Molecular Cloning and Expression Responses of Jarid2b to High-Temperature Treatment in Nile Tilapia (Oreochromis niloticus)

Nile tilapia is a GSD + TE (Genetic Sex Determination + Temperature Effect) fish, and high-temperature treatment during critical thermosensitive periods (TSP) can induce the sex reversal of Nile tilapia genetic females, and brain transcriptomes have revealed the upregulation of Jarid2 (Jumonji and AT-rich domain containing 2) expression after 36 °C high-temperature treatment for 12 days during TSP. It was shown that JARID2 forms a complex with polycomb repressive complex 2 (PRC2) that catalyzed H3K27me3, which was strongly associated with transcriptional repression. In this study, Jarid2b was cloned and characterized in Nile tilapia, which was highly conserved among the analyzed fish species. The expression of Jarid2b was upregulated in the gonad of 21 dpf XX genetic females after 12-day high-temperature treatment and reached a similar level to that of males. Similar responses to high-temperature treatment also appeared in the brain, heart, liver, muscle, eye, and skin tissues. Interestingly, Jarid2b expression was only in response to high-temperature treatment, and not to 17α-methyltestosterone (MT) or letrozole treatments; although, these treatments can also induce the sex reversal of genetic Nile tilapia females. Further studies revealed that Jarid2b responded rapidly at the 8th hour after high-temperature treatment. Considering that JARID2 can recruit PRC2 and establish H3K27me3, we speculated that it might be an upstream gene participating in the regulation of Nile tilapia GSD + TE through regulating the H3K27 methylation level at the locus of many sex differentiation-related genes.


Introduction
Sex determination refers to the process by which sexually reproducing organisms determine and initiate the differentiation from an early undifferentiated gonad toward the testis or ovary [1,2]. The sex of fish is remarkably plastic [3]. According to the role of genetic factors and environmental temperature in sex determination, the sex determination mechanisms in fish can be broadly divided into the following three major categories: genotypic sex determination (GSD), temperature-dependent sex determination (TSD), and genotypic sex determination with temperature effects (GSD + TE) [4]. For many fishes with GSD + TE, such as Nile tilapia (Oreochromis niloticus), the sex is jointly regulated by genetic factors and environmental temperature, and the environmental temperature can affect sex determination and differentiation only in a certain time interval, which is called the thermosensitive period (TSP). During TSP, the artificial treatments of high or low temperatures result in sex-ratio changes [5][6][7]. For example, artificially high-temperature treatment at 36 • C from approximately 9 days post-fertilization (dpf) and lasting from 10 to 30 days could induce the sex reversal of XX genotypic females to XX pseudomales in Nile tilapia [8][9][10], which masked the genetic XX/XY sex-determination system in Nile tilapia [11][12][13]. The molecular weight, theoretical isoelectric point, and average hydrophilicity were calculated by SMS2 [37]. Predictive analysis of Jarid2b signal peptide cleavage sites was performed using SignalP 3.0 [38]. The domain architecture prediction of Nile tilapia Jarid2b was performed using SMART [39]. The amino acid sequence of Nile tilapia Jarid2b was aligned with other species using the DNAMAN programs (Lynnon Biosoft, Quebec, QC, Canada). Phylogenetic analysis based on the amino acid sequences of Jarid2b was conducted using MEGA software (version 7.0.14, Mega Limited, Auckland, New Zealand) by the neighbor-joining method with 1000 bootstraps.

qRT-PCR Analysis
Jarid2b and β-actin specific primers (Table 1) for qRT-PCR were designed and the cDNA samples obtained above were used as the templates. qRT-PCR assays were performed using SYBR ® Green Premix Pro Taq HS qPCR Kit in a total 20 µL reaction volume according to the manufacturer's instructions (Accurate Biotechnology, Changsha, Hunan, China). qRT-PCR cycling conditions in Roche LightCycler ® 96 were followed as initial denaturation at 95 • C for 30 s, and then 40 cycles of denaturation at 95 • C for 5 s and extension for 30 s at 60 • C, followed by disassociation curve analysis to determine target specificity. The relative expression of the Jarid2b gene was calculated based on the delta-delta Ct method and normalized to the β-actin mRNA level [36,40]. PCR specificity was assessed by melting curve analysis.

