settled experimental work that is still missing. However, the development of genomic tools provided more consistent information. By whole genome bisulfite sequencing, Jiang et al (2013) demonstrated in zebrafish that the global methylation level at the 16 cells stage is matching the average methylation level of sperm (91 %) and oocyte (80 %), and that the percentage was increasing from this stage up to gastrulation. No stage earlier than 16 cells was assessed in this study. Potok et al (2013) reach almost the same conclusion although they mixed together the 2 to 16 cells embryos. Most importantly, these authors showed that in some genomic regions, this averaging pattern between sperm and oocyte level was not observed, and that instead the 2-16 cells embryos had a methylation pattern as low as the oocyte one. In all, no global demethylation to a ground zero state was reported.

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At a gene level, remethylation is not equally synchronized. The detailed analysis of remethylation in 5 genes (vasa, rassf1, tert, c-jun and c-myca) demonstrated that the timing was specific for individual

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genes (Fang et al 2013). Different research groups revealed that methylome is reprogrammed at

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embryogenesis following the paternal model, reaching at MBT a virtually identical pattern to the sperm (Andersen et al 2012, Potok et al 2013, Fang et al., 2013). Methylation patterns in most sperm

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promoters are similar to those of MBT-embryo (Lindeman et al 2010) thanks to the gradual reset of the maternal methylation pattern and the maintenance of the paternal one (Jiang et al 2013). Therefore, genes related to germline (vasa, piwi, dazl) or developmental genes such as most hox

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clusters, are hypermethylated in oocytes and hypomethylated both in sperm and embryos at midblastula stage (Potok et al 2013). At the ≈1000 cells stage, the maternal methylation state is similar to that of the sperm and cells preserve the totipotency (Jiang et al 2013). Nevertheless the

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paternal genome is not “copied” as a template during reprograming, as demonstrated the normal epigenetic remodeling suffered by partenogenetic embryos, lacking paternal contribution (Potok et al 2013). These discoveries show important differences in DNA reprogramming mechanisms between

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zebrafish and mammals after fertilization, because DNA methylation is not cleared in the sperm and

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seems to be directly inherited by the embryo upon MBT (Jiang et al 2013). The comparison of the methylome at different developmental stages revealed that, after midblastula, methylome is further

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modified in specific regions, but somatic cells show an important similarity to the spermatozoa, as they are both utterly differentiated cells. Lee et al (2015) have now provided a precise map of the zebrafish methylome during development, showing a huge number of genes which are differentially

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methylated during development. Most importantly, these authors showed that among the differentially methylated regions during development, only 10 % of them were located in gene promoter regions, the others being intronic or intergenic, nearby developmental genes. Last these regions proved to display enhacer activities. This most recent paper unravels a new insight into early development regulation by DNA methylation.

Regarding histones, early development has been investigated for several years (Vastenhouw et al, 2010; Andersen et al, 2013). Different profiles have been identified in the promoter regions of developmentally-regulated genes, showing sets of activating marks associated or not with repressing marks or bivalent marks (Lindeman et al 2010, 2011). The unmethylated DNA at these promoters support a transcriptionally permissive organization, whose expression can be modulated by the particular set of histone modifications, revealing a mechanism involved in the control of gene expression in specific stages of development. Albeit very repressed, multivalent chromatin in developmental loci is still able to be switched on for activation (Lindeman et al 2011). Wu et al (2011)

ACCEPTED MANUSCRIPT also reported a different expression timing of genes during embryo development according to the histone modifications in the spermatozoa, the genes enriched in activating marks -related to cell cycle and metabolism- being expressed before midblastula, and those with a lower level of activation

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being transcribed at later stages. Although no reports exist regarding the epigenetic profile of other

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fish species it is likely that similar differential modifications should exist in different blocks of genes, providing a mechanism to control gene expression both during spermatogenesis and, after

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fertilization, during early embryogenesis.

