Preliminary analysis of H3K4me3 in the gonads during gonadal differentiation in the olive flounder Paralichthys olivaceus

Abstract

Histone methylation is one of the most important epigenetic modifications. However, there are few studies on its role in fish gonadal differentiation. In this study, we analyzed the patterns of H3K4me3 and the expression of modifying- and de-modifying enzymes in the gonads of the juvenile olive flounder Paralichthys olivaceus during the gonadal differentiation process. Histological analysis showed that phenotypic female or male flounder were obtained by treatment with 17β-estradiol (E2) or testosterone (T), respectively. Real-time quantitative PCR (qPCR) results presented that the expression of kmt2b at 2 and 6 cm total length (TL) was higher in the T group than in the E2 group. The expression of kdm5a and kdm5c was higher in the E2 group at 10 cm TL. The results of in situ hybridization (ISH) indicated that kmt2b was mainly distributed in oocytes of the ovary at 10 cm TL. Western blot analysis showed that, H3K4me3 levels were higher in the T group than in the E2 group at 2 and 6 cm TL. As shown with Immunohistochemistry analysis, H3K4me3 was mainly located in oocytes in the ovaries. The signal in spermatogonia was weak in the testes at 10 cm TL. After incubation with an H3K4 methylase inhibitor (5’-deoxy-5’-methylthioadenosine, MTA), the levels of H3K4me3 in the gonads were significantly decreased. The qPCR results indicated that in the MTA group, the expression of sox9a and dmrt1 was significantly decreased in the testis. These results provide the basic data that H3K4me3 may be involved in fish gonadal differentiation.

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Preliminary analysis of H3K4me3 in the gonads
during gonadal differentiation in the olive ounder
Paralichthys olivaceus
Yan Liu
Institute of Oceanology, Chinese Academy of Sciences
Lijuan Wang
Institute of Oceanology, Chinese Academy of Sciences
Yuxia Zou
Institute of Oceanology, Chinese Academy of Sciences
Ze Li
University of Chinese Academy of Sciences
Zhihao Wu
University of Chinese Academy of Sciences
Congcong Zou
University of Chinese Academy of Sciences
Chang Shu
University of Chinese Academy of Sciences
Wenxiang Wang
University of Chinese Academy of Sciences
Shaoshuai Liang
Institute of Oceanology, Chinese Academy of Sciences
Feng You (  youfeng@qdio.ac.cn )
Institute of Oceanology, Chinese Academy of Sciences
Research Article
Keywords: epigenetic modication, H3K4me3, H3K4 methylase inhibitor (MTA), gonads and gonadal
differentiation, ounder
Posted Date: October 19th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-2143784/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
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Abstract
Histone methylation is one of the most important epigenetic modications. However, there are few
studies on its role in sh gonadal differentiation. In this study, we analyzed the patterns of H3K4me3 and
the expression of modifying- and de-modifying enzymes in the gonads of the juvenile olive ounder
Paralichthys olivaceus
during the gonadal differentiation process. Histological analysis showed that
phenotypic female or male ounder were obtained by treatment with 17β-estradiol (E2) or testosterone
(T), respectively. Real-time quantitative PCR (qPCR) results presented that the expression of
kmt2b
at 2
and 6 cm total length (TL) was higher in the T group than in the E2 group. The expression of
kdm5a
and
kdm5c
was higher in the E2 group at 10 cm TL. The results of
in situ
hybridization (ISH) indicated that
kmt2b
was mainly distributed in oocytes of the ovary at 10 cm TL. Western blot analysis showed that,
H3K4me3 levels were higher in the T group than in the E2 group at 2 and 6 cm TL. As shown with
Immunohistochemistry analysis, H3K4me3 was mainly located in oocytes in the ovaries. The signal in
spermatogonia was weak in the testes at 10 cm TL. After incubation with an H3K4 methylase inhibitor
(5’-deoxy-5’-methylthioadenosine, MTA), the levels of H3K4me3 in the gonads were signicantly
decreased. The qPCR results indicated that in the MTA group, the expression of
sox9a
and
dmrt1
was
signicantly decreased in the testis. These results provide the basic data that H3K4me3 may be involved
in sh gonadal differentiation.
Introduction
Improving aquaculture production requires an understanding of genetic and physiological mechanisms
that control the desired traits. In many commercial shes, economic values of growth rate and body
shape differ between males and females (Campbell et al., 2021). And sex determination is based on
gonadal differentiation. Therefore, studies on gonadal differentiation of sh are important for
aquaculture. Gonadal differentiation in many shes could be inuenced by genetic material and
exogenous environmental factors (Piferrer, 2013; Hayman et al., 2021), making it clearer that epigenetic
mechanism can provide a measurable link between environment and phenotype (Gavery and Roberts,
2017). Fish gonadal differentiation is closely related to sex steroid hormones (Frisch, 2004; Sun et al.,
2010), with many steroidogenic enzymes, such as steroidogenic acute regulatory protein (Star) and
cytochrome P450 (Cyp19), and related transcription factors, such as forkhead box protein L2 (Foxl2) and
sex determining region Y-box 9 (Sox9) (Sobhan et al., 2019; Bertho et al., 2016). Reports also showed that
gene expression of these enzymes and transcription factors is regulated by epigenetic modications (Liu
et al., 2020b). Epigenetic modications mainly include DNA methylation, histone modications, and non-
coding RNA activity. However, the relevant epigenetic studies in sh have focused almost exclusively on
roles of DNA methylation in the expression of these genes. For instance, high temperature could increase
DNA methylation levels of
cyp19a1a
and
dmrt1
, resulting in the suppression of gene expression (Navarro-
Martín et al., 2011, Shao et al., 2014).
Histone post-translational modications (PTMs) are key components of the critical epigenetic machinery
that provide protein-based regulatory information (Boukas et al., 2019). Histone lysine methylation is one

