Among the genes in the array, there were genes with “pro-male” functions, such as
amh, sox9,
dmrt1,
wt1,
nr5a2 and
nr0b1, which displayed conservation of testis-enhanced expression in the Asian seabass that was similar to other vertebrates (Table
2). For example, in humans, anti-Müllerian hormone (AMH), which is produced by fetal Sertoli cells, is responsible for the regression of Müllerian ducts during testis differentiation, and its mutation results in the presence of uteri and Fallopian tubes in males [
37]. Amh also inhibits follicular growth and aromatase expression in mouse ovaries [
38,
39]. In teleosts,
amh displayed a higher expression in zebrafish testes than in ovaries, and likewise, we detected a strong testis-enhanced expression of
amh (19.7-fold upregulation) in the Asian seabass [
9,
21]. Similarly,
sox9,
dmrt1,
wt1,
nr5a2 and
nr0b1 were also found to have increased expression in Asian seabass testes compared with ovaries, and earlier, their orthologs were shown to have roles in testis development in other teleosts and even in mammals [
7‐
13].
Germ cell markers
We have also included several germ cell markers in this array. One of these markers,
piwil1, has a role in maintaining transposon silencing in the germline genome and has higher expression in testes of adult zebrafish compared with the ovaries [
27,
30]. Similarly,
piwil1 was found to have higher expression in Asian seabass testes (Table
2). Another set of germ cell markers, Tudor domain containing proteins or Tdrds, are associated with the nuage or germinal granules of germ cells and have been found to be highly expressed in adult mouse testes [
40]. In our array,
tdrd1 and
tdrd7 also showed testis-enhanced expression in the Asian seabass.
The other four germ cell markers,
odf3,
sycp3l,
sept6 and
sept7, are known to be expressed in the spermatids and spermatozoa of other teleost and mammalian species. ODF proteins form the main cytoskeletal structure of the human sperm tail and are found to be essential for male fertility in the zebrafish [
29,
41]. Because of this structural function,
odf3 expression levels had the highest fold difference (2,628-fold) in Asian seabass testes compared with ovaries (Table
2). SCP3 is essential in meiosis progression and in the formation of spermatozoa in mice [
42]. In the medaka,
sycp has been shown to have higher expression in testes than ovaries, and likewise, we detected a 25.8-fold upregulation in Asian seabass testes (Table
2) [
28]. Septins are a family of GTP-binding proteins that forms a component of the cytoskeleton in eukaryotes [
43]. These proteins have been shown to be required for sperm development in mouse and have sexually dimorphic expression in zebrafish gonads [
22,
44]. In Asian seabass,
sept6 and
sept7 have 7.5-fold and 1.5-fold higher expression in testes compared with ovaries, respectively (Table
2). Additionally, the germ cell markers that we have investigated have displayed conservation in gene expression patterns with other vertebrate species.
In teleosts, 17β-estradiol (E2) and 11
-ketotestosterone (11-KT) are the key female and male hormones, respectively, and sex differentiation can be easily influenced by steroids or endocrine-disruptors, which result in sex reversal [
18,
19,
45,
46]. Both E2 and 11-KT are synthesized from testosterone, and
cyp11c1 converts testosterone to 11-KT [
45]. Therefore,
cyp11c1 has an important role in testicular development, and this role is reflected in its testis-enhanced expression in teleosts, including the European seabass (
Dicentrarchus labrax) and zebrafish [
20,
22]. Accordingly, in the Asian seabass,
cyp11c1 has over 300-fold upregulation in the testis (Table
2).
In contrast,
cyp19a1a, or ovarian aromatase, is involved in the conversion of testosterone to E2. During zebrafish gonadal transformation from juvenile ovaries to testes,
cyp19a1a is downregulated, whereas its expression remains high in adult ovaries [
9,
21,
23]. The overexpression of aromatase can also result in ovary development in genetically male chicken embryos [
47]. However, contrary to our expectation, an upregulation of gonadal aromatase was found in Asian seabass testes compared with ovaries (Table
2). One possible explanation is that, although
cyp19a1 was overexpressed by 5.5-fold in the testis,
cyp11c1, which is the gene that regulates the 11-KT level, was overexpressed by more than 300-fold. Given that both
cyp19a1 and
cyp11b act on the same precursor, which is testosterone, this result may indicate that estrogen levels may remain low relative to those levels of androgens in males. In addition, Cyp19a1 may also be regulated at the post-transcriptional level in the seabass gonads. It was also recently found that, in some cichlid lineages, both the ovarian and brain aromatases can have testicular function, and as a result, the sex steroid pathway has been suggested to be less conserved among teleosts [
48]. Nevertheless, the unexpected testis-enhanced expression of
cyp19a1 in the Asian seabass is worth further investigation in the future.
In recent years, cortisol has been shown to be involved in teleost sex differentiation. For example, in the Japanese flounder (
Paralichthys olivaceus) and in pejerrey (
Odontesthes bonariensis), elevated cortisol levels that are caused by increased temperature can result in female-to-male sex reversal [
24,
25]. Therefore, in this qPCR array, we have also included two genes that are involved in the metabolism of cortisol, which is also an important component of the stress response in fish [
49]. Although the interrenal tissues of the head kidney are known to be the major site of cortisol production in teleosts, it is, nevertheless, worthwhile to investigate the gonadal expression of the two genes
hsd11b2 and
hsd3b[
50].
