Background
The genetic machinery controlling gonad development is widely conserved, where downstream components tend to converge upon the regulation of common effectors. However, comparisons of the sex determination cascades in different organisms show an impressive diversity of ‘master sex-determining genes’ at the top of the genetic hierarchies [
1]. In most mammals, it is well known that this process involves the action of a Y chromosomal master gene, named
Sry
[
2]. In this case, once the sex is determined, it follows a unique path of development, producing testis or ovary [
3,
4]. In contrast, there are many exceptions to this rule in other vertebrates. In fish, gonad development can be influenced by fluctuations of intrinsic factors such as growth and behavior, and by extrinsic factors such as temperature, hormones and exposures to pollutants [
5‐
12]. The medaka fish, like in mammals, possesses an XX-XY sex determination system in which the male is heterogametic, although, like others species of fish, the sex chromosome cannot be morphologically recognized [
13]. A gene named
dmy or
dmrt1bY, that codes for a protein with the DM domain, was found in the sex determination region of the Y chromosome of medaka. It is a duplicated version of the
dmrt1a gene [
14,
15]. A study in medaka analyzed the main downstream sex determination genes, and showed major differences between mammals and medaka, notably amongst spatial and temporal expression patterns of the canonical signaling pathways, calling into question a strict conservation of regulatory and functional interactions of sexual development genes in vertebrates [
16].
The
dmrt1 gene (doublesex/mab-3 related transcription factor-1) belongs to the gene family first found in insects (
doublesex) and nematodes (
mab-3), both of which code for a DNA-binding protein with a zinc-finger-like motif, named DM domain [
17‐
19]. The
dmrt1 gene seems to be the only gene whose structure and role are conserved during differentiation and gonad development in males, and which has been found throughout vertebrate evolution [
19‐
26]. It is expressed in germ and Sertoli cells of mice testis, downstream from
Sry
[
27]. Mutant
Dmrt1
−/− mice present severe testis problems, showing abnormalities and loss of Sertoli cells function, which may possibly explain the loss of germ cell numbers in these animals. Thus, Dmrt1 is necessary for survival and differentiation of germ and somatic cells in mammals [
27].
Some species of turtles and all crocodiles do not have a sex chromosome, with sex being determined by the temperature at which the eggs are incubated [
28]. Kettlewell
et al. [
29] showed that expression of
dmrt1 in the genital ridge of turtle embryos was higher in those incubated at lower temperatures, which promotes the formation of male sex. Birds display the ZZ-ZW sex determination system, in which the female is heterogametic (ZW), and the best candidate for male sex determination is Dmrt1, located on the Z chromosome. This gene is expressed in bird embryos of both sexes, but at higher levels in (ZZ) males. Increased expression of this gene at the critical period of sex differentiation leads to testis development, showing a dose-dependent expression for male formation [
30,
31].
In fish, all modalities of sex determination have been found, with or without specific sex chromosomes [
32]. The
dmrt1 gene was found to be expressed exclusively during the early stages of testis differentiation, but not in the ovary [
21,
22,
33]. Induction of sex reversal with androgen in XX Nile tilapia increases the expression of
dmrt1 in the germ-cell-surrounding cells [
6]. In
Silurus meridionalis, two isoforms of
dmrt1 were isolated (
dmrt1a and
dmrt1b), with
dmrt1a being expressed exclusively in gonads, but at higher levels in testis, when compared with ovary. The same pattern was observed for
dmrt1b, but, besides the gonads, this isoform was also expressed in other tissues, such as kidney and intestine [
24].
It is well known that members of the SRY-box (Sox)-family show a role in the formation of gonads. This family encodes a transcription factor which displays a DNA-bind-motif, named SRY-like HMG (high mobility group).
Sox9 is a member of Sox-family that plays an essential role in testis determination besides other functions, e.g. in cartilage formation [
34,
35]. This gene seems to be the main effector gene of
Sry
[
36].
Sox9 mutations in an XY organism may lead to bone formation problems, gonad digenesis and sex reversal [
35,
37,
38]. The
sox9 gene is conserved in mammals and birds, as well as preserved its structure and function in teleost fish [
39‐
41]. In zebrafish, there are two copies of this gene -
sox9a and
sox9b- with
sox9a expressed in testis and
sox9b in adult ovary [
42]. On the other hand, in medaka,
sox9a is preferentially expressed in the brain and ovary, and
sox9b is more expressed in testis than in ovary [
43].
Astyanax altiparanae, popularly known as lambari, is a neotropical species with ecological and economical importance [
44,
45].
A. altiparanae was described as a new species in 2000 [
46], and little information is available about its reproductive biology and sex differentiation. Recently, we described the morphological alterations of the testis of this species based on the alterations of the germinal epithelium (GE) throughout the annual reproductive cycle [
47], but the alterations on the molecular level are not reported. Here, we report the isolation of
dmrt1 and
sox9 sequences and their gene expression patterns during gonad development and in different testes maturation phases in
Astyanax altiparanae.