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type of PTMs that mainly occur at K4, K9, K27, and K36 of histone H3. Among them, H3K4me3 is a
dynamic mark whose levels reect the balance between deposition by the lysine methyltransferase 2B
(KMT2B or Mll2) and removal by Lysine-specic demethylase 5 (KDM5 or JARID1) demethylase family
(Beacon et al., 2021; Denissov et al., 2014; Upadhyay et al., 2011). Some specic compounds have been
found that might inuence the level of epigenetic modication (Liu et al., 2020a). For example, 5-deoxy5-
methylthiosine (MTA) is a specic inhibitor of H3K4 methylation, which mainly inhibits H3K4 methylation,
especially trimethylation (Yuan et al., 2019). Methylated histones are recognized by chromatin effectors,
which recruit other molecules to alter chromatin structure and function (Taverna et al., 2007). And
H3K4me3 is associated with open chromatin and specically with genes that are potentially active
(Burlibaa et al., 2021). In the red-eared slider turtle
Trachemys scripta
, DNA hypomethylation and
H3K4me3 modication at the aromatase promoter may be a primary mechanism that releases a
transcriptional blockade of
cyp19a1a
to initiate a cascade of ovarian differentiation (Matsumoto et al.,
2016). However, there are few studies on the H3K4me3 of sexual differences in teleosts.
Olive ounder
Paralichthys olivaceus
is an important commercial marine sh that is widely cultured in
northern Asia. The growth rate of the female ounder is substantially faster than that of the male ones
(Yamamoto et al., 1999). And the gonadal differentiation of the ounder is inuenced by both genetic and
environmental factors. Therefore, analysis of the closely related epigenetic modication in the ounder
gonadal differentiation is essential. Studies have also shown that DNA methylation levels of the promoter
or coding regions of some sex-linked genes, including
cyp19a
,
cyp17-
,
dmrt1
, and
foxl2
, were inversely
related to their expression levels (Wen et al., 2014; Si et al., 2016; Fan et al., 2019), implying that DNA
methylation may regulate the expression of these genes. However, as in other shes, little is known about
the role of H3K4me3 in the ounder gonadal differentiation process. It was limited to our previous study
on sex differences between the ounder male and female gonads (Liu et al., 2021). In the present study,
we analyzed the patterns of H3K4me3 and the expression of modifying and de-modifying enzymes in the
gonads of the juvenile ounder during the gonadal differentiation process. Then, the level of H3K4me3
and the expression of genes related to sex steroid synthesis were detected in the gonads under
incubation with MTA. Our ndings provide a clue for exploring the role of histone methylation in gonadal
differentiation of teleosts.
Materials And Methods
Fish and sample collection
First, articial gynogenesis of the ounder was induced (You et al., 2001), and then phenotypic female or
male ounder were induced by 17β-estradiol (E2, Sigma) or testosterone (T, Solarbio) treatments,
respectively (Liang et al., 2018). E2 and T were dissolved in pure ethanol and mixed with commercial
feed. The feeds were dried under dark conditions. The juveniles at 1.2–1.5 cm total length (TL) were
randomly divided into three groups G, E2, and T groups, and were reared at 20–22°C. The juveniles were
fed two to six times daily with the diet containing T (5 µg/g diet) or E2 (5 µg/g diet) (Sun et al., 2010).
After anesthetization with 0.05% tricaine methanesulfonate (MS-222, Sigma, USA), the gonadal region