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4.3. Developmental events under epigenetic control

Beyond the establishment of the DNA methylation pattern in early embryogenesis, many different processes are regulated by changes in the epigenetic signatures, from modest gains and losses of

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global CpG methylation to changes in methylation of specific promoters or to intense remodeling of the associated histones. Cell differentiation, tissue specification, organogenesis, aging or sex differentiation, are some of the events related to epigenetic reprogramming. The knowledge of the

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mechanisms involved in specific processes is still very fragmentary, but rapidly increasing in some

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areas such as heart or pancreas development (Philippen et al 2015, Seki et al 2014, Arnes and Sussel 2015). Unraveling the mechanisms underlying cell differentiation, cell commitment and

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organogenesis is a very active research field nowadays (reviewed by Boland et al 2014) in which zebrafish has a role to play as a model species. Progression from embryo to mature tissues involves in zebrafish a redistribution of methylation signatures in CpG islands and repetitive sequences (LINEs,

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SINEs and LTRs) (McGaughey et al 2014), reaching a tissue-specific signature responsible for the particular cell phenotype.

5. Epigenetic trans-generational inheritance

Epigenome is more susceptible than genome to environmental changes (McCarrey 2014). Moreover epigenetic changes can be mitotically and/or meiotically inherited passing to the daughter cells after division or affecting the gametes and passing from parents to offspring. Inheritance of epigenetic changes is considered transgenerational when it is stabilized in the germinal cells and transmitted without variations, thus affecting the phenotype of multiple generations never exposed to the disrupting agent. Focusing on fishes, the F2 should show the epimutation to be considered as trangenerational inherited, because the first generation is directly derived from germinal cells that were affected by the agent during F0 exposure. As recently reviewed and discussed by Heard and

ACCEPTED MANUSCRIPT Martienssen, (2014), epigenetic inheritance was extensively described in plant where the germline keeps the memory of the environmental cues the somatic cells were exposed to. It is considered as a short-term adaptive mechanism to the environment that could be transmitted over several

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generations because of close connection between somatic and germline programming. On the

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contrary, epigenetic reprogramming during PGCs specification and gametogenesis in vertebrates, and later on during the first step of embryo development, offers an opportunity to correct aberrant

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marks. This should prevent the transmission of epimutations and allow that the full genetic potency of the next generation is restored. Therefore, the memory of the endured environmental factors is lost when the initial trigger is gone. Nevertheless, some reports tend to prove the inheritance of

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epigenetic modifications through generations in a sex-specific manner (Soubry et al, 2014, McCarrey, 2014). To some extent, some parental (or intergenerational) effects were reported as a consequence of in utero exposure of the embryo during germline programming, leading to changes affecting the

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grandchildren. Some rare examples of epimutation transmission were also described in vertebrates, although those were not triggered by environmental cues.

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Apart from theses changes directly affecting the mother or father’s germline through the grand-

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mother’s environment, it remains very difficult to prove that mechanisms resembling transgenerational epigenetic would not be due to cryptic sequence variation (Daxinger and Whitelaw, 2010; Heard and Martienssen, 2014), or to behavioral transmission (Skinner, 2014). A lot

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of research is developed today to master this question, as the implication is huge in the perspective of health control, selective breeding, or even evolution. The excitation and controversies around Dias’s striking paper (Dias and Ressler, 2014) about inheritance of parents olfactory experience in

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rats is one example of the high expectation, not only from the scientists, on the topic. Indeed, we know that some areas of the genome are constitutively silenced after embryo reprogramming, in order to ensure silencing of transposable elements including retroviruses (reviewed by Slotkin and Martienssen, 2007; Castañeda et al., 2011). One cannot exclude that some of the memory mechanisms at play in the faithful silencing restoration could, in a completely different context, transmit some environmental cues.