Hsd11b2 plays a role in the conversion of the physiologically active cortisol to inactive cortisone [
51]. It has been suggested that 11ß-hydroxysteroid dehydrogenases protect the teleost gonads from the inhibitory effects of cortisol, such as the inhibition of testicular androgen production [
52,
53]. In contrast,
hsd3b is involved in the early steps of the steroidogenic pathway, which results in the production of not only glucocorticoids but also mineralocorticoids and sex steroids [
54]. The
hsd3b gene was also found to be expressed in both the interrenal tissues of the head kidney and in the gonads of zebrafish [
54].
In the Asian seabass, both genes were overexpressed in testes compared with ovaries. The gene of the cortisol-producing enzyme
hsd3b displayed over 40-fold upregulation, whereas the gene of the cortisol-degrading enzyme,
hsd11b2, exhibited 12.8-fold overexpression (Table
2). Therefore, cortisol might also have reproduction-related roles in the Asian seabass because of the sexually dimorphic expression of both genes.
Wnt signaling pathway
Canonical Wnt signaling has been known to be involved in mammalian ovary development, and the overexpression of WNT4 in humans has been associated with XY sex reversal [
15,
16,
55]. Similarly, Wnt signaling has already been implicated in a reproductive role in teleosts, including the black porgy (
Acanthopagrus schlegeli), rainbow trout (
Oncorhynchus mykiss) and zebrafish [
14,
17,
56,
57]. It has been further shown that Wnt signaling could promote ovarian differentiation through the upregulation of gonadal aromatase [
56,
58].
Among the sexually dimorphic genes in the Asian seabass, there were three members of the Wnt family:
β-catenin 1 (ctnnb1),
dvl2 and
ck2a. β-catenin is the central molecule in the canonical Wnt signaling pathway. In the absence of Wnt ligand binding, cytoplasmic β-catenin is degraded by the ubiquitin-proteosome pathway and prevented from entering the nucleus and associating with
Lef1 to activate Wnt target genes [
59,
60].
Dishevelled is a positive transducer of Wnt signals and functions to activate both canonical and non-canonical Wnt signaling pathways [
61]. In mouse,
Ck2 phosphorylates β-catenin and, therefore, protects β-catenin from being degraded by the ubiquitin-proteosome pathway [
62].
Ck2 has also been found to be activated by Wnt3a [
63]. All three “pro-Wnt signaling” genes were upregulated in Asian seabass ovaries compared with testes, and this result suggested that, similar to mammals and other teleost species, the Wnt signaling pathway has a pro-female function in this species as well (Table
2).
Retinoic acid (RA) signaling pathway
RA is catabolized by both
Cyp26a1 and
Cyp26b1[
64]. In mice,
Cyp26b1 is required to retard or block germ cells from entry into meiosis in the testes and to prevent the apoptosis or conversion of the male germ cell fate [
65,
66].
Cyp26b1 expression in mice is also thought to be activated by Sox9 and Sf1 in the testes and inhibited by Foxl2 in the ovaries [
67]. Although little is known regarding the role of
Cyp26a1 in reproduction, the exposure of mice to RA resulted in the apoptosis of spermatogonia and in the increased expression of this gene [
68].
In the Asian seabass,
cyp26b1 was strongly upregulated in testes compared with ovaries, whereas there was only a slight downregulation of
cyp26a1 (Table
2). Thus, the sexually dimorphic expression of
cyp26b1 and
cyp26a1 suggest the possibility of retinoic acid signaling involvement in testes development in the Asian seabass.
During gonadal transformation in zebrafish, apoptosis is required to remove the unwanted female cells to ‘clear up space’ for the developing testicular cells [
69,
70]. In zebrafish, the main function of
fancl is to ensure the survival of female germ cells. As a result, its mutation causes zebrafish to develop as males, and its effects can be rescued through the mutation of the
tp53 tumor suppressor gene, which is a well-known pro-apoptotic gene [
71,
72]. Our results demonstrated that
tp53 was upregulated by two-fold in Asian seabass ovaries compared with testes. It is possible that this result is due to the atresia that occurs in the F4 ovaries and that the testes that are not undergoing transformation to ovaries are unlikely to have higher levels of apoptosis.
NF-κB pathway
NF-κB is a ubiquitous transcription factor that has been known to be involved in several biological processes, which include the immune response [
73‐
75]. In mammals, NF-κB was shown to interact with SF-1 and to prevent SF-1 from activating Amh, thus inhibiting Amh expression [
76]. In the zebrafish, NF-κB has been shown to inhibit apoptosis during gonad transformation, and thereby, to promote female bias [
77]. In contrast, NKAP is an activator of NF-κB, is also shown to repress Notch signaling and is necessary for T-cell development [
78,
79]. Although the NF-κB pathway seems to be a “pro-female” pathway, both genes (i.e.,
nfkb2 and
nkap) were downregulated in Asian seabass ovaries compared with testes. In the adult gonads of Asian seabass,
NF-κB may have additional reproduction-related roles as suggested by the sexually dimorphic expressions of
nfkb2 and
nkap.