Western Blot Analysis
Anti-Jarid2b polyclonal antibody was produced by Sangon Biotech Co., Ltd. (Shanghai, China), and commercial β-ACTIN (Sangon, Shanghai, China) and GAPDH (Sangon, Shanghai, China) antibodies were used in this study. The protein samples were extracted from the tissues mentioned above (each about 5 mg). Next, the protein concentration was detected using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China), and the samples reached the same concentrations by adding ddH 2 O before use. Then, the protein samples were run on a 6% SDS-PAGE gel and were transferred to PVDF membranes. Subsequently, the membranes were incubated with the rabbit anti-JARID2B, anti-β-ACTIN, or anti-GAPDH antibody diluted at a ratio of 1:1000 with 10 mM PBS (pH 7.4) overnight at 4 • C and then incubated with a horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (Beyotime Biotechnology, Shanghai, China) of 1:1000 at room tem-Genes 2022, 13, 1719 5 of 18 perature for 3 h. Finally, the JARID2B protein band was visualized using BeyoECL Plus (Beyotime Biotechnology, Shanghai, China) on the protein toning system (Vilber Lourmat, Paris, French).

Immunohistochemistry Analysis
The ovary and testis of 180 dpf Nile tilapia females and males were fixed in Bouin's solution to dehydrate for 24 h at room temperature, embedded in paraffin wax, and sectioned at 5 µm thickness for immunohistochemistry (IHC). Firstly, the sections were deparaffinized, hydrated, and blocked with 3% H 2 O 2 at room temperature for 1.5 h. Secondly, the slides were incubated with the anti-JARID2B antibody of 1:1000 overnight at 4 • C and then incubated with the HRP-labeled secondary antibody mentioned above at room temperature for 1 h. Next, an enhanced HRP-DAB Chromogenic Kit was applied for enzymatic reactions (Beyotime Biotechnology, Shanghai, China). Finally, the slides were stained in hematoxylin and visualized on Zeiss confocal microscope.

Statistical Analysis
All data were expressed as the average ± SD (n = 3). One-way analysis of variance (ANOVA) and Tukey's test were used to analyze the data using SPSS 21. Differences were considered significant when p < 0.05. Relative expression of Jarid2b gene was plotted using GraphPad Prism 8.0 software (version 8.0.2.263, GraphPad Software Inc., San Diego, CA, USA).

Molecular Cloning and Bioinformatic Analysis of Jarid2b
PCR using the specific primer pair was performed to obtain the complete open reading frame (ORF) of Jarid2b. The full-length Jarid2b ORF in Nile tilapia was 4239 bp and encoded a 1412-amino acid protein with a calculated molecular weight of 154.98 kDa and a theoretical isoelectric point of 10.14. The average hydrophilicity was −0.739 and the signal peptide was not found in Jarid2b. SMART online software analysis showed that three conserved and key structural features were found in Nile tilapia Jarid2b, including Jumonji N (JmjN) domain, BRIGHT and ARID domain, and JmjC domain ( Figure 1).
The predicted Nile tilapia JmjN domain (690-731) consists of 42 amino acid residues with the same sequence as O. a. and M. b. Alanine (A) residue at position 718 in predicted Nile tilapia JmjN domain was substituted for Serine (S) or Tyrosine (Y) only in A. centrarchus and A. o. Valine (V) residue at position 724 in predicted Nile tilapia JmjN domain was replaced with A in A. calliptera, M. z., P. n., S. d., and H. b. and both V and A occurred with the half probability among these ten species analyzed. The predicted JmjC domain (1032-1196) comprised 165 amino acid residues, which were the same as that in O. a. Aspartic acid (D) residue at position 1109 tended to be displaced with Asparagine (N) in half the analyzed species. In M. b. and A. o., the amino acid residues at position 1041 changed from N to A or Threonine (T), and at position 1100 changed from I to V.
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Tissue Expression Distribution of Jarid2b
The tissue expression distribution of Jarid2b was analyzed using cDNAs synthesizing from the total RNA isolated from 11 tissues of Nile tilapia at 180 dpf. β-actin expression in Nile tilapia was examined as an internal reference. The results showed that Jarid2b was constitutively expressed in all the examined tissues (eye, spleen, heart, liver, blood, XX brain, XY brain, muscle, skin, ovary, and testis) ( Figure 4A). The highest expression level