Discussion
Fish display high diversity of sexual differentiation strategies [
3,
52]. Therefore, it is important to study the molecular mechanism of sex determination. Brazil is known for its high abundance of fish species, and the
Astyanax genus is widely distributed throughout the neotropical territories. However, information about sex determination of this group is still scarce in the literature. In the present study, the complete mRNA sequence of
dmrt1 and
sox9 of
Astyanax altiparanae were cloned, and their expressions were analyzed during stages of gonad development and throughout the male reproductive cycle.
This group of fish displays high diversity of chromosomal systems, but no sex chromosome was described for
A. altiparanae, indicating absence of genetic sex determination or homomorphic sex chromosomes [
53]. The
doublesex and mab-3 transcription factor 1 gene was shown to be conserved during evolution, as described in mammals, reptiles, amphibians, fish and invertebrate [
19‐
24,
54]. Analyzing the DM domain structure and the phylogenetic tree of the deduced Dmrt1 amino acid sequence of lambari, we found that its sequence is similar to species that belong to the Superorder Ostariophysi. The Ostariophysi is composed by four orders: Characiformes (tetras, piranhas), Siluriformes (catfishes), Cypriniformes (carps, zebrafish) and Gymnotiformes (electric fish). The DM domain of lambari varies in only one amino acid, when compared with other vertebrate species (A-S). Interestingly this same amino acid is changed in fugu (A-V). However, this amino acid change is conservative in fugu, but this is not the case for
A. altiparanae. Biochemical experiments have to be done to see if this single change has an influence on the DNA-binding properties of the protein.
The
dmrt1 gene is not only characterized by its DM-domain, but also by its male-specific region. Two types of DSX genes are present in
Drosophila, namely the male type (DSX
m) and the female type (DSX
f). The male-specific motif is present only in the DSX
m. The male specific motif has also been characterized by molecular cloning in vertebrates [
21,
24,
33]. By molecular cloning of lambari
dmrt1, we collected additional evidence of the evolutionary conservation of this domain, since only two amino acid changes were observed when compared with the African catfish sequence.
More than one isoform of
dmrt1 in fish species have been described [
24,
25]. Liu
et al. [
24] described an alternative splicing isoform of
dmrt1, with the DM domain being similar for both isoforms. Although only one isoform was isolated in the present work, it is possible that more isoforms may be found in
Astyanax altiparanae.
The
sox9 gene is another sex determination related gene, which has been well described in mammals. However, this gene is also important for cartilage formation [
34,
35,
55]. This gene also contains a DNA binding domain, known as HMG domain, which is characteristic of the Sox family [
56,
57]. By sequence comparison, we found that the
sox9 gene from
Astyanax altiparanae belongs to the SoxE subfamily. Analysis of gene sequences and of the Sox9 phylogenetic tree shows that
Astyanax altiparanae clusters in the basal teleost fish, confirming the previously described morphological data. Another characteristic of the Sox9 protein is the presence of the transactivation domain in the C-terminal region. This domain is also conserved being rich in proline, glutamine and serine. Mutations in this transactivation domain lead to sex reversal in humans, suggesting that mutations probably inhibit transactivation of
sox9 downstream genes in mammals [
58]. Analyzing the transactivation domain of lambari, we show a high degree of similarity with other teleost fish, particularly, with the
sox9b gene copy.
It has been proposed that gene duplication facilitates the evolution of gene functions, via mechanisms of neofunctionalization and subfunctionalization [
59]. Some human gene families demonstrate the history of two rounds of gene duplication during early vertebrate evolution, and fish genomes have often two co-orthologs for many human genes, as a result of a third round of genome duplication that occurred at the base of the teleost radiation [
60]. Cresko
et al. [
61] described in zebrafish that the combined expression pattern of the two
sox9 genes approximately corresponds to that of the single
Sox9 in mouse, which is indicative of a partitioning of an ancestral function. In this work we isolated only one copy of
sox9 in lambari, derived from testis samples, which also showed male specific expression in gonads. The genome of
A. mexicanus provides us a predicted sequence of a second copy of the
sox9 gene, and together with our phylogenetic analyses, the
sox9 gene of lambari shows higher identity with other teleost
sox9b genes. However, the presence of another
sox9 gene copy in lambari can only be confirmed by isolation and characterization of this gene in this species. The
sox9a and
sox9b of zebrafish are more divergent in comparison to other teleost sequences, and even lambari being closer phylogenetically to zebrafish, the Sox9 sequence does not cluster together with the zebrafish sequence. But the sequence of Sox9 from lambari still clusters in the group of basal teleost, going along with morphological analyses [
62].