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samples at 2, 3, and 4 cm TL and the gonads at 6, 8, and 10 cm TL in each group were collected.
Approximately 30 juveniles were used per sampling site in each group. A part of the samples were xed
individually in Davision xative solution for histological,
in situ
hybridization (ISH), and
immunohistochemistry (IHC) analyses. The other part of the samples were immediately frozen in liquid
nitrogen and stored at -80°C for RNA and protein isolation.
The adult ounder used for tissue incubation were purchased from Nanshan sh market (Qingdao,
China) (18–20 cm TL) and acclimated for more than 3 days in the aquarium of the institute. Three males
and 3 females were collected and anesthetized with MS-222 (Sigma, USA), and the gonadal tissue was
dissected for tissue incubation.
All animal experiments were performed according to relevant national and international guidelines. The
animal protocols were approved by the Institute of Oceanology, Chinese Academy of Sciences.
Incubation Of The Gonads
The collected whole gonadal tissue was repeatedly cleaned with PBS containing 1% Penicillin-
Streptomycin-Amphotericin B Solution (BI, Israel) and cut into tissue blocks of 50–100 mg. One tissue
block was xed in Davision xative solution for histological analysis. The other tissue blocks were placed
in a 24-well plate, with each well containing two tissue blocks. Pre-incubation with 600 µl L15 medium
containing 1% antibiotics was performed for 6 h in an incubator at 25 ℃. Then we divided the incubated
samples into two groups, the MTA group and control group. For group treated with the H3K4 methylase
inhibitor, 1 mM MTA (Sigma) was added to the medium. For the control group, only the culture medium
was added to the wells. Both groups were incubated at 25 ℃ for 24 h. All incubation for each group was
made in triplicates. After incubation, the tissue blocks were stored at -80℃ for following RNA and protein
extraction.
Gonadal Histology
To learn the sex and developmental stages of the gonads from the ounders, histological analysis of the
gonadal samples was performed through tissue sections stained with hematoxylin/eosin (HE) as
previously described (Fan et al., 2017).
Reverse Transcription And Quantitative Expression Analysis
Total RNA was extracted from the gonadal region samples or gonads using Trizol reagent (Toroivd, UK).
The quality and concentrations of the total RNAs were checked with electrophoresis of a 1.5% agarose
gel and a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientic Inc., USA). Total RNAs were used
to synthesize cDNAs with the PrimeScript™ RT reagent kit including gDNA Eraser (Takara, Japan)
according to the manufacturer’s instructions.