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Impact of the reproductive biotechnologies

Selection of breeders has been traditionally performed by choosing parental phenotypic desirable characteristics, and different reproductive technologies were developed in order to improve and facilitate animal breeding, farming, and genetic resources management: superovulation, in vitro oocyte maturation, in vitro fertilization, embryo culture, intracytoplasmic sperm injection, X

ACCEPTED MANUSCRIPT spermatozoa sorting, sex reversion, nuclear transfer and cryopreservation are also available. We exposed that the phenotype is resulting from the genotype associated with epigenetic information, and that the gametes are carrying not only genetic but also epigenetic information that will be

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transferred to the progeny. Therefore, any undesirable alteration due to gamete and embryo

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manipulation must be avoided. The impact of these artificial reproductive technologies (ARTs) is being increasingly studied in domestic mammals (Urrego et al, 2014), in model species and in human

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(Jammes et al, 2011). In humans, artificial reproductive technologies (ARTs) have been associated with a higher risk of epigenetic-linked syndromes involving imprinted genes (van Montfoort et al., 2012) such as Beckwith-Wiedemann, Angelman and Prader- Willi syndrome (Carrell and Hammoud,

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2010; Amor and Halliday, 2008; Maher et al., 2003b). Whatever the species, most studies point to embryo in vitro culture as the most sensitive period during which these alterations could be

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produced.

Farmed fish are exposed to the same reproductive technologies, although embryo culture should be less problematic as most fish embryos develop naturally in an external environment. Sex reversion,

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photoperiodic control of gametogenesis, induction of ovulation, and sperm cryopreservation are the

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most used technologies and are the one we should focus on the most with regards to possible epigenetic alteration. To our knowledge, cryopreservation is the only technology for which some

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data are emerging in fish. One reason is that sperm cryopreservation is developed to ensure a proper phenotypic transmission of characters, and therefore the field where epigenetic concerns are the

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most developed.

Sperm cryopreservation is still little studied in mammals, likely because a long standing use of the method in human and cattle did not raise any concern about possible epigenetic alteration. For example, Klaver et al (2012) showed in human that a selection of nine genes including imprinted genes had the same methylation pattern before and after cryopreservation. Another study in mice showed that when embryos were produced by intracytoplasmic injection of sperm cryopreserved at 20°C, they bore the same labeling pattern as the embryos from fresh sperm for DNA methylation, H3K4me3 and H4K12ac marks (Chao et al, 2012). In fish, sperm cryopreservation often relies on the use of methylated cryoprotectant, such as dimethyl sulfoxide (DMSO) and methanol. Originally, some concern arose from the work of Kawai et al, (2010) who demonstrated in vitro that DMSO could produce reactive methyl groups in the presence of reactive oxygen species (ROS), leading to cytosine methylation of purified DNA. Because damaged mitochondria are releasing ROS in the sperm medium upon freezing and thawing, it was important to test whether some hypermethylation could arise from cryopreservation with methylated cryoprotectant. The first published study showed

ACCEPTED MANUSCRIPT indeed some effect of cryopreservation on the genital ridges of zebrafish embryos (Riesco and Robles, 2013): bisulphite sequencing analysis of CpG methylation in the promoter of different gene showed that some regions where hypermethylated after cryopreservation, whereas others were not

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affected. The alteration in the epigenetic status was induced by cryopreservation itself rather than by

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the incubation in the cryoprotective solutions. In mature sperm, preliminary studies (Labbé et al, 2013) showed that the effect of cryopreservation was not always straightforward, and that instead,

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some individual sperm would be globally hypermethylated, and others hypomethylated after cryopreservation. It was the cryopreservation procedure which was damaging, as sperm snap freezing without any additives was not affecting methylation, although all cells were dead. Besides,

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some species would be less sensitive than others to cryopreservation-induced epigenetic alteration. Several questions should be addressed with regards to the cryopreservation technology: i) does species-specific nucleus organization/packing make some species more or less resistant to epigenetic

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alteration, ii) are some genome areas more sensitive to epigenetic alteration than others, or is it only a random process, and finally iii) are these changes triggering any consequences on the offspring. It is known that in some species, fertilization with cryopreserved sperm will induce some developmental

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alteration of the embryos, but the role of epigenetics in this pattern still has to be demonstrated.