Tissue Expression Distribution of Jarid2b
The tissue expression distribution of Jarid2b was analyzed using cDNAs synthesizing from the total RNA isolated from 11 tissues of Nile tilapia at 180 dpf. β-actin expression in Nile tilapia was examined as an internal reference. The results showed that Jarid2b was constitutively expressed in all the examined tissues (eye, spleen, heart, liver, blood, XX brain, XY brain, muscle, skin, ovary, and testis) ( Figure 4A). The highest expression level of Jaird2b was observed in Nile tilapia blood, followed by brain and testis, while the lowest was in the ovary. Moderate expression levels were detected in the eye, spleen, heart, liver, muscle, and skin. Western blot analysis of 21 dpf Nile tilapia gonads showed that the anti-JARID2B polyclonal antibody only stained the 155 kD protein band in the XX, XX + HT, and XY samples as expected, which verified the specificity of the JARID2B antibody ( Figure 5B). Furthermore, the titer of the antibody reached 1:512,000. The specific JARID2B antibody was used for Western blot analysis and the results showed that the JARID2B protein expression profile was similar to that obtained from the qRT-PCR ( Figure 4B).

High-Temperature, but Not MT/letrozole Treatment, Upregulates Nile Tilapia Gonadal Jarid2b mRNA and Protein Expression
To investigate the expression pattern of Jarid2b after various treatments, the Jarid2b levels in the Nile tilapia gonad at 21 dpf after treatment for 12 days were examined in this study. According to qRT-PCR, the expression of the Jarid2b gene in the XX + HT and XY groups was significantly higher than that in the XX group at 21 dpf ( Figure 5A). There was no significant difference between the XX + HT and XY group, showing the important role of high-temperature treatment in affecting the expression of Jarid2b. In addition, Western blot analysis showed a similar JARID2B protein expression profile to those obtained from the qRT-PCR (Figure 5B), and the high-temperature treatment also up-regulated the JARID2B protein expression level. Consistent with the Western blot data, the IHC results showed that weak positive signals were observed in somatic cells in the XX gonad, whereas strong positive signals were observed in Sertoli cells in the XX + HT and XY gonads ( Figure 5C). Collectively, high-temperature treatment during TSP resulted in a significant upregulation of Nile tilapia Jaird2b mRNA and protein expression.  To investigate the expression pattern of Jarid2b after various treatments, the Jarid2b levels in the Nile tilapia gonad at 21 dpf after treatment for 12 days were examined in this study. According to qRT-PCR, the expression of the Jarid2b gene in the XX + HT and XY groups was significantly higher than that in the XX group at 21 dpf ( Figure 5A). There was no significant difference between the XX + HT and XY group, showing the important role of high-temperature treatment in affecting the expression of Jarid2b. In addition, Western blot analysis showed a similar JARID2B protein expression profile to those obtained from the qRT-PCR ( Figure 5B), and the high-temperature treatment also up-regulated the JARID2B protein expression level. Consistent with the Western blot data, the IHC results showed that weak positive signals were observed in somatic cells in the XX gonad, whereas strong positive signals were observed in Sertoli cells in the XX + HT and XY gonads ( Figure 5C). Collectively, high-temperature treatment during TSP resulted in a significant upregulation of Nile tilapia Jaird2b mRNA and protein expression.
Sex steroid hormones and their inhibitors can affect the expression of a large number of genes in many species by activating related nuclear receptors. To explore whether MT and letrozole can affect Jarid2b expression, qRT-PCR assays were performed using the sampled gonads after various treatments. Letrozole treatment (XX+ AI) of XX genotypic females of Nile tilapia larvae at 9 dpf for 12 days (21 dpf) did not affect the mRNA level of Jarid2b in the gonads compared to the XX control group (Figure 6). Similarly, MT treatment (XX+ MT) did not also affect the expression of Jarid2b. Collectively, contrary to the effects of high-temperature treatment on the expression of Jarid2b in the gonads (XX + HT), MT and letrozole treatments do not affect the expression of Jarid2b in the gonads. Therefore, the expression of Jarid2b in the Nile tilapia gonads was specifically affected by high-temperature treatment during TSP.