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The relative transcript levels of histone methyltransferase and genes related to gonadal differentiation
were detected with real-time quantitative polymerase chain reaction (qPCR) method. The primers used in
this study were from the previous studies (Table S1; Liu et al., 2021; Fan et al., 2019). Relative transcript
levels were normalized to
β-actin
(Zheng and Sun, 2011) and calculated using the 2 −△△Ct method (Livak
and Schmittgen, 2001).
Ish Assay
The primers for the cDNA fragments of the target genes used in ISH assay were referred to our previous
study (Table S2; Liu et al., 2021). The SP6 promoter in the sense primers and the T7 promoter in the
antisense primers were all underlined. The digoxigenin-labeled antisense probes from these fragments
were synthesized as described previously (Tan and Du, 2002). Sense RNA probes were synthesized in the
same way. The gonads were embedded in paran, then cut into 6 µm slices and mounted on poly-L-
lysine-coated glass microscope slides. ISH was performed using the labeled probes as described
previously (Feng et al., 2011).
Western Blot
Western blot (WB) was carried out to detect the specicity of H3K4me3 in the ounder gonads with H3 as
a reference. Proteins from the gonads were prepared with ice-cold lysis buffer containing 50 mM Tris-HCl
(pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 1
mM EDTA, and 1 mM phenylmethylsulfonyl uoride (PMSF). They were then separated with 12% SDS-
PAGE and electro-transferred onto polyvinylidene uoride membranes (Millipore, Billerica, USA).Then,
H3K4me3 and H3 were probed with the corresponding antibodies (H3K4me3, 1:1000, Abcam, USA; H3,
1:1000, Transgene, China) overnight. The secondary antibody (anti-mouse, Transgene, China) was
incubated with the membrane for 2 h at room temperature. The immunoreactive bands were visualized
with the enhanced chemiluminescence reagents by using a chemiluminescence imaging system, and
analyzed using Image J software (http://imagej.nih.gov/ij).
Ihc Assay
IHC was conducted to determine the distribution of H3K4me3 in the gonads. The gonads were embedded
in paran, cut into 6-µm sections, and placed on glass slides. The subsequent experimental procedures
refer to our previous article (Liu et al., 2021). Briey, the sections were dewaxed, rehydrated, blocked, and
processed in sequence. Then, the sections were incubated with the 1 : 100 mouse anti-H3K4me3 (Abcam,
China). After incubation with the secondary antibodies (anti-mouse, Transgene, China), the sections were
exposed to 3′, 3′-diaminobenzidine (DAB) and stained with hematoxylin to visualize the nuclei of cells in
the gonads. Sections processed without primary antibodies served as the negative controls.

Page 6/19
Statistical analysis
All data were shown as the mean value ± S.E.M. Data on gene expression and H3K4me3 levels during the
gonadal differentiation were subjected to one-way analysis of variance (ANOVA), followed by Duncan’s
multiple range test. Data on gene expression and H3K4me3 levels during tissue incubation were
subjected to paired-sample T test. The association analysis between levels of H3K4me3 and
kmt2b
expression was carried out by Pearson correlation analysis. All statistical analyses were calculated by
SPSS statistics software (version 20.0, SPSS, USA).
P
value of < 0.05 was considered statistically
signicant difference.
Results
Spatiotemporal expression analysis of kmt2b and kdm5s during the ounder gonadal differentiation
Sex ratios were determined by histological analysis. The male proportions in the T, E2, and G groups were
100%, 0%, and 0%, respectively.
The expression of
kmt2b
and
kdm5s
in the ounder gonads during the gonadal differentiation process
was analyzed by using qPCR assay (Fig. 1). In general, the expression of
kmt2b
and
kdm5s
uctuated
dynamically during this period. However, the expression trends of
kmt2b
and
kdm5s
in the juvenile
gonads at 2 to 8 cm TL were similar in the E2 and G groups. At 2 and 6 cm TL, the expression level of
kmt2b
was signicantly higher in the T group than in the E2 and G groups (
P
< 0.05). At 10 cm TL, there
was no signicant difference between the expression levels of
kmt2b
in the E2 and T groups, whereas its
expression was higher in the G group than in the E2 and T groups. The expression of
kdm5a
and
kdm5c
was higher in the E2 group than in the T group.
Cellular localization of
kmt2b
and
kdm5a
in the gonads of juveniles at 6 and 10 cm TL was analyzed by
using ISH assay and are shown in Fig. 2. In the ovary, almost no signal of
kmt2b
was detected in the
nuclei of oogonia at 6 cm TL, whereas strong signals of
kmt2b
were observed in both oocytes and
oogonia at 10 cm TL. For
kdm5a
, no signal was detected in the ovary at 6 cm TL, whereas positive
signals were detected in all cell types at 10 cm TL. In the testis,
kmt2b
and
kdm5a
genes were widely
distributed in all cell types at both 6 and 10 cm TL.
Spatiotemporal Expression Analysis Of H3k4me3 During The Gonadal
Differentiation
Total H3K4me3 levels in the gonads during the gonadal differentiation process were analyzed by using
WB assays and are shown in Fig. 3. The levels of H3K4me3 changed dynamically during this process. In
the juveniles at 2 cm TL, there was no signicant difference between the H3K4me3 levels in the E2 and T
groups, whereas the level in the T group was higher than in the G group (
P
< 0.05). In the juveniles at 6 cm
TL, the level of H3K4me3 was higher in the T group than in the G (
P
< 0.05) and E2 groups (
P
< 0.01),