As developed by Martinez et al (2016, this special issue), genetic resources preservation relies on

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other technologies: germinal stem cell cryopreservation, proliferation and transplantation; somatic cell cryopreservation and transfer in oocytes. These technologies obviously need to be assessed at the epigenetic level. As reviewed by Armstrong et al (2006) in mammals, nuclear transfer requires

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extensive epigenetic reprogramming, and we have demonstrated recently that as in mammals, epigenetic reprogramming is still not completely mastered in fish (Depincé et al, 2015).

7. Impact of the environment on fish epigenetic changes The impact of environment is of utter importance in fish, an animal group directly exposed to temperature changes, and to water quality. This chapter is at the heart of the trans-generational inheritance perspective exposed earlier, although we have today very little example of environmental effect mediation via the gametes epigenetic pattern.

7.1. Domestication Although not yet at the stage of publication, we know that fish domestication is one process more and more under epigenetic investigation. Domestication is referred to as the multi-step process where a species is taken in the wild and submitted to farmed environment for its growth, maturation, reproduction and development. In other words, domestication takes the whole genetic

ACCEPTED MANUSCRIPT background of a species, and tries to adapt a subset of the population to farming conditions (Teletchea and Fontaine, 2014). We do not know yet whether the process involves the selection of the most suited (and the death of the most fragile), in which uncontrolled genetic selection would be

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at stakes, or whether it relies on the adaptation of some individuals via epigenetic modulation.

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Therefore, epigenetic mechanisms in response to these drastic environmental changes deserve some specific attention. As a single example, embryo incubation conditions can affect at early stages the

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ability of the fish to cope with the farming environment, and as demonstrated in mammals, it is likely that the information is passed on to the next generation via epigenetic memory in the gametes for later on modulation of gene expression. This question is almost unique to fish, because among

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farmed animals, fish is almost the only group where domestication is still at stake. 7.2. Temperature

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The most well studied effect of temperature on fish gametes is sex differentiation. Epigenetics of sex differentiation has been analyzed in fish whose mechanisms are particularly variable, ranging for genotypic to environmental sex determination models (Penman and Piferrer 2008). Gonads are the

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only somatic tissues that, according to the activated signaling pathways, can develop in two totally

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different mature organs, ovary or testes, according to the activated or repressed genes long after the establishment of the genotype at fertilization (Munger and Capel 2012), the epigenetic mechanisms becoming of outmost importance. Sex determination is dependent, in several aquacultured fish

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species, on temperature, changes in temperature inducing masculinization via aromatase repression (Lance, 2009). The epigenetic basis of the process has been studied in European sea bass, which is thermosensitive at larval stages. Methylation of aromatase promoter is double in juvenile males than

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in females (Navarro-Martín et al 2011) repressing gene expression. Moreover, the methylation status of the promoter is, only in the gonadal tissue, affected by the temperature during the thermosensitive period. Therefore, high temperature exposure increases the methylation level in females, inducing gene silencing and masculinization (Navarro-Martín et al. 2011). This study provided the first evidence of the link between the environmental ability to modify the epigenetic marks in a particular gene and the changes promoted by temperature in the sex differentiation process, explaining how genetic and environmental sex differentiation mechanisms cooperates in this species. More epigenetic mechanisms related to sex differentiation in fish have been reviewed by Piferrer (2013), helping to understand how environmental conditions during the sex differentiation period can influence the gonadal fate. Based on this striking example, it is to be expected that the known effect of fish rearing temperature during gametogenesis and embryo development will involve gene epigenetic mediation which remains to be studied.

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7.3. Nutrition

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The effect that maternal nutrition has on the progeny is well established in mammals. It is known

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that both, starvation or overnutrition, could produce adverse effects that could be transmitted multigenerationally (Susiarjo and Bartolomei 2014). Radford et al (2014) demonstrated that F1

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mouse embryos´ nutritional environment in the utero alters, once they are adults, the DNA methylome in the germline. This study concludes that metabolic disease in offspring could be explained attending to the nutritional environment in utero during a specific time of germ cell

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development. However, although they found hypomethylated regions in the F1 sperm, their studies in F2 generation suggest that DNA methylation should not be the principal epigenetic mechanism in explaining F2 phenotypes. DNA methylation is only one of the components of the epigenome, and

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other components such as proteins (histone modification) and RNA (non-coding RNAs) could be acting directly or indirectly on the epigenome (McGraw et al 2013). As an example, rats subjected to a diet restricted in protein content produced high cholesterol levels in the progeny that coincided

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with repressive histone modifications at the cholesterol 7 alpha hydroxylase promoter (Sohj et al.