The pattern and timing of gonad differentiation and sex determination have been studied in fish [
63‐
68]. In most gonochoric teleost species, the ovary develops first while in male the gonad remains undifferentiated and a few days, weeks or even months later the formation of testis occurs [
67,
68]. Apparently, this pattern is observed during the development of lambari gonads, with the first sign of ovary (at 58 dah) being observed in female. In males the gonad remains undifferentiated. Only two weeks later the testis formation was observed in males (at 73 dah). However, the testes showed some cysts in advanced stages, indicating that the formation of the organ occurred sometime before that observed. The origin of germ cells occurs independently from the gonadal tissue, and, during development, these cells migrate to the genital ridge [
69‐
71]. The histological analysis of gonads during lambari development showed that at 5 dah the germ cells are already located in the genital ridge, where the future gonad will be located.
During gonad development, the exact timing of
dmrt1 and
sox9 expression and sex determination varies between species, but, in general, their expression is correlated to the formation of testis [
72]. In zebrafish and medaka, the onset of
dmrt1 expression occurs in the first days after hatching (10 dah), and in medaka this gene is the first that is differentially expressed, when male and female are compared [
73,
74]. The
dmrt1 gene has been shown to be the main sex determination gene, being critical for male determination [
75]. In lambari, the expression of
dmrt1 was just observed in the juvenile gonads where no testis structure was observed (at 58 dah), indicating that the role of
dmrt1 in the testis is most likely prior to visible sex differentiation.
In amniotes, the expression of
dmrt1 is restricted to gonads, being more highly expressed in testis than in ovary. Our data go together with other fish species, where the expression of
dmrt1 is apparently restricted to gonads, similarly to amniotes. The testicular form of
sox9 is an upstream gene in the sex differentiation cascade being conserved in vertebrates [
57]. In the gonad of lambari, the
sox9 expression is male-specific. However, our data show expression of
sox9 in other adult tissues of lambari not only in gonad, a result that has also been observed in zebrafish [
42,
74]. However, the higher expression of
sox9 observed in gut of both males and females and the higher expression in female gills when compared to male gills, are totally uncommon in fish (Table
2). In chicken and
Lepidochelys olivacea turtle,
Sox9 expression was identified in undifferentiated gonads, being upregulated during testis formation, and downregulated when it differentiates into ovary [
76,
77]. The function of
sox9 in fish gonad differentiation remains unknown. In zebrafish, a high expression of the
sox9a variant has been shown during gonadal differentiation to male [
74]. In medaka no
sox9 expression was shown in the early developing gonad, when comparing male and female embryos. This shows that the role of this gene is not related to the early sex determination in medaka [
78]. In lambari, there is also no difference of
sox9 expression before and after the differentiation of testis, indicating that
dmrt1 is apparently more crucial for this role than
sox9. However, the expression level of
sox9 is already observed in the larvae of lambari, showing higher levels at 5 dah, being down-regulated at 12, 19 and 26 dah. Since the expression levels of
sox9 was determined from the whole larvae body, those early expression levels are not necessarily related to gonad formation or sex determination. To confirm if the expression pattern of
dmrt1 and sox9 in adults and during the development of the gonad is directly related to sex determination and differentiation, future functional experiments have to be performed.
Table 2
Gene expression of
sox9a
and
sox9b
in adult tissues of different teleost specie
Astyanax altiparanae
| - | ++ | - | - | - | ++ | ++ | + | - |
Oncorhynchus mykiss
*
| + | N.R. | N.R. | + | N.R. | N.R. | + | + | + |
Oryzias latipes
| - | - | + | + | - | - | - | + | - | - | N.R. | ++ | ++ | + | +++ | +++ | - |
Danio rerio
| - | - | + | + | + | - | + | - | + | + | N.R. | ++ | + | ++ | - | - | +++ |
The morphology of
A. altiparanae testis shows that during the annual reproductive cycle the male gonad passes through complex changes in the structure of the germinal epithelium [
47]. Our data show that the expression pattern of
dmrt1 and
sox9 also change during the process of gonad maturation, especially at the late GE development phase, where the lumen was filled with spermatids and spermatozoa. In tilapia with
dmrt1-deficient testes, a significant testicular regression was observed including deformed efferent ducts, degenerated spermatogonia or even loss of germ cells, and proliferation of steroidogenic cells [
79]. In medaka, a loss-of-function mutation in
dmrt1 leads to a complete male-to-female sex reversal during the phase of maintenance of the gonad [
80]. Expression analyses of
dmrt1 in rainbow and pejerrey during the different spermatogenesis stages showed a similar situation in lambari, where
dmrt1 expression is extremely decreased after spermiation. Analysis of medaka
sox9b reveals that this gene has a important role in the germ cell maintenance in males and females, which could explain the differences of lambari
sox9 gene expression throughout the testis maturation cycle [
81].
Authors’ contributions
MCA carried out the molecular analyzes, sampling of the larvae and juveniles, statistical analysis and drafted the manuscript. ACOC and MCS supervised the molecular analyses, and helped to draft the manuscript. LWOJ collaborated with the molecular analyses and participated in the sampling of adult animals. JB conceived the molecular genetic studies and participated in the design of the study. RMF performed all histological preparation. MS conceived of the study and helped to draft the manuscript. MIB defined the research theme, coordinated all steps of the study and reviewed all version of the manuscript. All authors read and approved the final manuscript.