Page 7/19
whereas it was higher in the G group than in the E2 group (
P
< 0.01). In the juveniles at 10 cm TL, there
was no signicant difference between the level of H3K4me3 in the E2 and T groups, but the level was
higher in the G group than in the E2 group (
P
< 0.05). During the whole process of the gonadal
differentiation, the level of H3K4me3 in the T group was the highest at 6 cm TL (
P
< 0.05), and that in the
G group was higher at 6 and 10 cm TL than at 2 cm TL (
P
< 0.05). And we found that the total levels of
H3K4me3 during this process were positively associated with the expression of
kmt2b
(R2 = 7.45,
P
<
0.05).
Cellular localization of H3K4me3 in the gonads was analyzed by using IHC assay and is shown in Fig. 4.
Weak signals were observed in nuclei of oogonia in the ovaries of the juveniles at 6 and 10 cm TL. The
signals in the nuclei of spermatogonia were weak and almost no signal was detected in Sertoli cells in
the testes at 6 cm TL. There were strong signals in the nuclei of spermatocytes in the testes at 10 cm TL.
Effects Of Mta On The Flounder Gonads
The H&E staining results of the ounder gonads used for tissue incubation are shown in Fig. 5.
Histological analysis revealed that both the testes and ovaries were at stage .
Total levels of H3K4me3 in the gonads incubated with MTA are shown in Fig. 6. The WB analysis
presented that, compared with the control group, H3K4me3 levels signicantly decreased in the both
ovaries (
P
< 0.01) and testes (
P
< 0.05) of the MTA treatment group.
The expression of genes related to sex steroid hormone synthesis in the ounder gonads is displayed in
Fig. 7. The qPCR results indicated that in the MTA incubation group, the expression of
sox9a
and
dmrt1
was signicantly decreased (
P
< 0.05) and the expression of
star2a
tended to decrease in the testis, while
the expression of
star2b
was signicantly increased (
P
< 0.05). In the ovary, under the MTA treatment, the
expression of
foxl2
was signicantly elevated (
P
< 0.05), and the expression of
nr5a2
tended to decrease
while the expression of
cyp19a
tended to increase without signicant difference. Expression of
nr0b1
did
not change both in testis and ovary.
Discussion
The ounder
P. olivaceus
is a gonochoristic sh with a XX/XY sex determination system. The gynogenetic
ounder, which shares the same X genome, is capable of producing two distinct sexual phenotypes in
response to environmental factors (Sun et al., 2010). To date, studies on H3K4me3 in gonadal
differentiation have been mainly limited to a few species such as mammals and reptiles, but have not
been reported in sh (Matsumoto et al., 2016; Yang and Wilson, 2019). Given the lack of knowledge on
epigenetic mechanisms in sh gonadal differentiation, we here analyzed H4K4me3 levels and
methyltransferase gene expression patterns during this period and examined H3K4me3 levels and
expression of gonadal differentiation related genes under MTA incubation in the ounder. The results
showed that H3K4me3 levels and the expression of
kmt2b
and
kdm5
genes changed dynamically during