High-Temperature Treatment Also Upregulates Jarid2b Expression in Other Tissues in Nile Tilapia
High-temperature treatments during TSP upregulated the mRNA and protein expression of Jaird2b in the gonad of Nile tilapia. However, whether the expression of Jaird2b in various tissues was affected by high-temperature treatment remains to be elucidated. The results showed that the expression of gene-encoding Jarid2b in the heart, brain, liver, muscle, eye, and skin in the XX + HT group was significantly increased compared with that in the XX group (Figure 7). The highest upregulation of Jarid2b was observed in the eye of the XX + HT group with about 52-fold increases, while the lowest upregulation was in the skin with about 2.5-fold increases. However, no significant change in Jarid2b expres-

High-Temperature Treatment Also Upregulates Jarid2b Expression in Other Tissues in Nile Tilapia
High-temperature treatments during TSP upregulated the mRNA and protein expression of Jaird2b in the gonad of Nile tilapia. However, whether the expression of Jaird2b in various tissues was affected by high-temperature treatment remains to be elucidated. The results showed that the expression of gene-encoding Jarid2b in the heart, brain, liver, muscle, eye, and skin in the XX + HT group was significantly increased compared with that in the XX group (Figure 7). The highest upregulation of Jarid2b was observed in the eye of the XX + HT group with about 52-fold increases, while the lowest upregulation was in the skin with about 2.5-fold increases. However, no significant change in Jarid2b expression was observed in the gill after high-temperature treatment (Figure 7). In conclusion, the expression of gene-encoding Jarid2b in various tissues except for gill was significantly upregulated after high-temperature treatment. Figure 6. Gonadal Jarid2b expression responds to 17-Methytestosterone (MT) and letrozole (AI) treatment at 21 dpf. XX + MT: genetically XX Nile tilapia with 17-Methyltestosterone treatment under 28 °C temperature water; XX + AI: genetically XX Nile tilapia with letrozole (AI) treatment under 28 °C temperature water; XX: control female group; XX + HT: high-temperature-treated female group; XY: control male group. The expression level in the gonad of the XX group was defined as 1, following normalization to the β-actin expression. The different letters indicate significant differences among treatments (p < 0.05).

High-Temperature Treatment Also Upregulates Jarid2b Expression in Other Tissues in Nile Tilapia
High-temperature treatments during TSP upregulated the mRNA and protein expression of Jaird2b in the gonad of Nile tilapia. However, whether the expression of Jaird2b in various tissues was affected by high-temperature treatment remains to be elucidated. The results showed that the expression of gene-encoding Jarid2b in the heart, brain, liver, muscle, eye, and skin in the XX + HT group was significantly increased compared with that in the XX group (Figure 7). The highest upregulation of Jarid2b was observed in the eye of the XX + HT group with about 52-fold increases, while the lowest upregulation was in the skin with about 2.5-fold increases. However, no significant change in Jarid2b expression was observed in the gill after high-temperature treatment (Figure 7). In conclusion, the expression of gene-encoding Jarid2b in various tissues except for gill was significantly upregulated after high-temperature treatment.

Jarid2b Expression Responds Early to High-Temperature Treatment
Because high-temperature treatment affected the expression of Jarid2b in most tissues in Nile tilapia, the brain tissue was selected to investigate whether the Jarid2b expression responds early to high-temperature treatment. Temporal changes in brain Jarid2b transcript levels within 21 h after high-temperature treatment were examined and the result showed that the expression levels of Jarid2b had no significant differences at 4 and 6 h after hightemperature treatment in the XX + HT group compared with the XX group, and then turned to extremely significant upregulation from 8 to 21 h (Figure 8). Taken together, this result indicated that high-temperature treatment during TSP could affect the transcript level of Jarid2b as early as 8 h.