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2011).

Although pregnancy is a sensitive period to altered nutritional conditions, dietary transgenerational

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effects are not only limited to the maternal factor. It is known that paternal malnutrition is able of programming metabolic disease in the offspring (DelCurto et al. 2013). In mice, Fullston et al (2012, 2013) reported that paternal obesity produces epigenetic alterations in their spermatozoa and

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metabolic alterations in progeny; and paternal low protein diets produced changes in promoter methylation status and gene expression levels, in particular those related with lipid and cholesterol biosynthesis in the progeny (Carone et al. 2010). But, what nutritional compounds could be affecting gamete epigenome, and consequently being potential causes of transgenerational effects? It is known that several dietary compounds could affect DNA methylation patterns: geistein, a phytoestrogen present in soy, which is known to have negative effects on mice male reproductive development (Eustache et al 2009). It is also known that deficiencies in some vitamins such as folates could produce DNA methylation alteration in the offspring in rats (Mejos et al 2013). Although most of these evidences have been found in mammals, particularly in mice and rats, there are some data in fish that points to the same evidences. As an example, in Zebrafish, low B-vitamin levels in the feed induced higher inclusion of lipids in the hepatocytes of F1 livers (Skaerven et al. 2014). Considering the reversible condition of epigenetic changes, nutrients must be considered as epigenetic regulatory factors that either could alter or rescue the epigenetic status. In this line, during recent years, different studies are focusing their

ACCEPTED MANUSCRIPT interest in the potential correlation between nutrition, epigenome and better larval performances, particularly in species with commercial interest such as Senegalese sole (Canada et al. 2014).

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7.4. Xenobiotics

Gamete epigenetics could be seriously affected also by other environmental factors such as toxics. It

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is well known that parental exposure to pesticides or other toxic compounds present in the environment could lead to different abnormalities in the progeny produced by gamete defects. It is reported that paternal exposure to polycyclic aromatic hydrocarbons are related to leukemia in the

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progeny (Castro-Jimenez et al. 2011). Moreover, the exposure to such compounds has been also correlated with poor sperm quality (Jeng et al. 2013). Does epigenetics play an important role in such transgeneraltional effect? In a recent review, Soubry et al (2014) revised several compounds such as

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fungicides (ex. Vinclozolin) herbicides (ex. Simazine) pesticides (Roundup) that adversely affected the progeny in animal models even at low dose. Several pollutants, heavy metals are known to disrupt spermatogenesis and compounds such as bisphenol A (BPA) and bis(2ethyl-hexyl)phthalate (DEHP)

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and dibutyl phthalate caused DNA methylation alteration in several generations of animals. It has

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been established that BPA concentration in river water are less than 21 µg/L and in landfill leacheate less than 17.2 mg/L (Crain et al. 2007). In fish it has been reported that low concentrations of BPA

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can cause testicular changes, decreasing mature spermatozoa count and increasing immature sperm stages (Crain et al. 2007). It is known that DEHP impaired reproduction in zebrafish (Corradetti et al. 2013) and a significant reduction of fecundity in fish exposed to DEHP was observed affecting signals

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involved in oocyte growth, maturation and ovulation (Carnevali O et al. 2010). Studies performed in our lab demonstrated that BPA exposure, decreased regeneration ability after zebrafish fin amputation, downregulating genes crucial for regeneration process. In some of the studied genes, downregulation correlated with promoter demethylation (Navarro et al. sent for publication). Moreover, zebrafish paternal BPA exposure demonstrated an increase in hear failures in the progeny up to the F2 and a decrease in remnant mRNA related to early development in spermatozoa from F0 and F1 individuals (Lombo et al. 2015). The estrogenic effect of BPA has been also confirmed in other teleost such as seabream (Sparus aurata) (Maradonna et al. 2014) indicating that this compound could adversely affects to reproductive performance. In a very recent work, Santangeli et al. (2016) observed that BPA down-regulated oocyte maturation promoting signals and promoted apoptosis in mature zebrafish follicles. The authors concluded that these negative effects may be due to epigenetic deregulation.