Page 8/19
the gonadal differentiation, and the trends differed among the different groups. The H3K4me3 levels in
the gonads and the expression levels of
sox9
and
dmrt1
decreased after the MTA incubation. The results
suggest that H3K4me3 modication might regulate gene expression and determine the direction of the
gonadal differentiation in
P. olivaceus
.
The H3k4me3 Pattern During The Gonadal Differentiation
The direction of the gonadal differentiation in the ounder might be controlled by exogenous hormones
such as estradiol and testosterone (Sun et al., 2010). Epigenetic regulation gives organisms the ability to
generate specic epigenotypes in response to changes in the internal or external environment. The
combination of specic epigenetics and genotypes results in specic phenotypes (Tachibana, 2016).
Histone methylation is one of the most widely studied epigenetic modications, but there are few related
studies on the process of gonadal differentiation. However, H3K4me3 has been found to be distributed on
chromosomes with sexual dimorphism in mouse
Mus Musculus
germ cells (Yang and Wilson, 2019). In
red-eared slider turtle
T. scripta
, hypomethylation and high H3K4me3 levels in the promoter region of gene
cyp19a1a
induced by temperature changes could initiate ovarian differentiation (Matsumoto et al., 2016).
And we also found a similar result that H3K4me3 levels differed signicantly between the testis and
ovary (Liu et al., 2021). This suggests that H3K4me3 might be involved in gonadal differentiation. In this
study, H3K4me3 levels at 6 cm TL were signicantly higher in the T group than in the G and E2 groups.
When the juvenile ounders are cultured at 18–20 ℃, the rst sign of testicular differentiation is at about
6.3 cm TL (Sun et al., 2010). The H3K4me3 levels were higher in the gonads before testicular
differentiation. Therefore, we speculate that H3K4me3 plays an important role in regulating the ounder
testicular differentiation.
Histone methylation is a dynamic modication regulated by lysine-specic methyltransferase and
demethylase (Hyun et al., 2017). Transcriptomic analysis showed that the expression levels of histone
methyltransferase genes
ehmt2
and
Jarid2a
were signicantly increased during high-temperature
masculinization of
Dicentrarchus labrax
(Diaz and Piferrer, 2015). As for H3K4me3, histone methylated
methyltransferase KMT2B and demethylase KDM5 are essential for normal vertebrate development
(Zhang et al., 2014; Hanna et al., 2022). In this study, the changes in
kmt2b
expression were similar in the
female groups (G and E2 groups), increasing from 6 cm TL to 10 cm TL. At the same time, its signal was
weak in the ovaries at 6 cm TL, whereas stronger signals were detected in oocytes of the ovary at 10 cm
TL in these two groups. These results suggest that
kmt2b
may be involved in ovarian development after
differentiation, rather than during ovarian differentiation. The expression of
kmt2b
, which correlated
positively with H3K4me3, was the highest in the T group at 6 cm TL when the testicular differentiation
began, and was also higher than in the female groups (G and E2 groups). We hypothesize that
kmt2b
may affect the ounder testicular differentiation by regulating H3K4me3 levels. However, the expression
of
kdm5a
and
kmt5c
at 10 cm TL was higher in the T group than in the E2 group, implying that
kdm5s
might have other function other than function for gonadal differentiation. Further studies are needed to
conrm these hypotheses.

Page 9/19
Effects Of Mta On The Gonads Of The Flounder
The exogenous environmental factors could inuence the activity of gene expression through epigenetic
modication, and increase the plasticity of sex phenotype formation in sh (Hammond et al., 2016). As
mentioned above, H3K4me3 might be involved in the gonadal differentiation of the ounder. In
T. scripta
,
hypomethylation and high H3K4me3 levels in the gene promoter region induced by temperature changes
could promote gene expression of
cyp19a1a
, and then initiate ovarian differentiation (Matsumoto et al.,
2016). Currently, although there is little direct evidence that H3K4me3 regulates gonadal differentiation,
genes related to sex hormone synthesis have been found to be regulated by H3K4 methylation. For
example, extracellular regulated kinase (ERK) inhibitors inhibit
star
mRNA expression by decreasing
H3K4me3 levels in rat ovarian granulosa cells (Maekawa et al., 2016). The STAR transports cholesterol
across the mitochondrial membrane to the inner mitochondrial membrane, which is a rate-limiting step of
sex hormone synthesis (Stocco, 2000). MTA, a specic H3K4 methylation inhibitor, mainly inhibits H3K4
methylation, especially trimethylation, by downregulating Set1 expression (Huang et al., 2006). In the
present study, we incubated the ounder gonads with MTA and found that MTA signicantly reduced
H3K4me3 levels in the incubated testes and ovaries, as well as the expression of
sox9a
and
dmrt1
genes
in the incubated testes. It is suggested that H3K4me3 might be involved in the regulation of
sox9a
and
dmrt1
genes. Sox9a and Dmrt1 are important transcription factors of Cyp19a, a key enzyme for the
conversion of male and female hormones, and can repress the expression of
cyp19a
(Yuan et al., 2020;
Fan et al., 2019). And sex hormones play an essential role in the process of gonadal differentiation.
Therefore, we further speculate that H3K4 might be involved in gonadal differentiation by regulating the
expression of genes related to sex steroid synthesis in the ounder. As expected, the changes of gene
expression in the incubated ovaries were different from those in the incubated testes. In the ovary, the
expression of
foxl2
was signicantly elevated and the expression of
nr5a2
tended to decrease, while the
expression of
cyp19a
tended to increase under the MTA treatment. In the testis, the expression of these
three genes showed almost no changes. These results suggest that MTA has different effects on testis
and ovary. Further study needs to be performed to explore the detailed difference and function.
In conclusion, the levels of H3K4me3 and
kmt2b
varied dynamically during the ounder gonadal
differentiation, and the trends of variation in the T and E2 treatment groups were different. The
expression of
kmt2b
was positively correlated with the level of H3K4me3, and both were higher in the T
group before the testicular differentiation. The expression of genes related to testicular differentiation
and sex hormone synthesis, such as
sox9a
and
dmrt1
, was downregulated with the decrease of
H3K4me3 level when the incubated testes were treated with MTA. These results suggest that H3K4me3
mediated by KMT2B may affect the testicular differentiation by regulating the expression of genes
related to sex hormone synthesis. In the future, the nature of H3K4me3 modication in the gonadal
differentiation needs to be analyzed by high-throughput sequencing.
Declarations