Jarid2b Expression Responds Early to High-Temperature Treatment
Because high-temperature treatment affected the expression of Jarid2b in most tissues in Nile tilapia, the brain tissue was selected to investigate whether the Jarid2b expression responds early to high-temperature treatment. Temporal changes in brain Jarid2b transcript levels within 21 h after high-temperature treatment were examined and the result showed that the expression levels of Jarid2b had no significant differences at 4 and 6 h after high-temperature treatment in the XX + HT group compared with the XX group, and then turned to extremely significant upregulation from 8 to 21 h (Figure 8). Taken together, this result indicated that high-temperature treatment during TSP could affect the transcript level of Jarid2b as early as 8 h.

The Identification and Tissue Distribution of Jarid2b
In this study, we successfully isolated and sequenced Jarid2b in the Nile tilapia. The result of bioinformatic analysis and multiple sequence alignments indicated that Jarid2b belonged to the JmjC gene family and had a highly conserved JmjC domain located at residues 1032-1196. The JmjC domain was first defined based on the amino-acid similarities in the JARID2 (Jumonji), JARID1C (Smcx), and JARID1A (RBP2) proteins [41][42][43]. JmjC-domain-containing proteins were classed as seven evolutionarily conserved groups including the JHDM1, PHM2/PHF8, JARID, JHDM3/JMJD2, UTX/UTY, JHDM2, and JmjC domain only [21]. Nowadays, it is considered that JmjC-domain-containing proteins

The Identification and Tissue Distribution of Jarid2b
In this study, we successfully isolated and sequenced Jarid2b in the Nile tilapia. The result of bioinformatic analysis and multiple sequence alignments indicated that Jarid2b belonged to the JmjC gene family and had a highly conserved JmjC domain located at residues 1032-1196. The JmjC domain was first defined based on the amino-acid similarities in the JARID2 (Jumonji), JARID1C (Smcx), and JARID1A (RBP2) proteins [41][42][43]. JmjC-domain-containing proteins were classed as seven evolutionarily conserved groups including the JHDM1, PHM2/PHF8, JARID, JHDM3/JMJD2, UTX/UTY, JHDM2, and JmjC domain only [21]. Nowadays, it is considered that JmjC-domain-containing proteins might be involved in demethylation within histones [44,45]. However, JARID2, one subgroup of the JARID group, was predicted to have no histone demethylase activity because it did not share the conserved residues that were essential for histone demethylase activity compared to other JmjC-domain-containing proteins [21,46]. Moreover, the function of the JmjC domain also seemed to be different within the two JARID subgroups, as the amino acids required for enzymatic function are intact in most members of the JARID1 subgroup but completely lacking in the JARID2 subgroup [46]. Further study showed that Jarid2 was certified to constitute a subunit of PRC2 and related to the catalytical activity of histone methylation [14]. In this study, Phylogenetic analysis showed that the Jarid2b-deduced amino acid sequence was conserved among the analyzed fish species and Nile tilapia Jarid2b was most similar with that of O. a.. JARID2B was relatively conservative between different species, which implied that its potential histone methylation activity may be conserved across multiple species. JmjN is one of the conserved Jmj domains and the function of the JmjN domain remains largely undetermined. Research has demonstrated that JmjN and JmjC interact physically to form a structural unit that ensures the stability activity of Gis1 [47]. Moreover, the JmjN in Jhd2 is also important for its protein stability [48]. So far, there were no reports related to the function difference between Jarid2a and Jarid2b. Nile tilapia Jarid2a gene has also not been reported.
Herein, we investigated Jarid2b gene expression patterns in adult Nile tilapia tissues and revealed differential expression levels in various tissues, which is consistent with the obtained expression pattern based on transcriptome data in Nile tilapia [49]. There is a variety of evidence that Jarid2 is widely expressed and has different biological functions in other species. Studies have shown that miR-130a, an Etv2 downstream target, was defined an important role in the mediation of vascular patterning and angiogenesis. Mechanistically, miR-130a directly regulated Jarid2 expression by binding to its 3 -UTR region, and its expression was increased in zebrafish miR-130a morphants. Further study showed that over-expression of Jarid2 in HUVEC cells led to defective tube formation indicating its inhibitory role in angiogenesis. These findings demonstrated a critical role for Etv2-miR-130a-Jarid2 in vascular patterning [50]. Additionally, the Jarid2 expression profile during embryonic development and in adult tissues in mangrove rivulus fish suggested that it might be important in development, gametogenesis, and neurogenesis, which may be related to the epigenetic regulation role of Jarid2 [51]. Moreover, Jarid2 expression at high levels was detected in the heart, brain, thymus, and skeletal muscle in adult mouse and in the brain and heart in adult human, indicating that it might be involved in multiple organ development [21,[52][53][54].
In this study, we found that Jarid2b was expressed in the gonads of both sexes during the TSP and the expression pattern exhibited sexual dimorphism, which suggested that it may be involved in the early development and differentiation of Nile tilapia gonads. Remarkably, the sexually dimorphic patterns of Jarid2b transcripts appeared to be maintained to adulthood as demonstrated by the tissue distribution analysis in this study, suggesting that it may be important for gonadal differentiation and maintaining the adult sexual phenotype in Nile tilapia.