ACCEPTED MANUSCRIPT Nowadays the relation between environmental contaminants and alterations in organism´s epigenome is commonly accepted; recently it has been proposed to use the epigenetic status of an organism as “foot-printing” to discover the contaminant exposure throughout the organism life time

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(Mirbahai and Chipman, 2014). Fish, and the model species zebrafish, are of major interest in this

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new research area (Kamstra et al, 2014).

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8. Future perspectives

As developed in this review, a huge amount of information about gamete and embryo epigenetics is becoming available in fish, with more outstanding papers every year. The fish community more

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involved in fish breeding and gamete manipulation can now take advantage of this literature to expand to the epigenetic perspective their specific scientific questions about gamete quality, gamete

possible hotspots are proposed here:

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ability for cryopreservation, gametogenesis, sex control, spawning induction and so on. Some

We showed that little information is available about parental imprinting in fish, at odds with the

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extensive research developed in mammals on this topic. Deciphering the existence of a putative

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allelic dosage from parental origin is an important issue. Indeed, several fish farms are using neomales, ie XX sex reversed males, or super males YY sired by XY sex-reversed females. Both broodstock will allow the production of monosexe population, respectively all female and all male (at

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least for species with a male heterozygotie). In this context, we do not know the impact of sex reversion, or fertilization with YY genome, on gene reprogramming after fertilization. Beyond the applied interest of this question, fish sex plasticity offers an outstanding model for studying

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epigenetic and parental imprinting during evolution. Moreover, allelic dosage mechanisms, or the lack of, are interesting in fish with regards to the several whole genome duplications undergone in teleosts (Jaillon et al, 2004, Berthelot et al, 2014). It is known that the genome duplication is followed by duplicated genes neo functionalization, disappearance, or functional maintenance. Whether the plasticity in multiple allele expression relies on epigenetic mechanisms has not been explored to our knowledge. Another question of interest lies in the high diversity found between fish species with regards to sperm chromatin packing and organization. This diversity provides a unique opportunity to study genome stability after sperm cryopreservation (see the review by Herraez et al, 2016 in this special issue), and the associated epigenome pattern and stability. One great advantage in fish is that hundreds embryos can be produced from a single egg batch and sperm sample, and this should be an opportunity to unravel how parents gametogenesis and gamete exposure to biotechnologies would influence offspring performance via epigenetic mechanisms.

ACCEPTED MANUSCRIPT Fish are already widely used models to investigate environmental effects, and the short intergeneration time in some species makes them even more suitable to tackle any potential intergenerational effect. In husbandry practices, photoperiodic, hormonal and thermic control of

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reproduction can be used, and the consequence on gamete epigenetic pattern would be interesting

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to assess with regards to change in gametogenesis and maturation duration.

The impossibility to restore a population from cryopreserved ova or embryos in fish prompted the

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development of promising regeneration technologies using primordial germ cells, germinal stem cells, or somatic cells (see Martinez-Paramo et al, 2016, in this special issue). Possible epigenetic alterations are to be assessed and the risk is to be carefully thought over between genetic

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preservation and transmission of stable epimutations. Additionally, nuclear transfer with somatic cells offers a unique opportunity to better understand the role of gamete epigenetic blueprint, as

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somatic cells are cells bearing a high differentiation pattern which is not matching that of the differentiates gametes.