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Funding informationThis study was nancially supported by the National Key R & D Program of China
(No. 2018YFD0900202), National Natural Science Foundation of China (Nos. 31702337, 31772834, and
31872558), National Marine Genetic Resource Center, National Postdoctoral Science Foundation (No.
2020M672149), Shandong Postdoctoral Innovation Foundation (No. 202003017), and Qingdao
Postdoctoral Science Foundation.
Conicts of interestThe authors declare that they have no conicts of interest.
Ethical approvalAll experiments were conducted according to the regulation of local and central
government of China, and approved by Institute of Oceanology, Chinese Academy of Sciences.
Availability of data and material Not applicable.
Code availability Not applicable.
Authors' contributionsYan Liu, Feng You, and Yuxia Zou conceived and designed the experiments. Yan
Liu, Lijuan Wang, Ze Li, and Congcong Zou performed the experiments. Zhihao Wu and Lijuan Wang
induced and incubated the meio-gynogenetic ounder fry. Wenxiang Wang, Shaoshuai Liang, Ze Li and
Chang Shu cultured the samples. Yan Liu, Wenxiang Wang, Shaoshuai Liang, and Lijuan Wang analyzed
the data. Yan Liu wrote the original draft. Yan Liu and Feng You reviewed the manuscript. Feng You
supervised the study.
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Figures

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Figure 1
Expression pattern of
kmt2b
and
kdm5s
in the gonadal region samples or gonads. Values are the mean
value ± S.E.M; n = 3. Different letters indicate a statistically signicant difference in the same gene (
P <
0.05).

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Figure 2
Cellular localization of
kmt2b
and
kdm5a
in the ounder gonads. Sg, spermatogonium; Og, oogonia. Bar,
50 μm.

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Figure 3
Total level of H3K4me3 in the ounder gonads. Data are expressed as the mean value ± S.E.M; n = 3.
Different letters indicate a signicant difference during the gonadal differentiation process (
P
< 0.05). *
indicates a signicant difference between the groups (
P
< 0.05). ** indicates an extremely signicant
difference between the groups (
P
< 0.01).

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Figure 4
Cellular localization of H3K4me3 in the ounder gonads. Sg, spermatogonium; Se, Sertoli cell; Sc,
spermatocyte; Og, oogonium. Bar, 30 μm.
Figure 5

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Developmental stage of the founder gonads. Sg, spermatogonium; Se, Sertoli cell; Sc, spermatocyte; Oo,
oocyte. Og, oogonium. Bar, 30 μm
Figure 6
Total level of H3K4me3 in the ounder gonads incubated with MTA. Data are expressed as the mean
value ± S.E.M; n = 3. * indicates a statistically signicant difference between the groups (
P
< 0.05). **

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indicates an extremely signicant difference between the groups (
P
< 0.01).
Figure 7
Expression patterns of relative genes in the gonads under the MTA incubation. Values are the mean value
± S.E.M; n = 3. Different letters indicate a statistically signicant difference in the same gene (
P
< 0.05).
Supplementary Files
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tableS1.docx
tableS2.docx