High-Temperature, but Not MT/letrozole Treatment, Affects Jarid2b Expression
In this study, the effect of high-temperature treatment on Jarid2 expression was first studied in detail in GSD + TE fish. The results showed that Jarid2b expression was notably high in the gonad of XX tilapia after high-temperature treatment compared to the XX control at 21 dpf, which suggests that Jarid2b may play an important role in the high-temperature induced sex reversal of Nile tilapia females into pseudomales. The use of specific androgen or aromatase inhibitors can cause the sex reversal of genetic females into phenotypic males in various fish species exhibiting GSD + TE or GSD, and affect the expression of many hormone receptor or steroid synthase-related genes [35,36,[55][56][57]. For instance, genetic all-female rainbow trout were treated with the androgen 11beta-hydroxyandrostenedione, which resulted in 100% males at a dosage of 1 mg/kg in food. Steroid enzyme P450scc was clearly up-regulated, and 3betaHSD and P450aro were down-regulated during the treatment [55]. In zebrafish, an all-male population was observed after exposure to 9.7 ng/L synthetic androgen trenbolone and above from 20 to 59 days post-hatch (dph) [58]. Another study showed that batches of tilapia fry treated with aromatase inhibitor during the first 30 days following yolk-sac resorption (7-37 dph) and the percentage of males remained approximately constant (92.5-96.0%) from 200 to 500 mg/kg [59]. Quantitative analysis showed that a certain concentration of MT or letrozole treatment resulted in a similar sex reversal rate of Nile tilapia XX genetic females as the high-temperature treatment [35,36]. However, the results in this study showed that the same doses of MT or letrozole treatment as used by Teng et al. [36] and Wang et al. [35] did not affect Jarid2b expression, which is different from the result of high-temperature treatment. So far, the effect of MT or letrozole treatment on Jarid2 expression has not been reported. High-temperature, but not MT/letrozole treatment, affects Jarid2b expression, which suggests that the molecular mechanism and the pathways of sex reversal in Nile tilapia females induced by androgen or aromatase inhibitor treatment perhaps are partially different from that of high-temperature treatment. We speculated that high-temperature and MT/letrozole treatment may mutually act on some downstream genes [36], but the upstream genes affected by high-temperature and MT/letrozole treatment may be completely different.
After high-temperature treatment during TSP, the level of Jarid2b transcripts in most tissues of Nile tilapia were significantly upregulated. For example, the average Jarid2b mRNA level in juvenile tilapia blood in the XX + HT group was 52 times higher than that in the XX group. High-temperature treatment also affected the expression of jarid2b in multiple tissues except for gills, such as the heart and brain, suggesting that Jarid2b may be a constitutively expressed gene and play an important role in coping with temperature treatment in most tissues. We speculated that the molecular mechanism of Jarid2 expression regulated by high-temperature treatment is basically the same in multiple tissues. Similarly, it was shown that the expression of Hsp70 and Hsp90 was up-or down-regulated in gill, liver, and muscle when Kaluga (Huso dauricus) was treated with different temperatures or salinities [60]. qRT-PCR showed that the upregulated response of turbot (Scophthalmus maximus) PRLR at multiple time points (1 h, 6 h, 12 h, 24 h, 3 m, and 9 m) was similar in gill, kidney, and intestinal tissues after low salt (5, 10, or 30 ppt) treatment [61].
Previous studies showed that the treatment with the glioma inhibitory drug temozolomide (TMZ) resulted in Jarid2 downregulation and CCND1 upregulation within glioma tissues of different grades, and further studies showed that JARID2 negatively regulates CCND1 expression by increasing the H3K27me3 level on the CCND1 promoter in leukemia cell [62,63]. In addition, Jarid2 expression was increased in bladder cancer tissues and cells, and upregulation of Jarid2 increased the H3K27me3 level at the PTEN promoter, thus enhancing the progression of bladder cancer through regulating PTEN/AKT signaling [64]. Given that Jarid2 could generally regulate H3K27me3 status, we speculate that Jarid2b may affect the methylation level of genes in the female differentiation pathway and thereby suppress their expression during the sex differentiation in Nile tilapia of GSD + TE. Recently, Zhong et al. [65] found a high H3K27me3 level could transcriptionally repress the expression of RUNX1 (the runt-related transcription factor 1), a transcription factor influencing granulosa cells' growth and ovulation, whereas RUNX1 acts as an activator of steroidogenesis-related genes Cyp19a1, promoting the production of estrogen in porcine. Similarly, Lee et al. [66] provided in vivo evidence that the level of H3K27me3 is involved in the rapid changes in Cyp19a1 expression by altering the chromatin structure of the promoters. Furthermore, CBX2, a subunit of the Polycomb Repressive Complex 1 (PRC1), which can mainly regulate the level of H3K27me3, can directly bind the ovary-promoting gene Lef1, resulting in the bivalent and repressed status in Sertoli cells of the XY fetal gonad. These results suggested that stabilization of the testis fate requires H3K27me3-mediated repression of ovary-determining genes, which would otherwise block testis development in mice [67].