Last although not least, the erasure-establishment of the epigenetic marks during gametogenesis in

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fish is still not described. Fish have a germ-line determination process which is very different from

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mammals in that the germline is programmed very early during embryo development, when incorporating the germplasm already present in the egg. It is of utter scientific interest to understand

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how the germplasm will be possibly driving germ cell epigenetic differentiation, and how distant from the mammalian process the mechanisms are. Unraveling these mechanisms would also help researcher to understand to what extent some intergenerational epigenetic inheritance can be

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passed on from parents to offspring via the gametes, and whether the environment under which gametogenesis is taking place should be considered as having some programming potential for the embryo and the adult to come. ACKNOWLEDGEMENTS. Authors thank Marta Lombó for designing the Figure 1. Work was partially supported by the projects from the Spanish Ministry of Economy and Competitiveness AGL201127787, AGL2015-68330-C2-1-R , and by the French CRB Anim project «Investissements d’avenir», ANR-11-INBS-0003. The support of the EU COST Actions FA1205: AQUAGAMETE, and FA 1201: EPICONCEPT for training courses, short term scientific mission and workshop participation is gratefully acknowledged.

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ACCEPTED MANUSCRIPT Figure captions Figure 1: Schematic representation of the main epigenetic marks studied in fish gametes. A: DNA methylation: hyper or hypo-methylation of the gene promoter region signs a repressive or permissive

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status of transcription, B: Chromatin modifications on the nucleosome. 5mC: 5-methyl cytosine

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group; H: histone monomers; ac: acetyl group on lysine or arginine residue of the amino acid tail of

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the histone; me3: trimethyl group.

Figure 2: Theoretical scheme of DNA methylation profiles during development and gametogenesis. Methylation in male germ cells is shown in blue and in female germ cells in orange. Figure is based on the scheme from McCarrey (2014) in mice. Data are still missing to draw this scheme from sequential

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experimental data in fish species.

Figure 3: DNA methylation pattern during development in goldfish. Embryos at 1 cell to 24 day post

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fertilization (dpf) stage were fixed with 4% paraformaldehyde and processed for 7 µm sections after paraffin embedding. Sections were stained with anti 5-methyl cytosine (mouse monoclonal IgG1) (5-

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meC) and counterstained with propidium iodide (ADN). From the 1 cell stages on, the blastomeres showed an intense 5-meC labeling. Intensity changes during development could not be established

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from these images.

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Figure 1

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Figure 2 embryo reprogramming

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Figure 3

ACCEPTED MANUSCRIPT Table 1: Summary of the main chromatin-linked epigenetic marks.

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For a more extensive list of histone modifications, refer to Musselman et al (2012). For a more extensive list of writers and erasers, refer to Sadakierska-Chudy and Filip (2015). For a better understanding of the interplay between polycomb and trithorax complexes, refer to Noordermeer and Duboule (2013).

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5mC: methyl cytosine; 5hmC: hydroxymethyl cytosine; C: unmethylated cytosine; H3,4: histone; K: lysine; ac: acetyl; me3: trimethyl; DNMT: DNA methyl transferase; HMT: histone methyl transferase; PRC: polycomb repressive complex; HDM: Histone demethylase; HAT : histone acetyl transferase; HDAC: histone deacetylase;.

Mark

Gene expression status

Writer

DNA

5mC

-: stabilization of gene repression

DNMT1

5hmC

-: transient mark towards cytosine demethylation (repression removal)

Enzyme of demethylation pathway (TET family among others)

C

+: permissive (allows gene transcription)

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Site

Enzyme of demethylation pathway (TET family among others)

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DNMT3A/3B

Eraser

H3K9me3

-: Repressive

HMT

HDM

Histone

H3K64me3

tail

H4k20me3

H3K27 me3

-: Transient repression, found with H3K4me3 on silent genes

HMT (including proteins in the PRC2/1)

HDM

H3K4me3

+: active gene transcription

HMT (including proteins in the Trithorax –TRXcomplex)

HDM

AC

Core

HAT H3K9 ac

HAT

HDAC

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HAT

HDAC

H3K27 ac

HAT

HDAC HDAC

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H4K9 ac