Jarid2b Expression Was Affected Early by High-Temperature Treatment
Jarid2b expression in the brain responded sharply in the early period of high-temperature treatment during TSP and its expression was significantly upregulated from 8 h. In Arabidopsis, the cold-related COR (cold-regulated) gene was mediated by CRT (C-repeat)/DRE (dehydration-responsive element). CBF1, a transcriptional activator, was found to bind to the CRT/DRE and its overexpression could induce COR gene expression and increase freezing tolerance [68][69][70]. The transcript levels for CBF increased within 15 min after transferring plants to a low temperature, followed by the accumulation of COR gene transcripts at about 2 h, which indicated that CBF gene induction is an early event in the low temperature-stimulated signaling cascade [71,72]. Similarly, the rapid expression responses of barley clock genes to temperature were examined and the clock genes such as CCA1 and PRR73 responded rapidly to the changes in temperature within 6 h [73,74]. Pufferfish (Takifugu rubripes) HSP70 showed a rapid response to temperature treatment (from 24 to 28 • C or 24 to 20 • C) at the 3rd h in the gill, muscle, and liver, speculating that HSP70 might act as the main gene to regulate fish adaptive capacity with changed temperature [75]. Thus, these genes, which rapidly respond to temperature changes, may be the upstream gene responding to high-temperature treatment and the connection point between temperature treatment and downstream genes affected by temperature treatment. Therefore, we speculate that Jarid2b may be an upstream gene responding to high-temperature treatment and play an important role in the regulation of Nile tilapia GSD + TE.

Conclusions
In this study, Jarid2b was characterized in Nile tilapia, and Jarid2b was observed to be commonly expressed in multiple tissues in adult tilapia and exhibited a male-biased expression pattern. We have shown for the first time that high-temperature treatment, but not MT or AI treatment, upregulated Jarid2b levels in the gonads of juvenile Nile tilapia at 21 dpf, and this upregulation was consistent across multiple tissues. Jarid2b expression was found to rapidly respond to high-temperature treatment. Our results suggest that Jarid2b may play an important role in the regulation of Nile tilapia GSD + TE.