De novo transcriptome analysis to search for sex-differentiation genes in the Siberian sturgeon
Introduction
Sturgeons are a family of ray-finned, non-teleost fishes (Actinopterygii), including several species used for caviar production. These species are among the most lucrative commercial fishes in the world. Unfortunately, some sturgeons face critical conservation problems (Birstein, 1993, Sokolov and Vasil’ev, 1989) due to overfishing, incidental capture, river pollution, dam construction, other environmental disruptions, and poorly-managed fishery systems (Bacalbaşa-Dobrovici, 1997, Bemis et al., 1997, Sokolov and Vasil’ev, 1989, Wei et al., 1997). Elucidating the sex-differentiation mechanisms of sturgeons will add to our knowledge on the biological cycle of this valuable and threatened fish, supporting efforts to maintain the species.
The majority of existing information on the molecular factors involved in sex differentiation in fish comes from studies on teleosts (Baroiller et al., 1999, Devlin and Nagahama, 2002, Guiguen et al., 2010, Piferrer and Guiguen, 2008), in which sex is determined by various non-conserved master sex-determining genes (SDG) linked to the Y-chromosome (Hattori et al., 2012, Matsuda et al., 2002, Myosho et al., 2012, Takehana et al., 2014, Yamamoto et al., 2014). However, a SDG has not been identified in fish with a ZZ/ZW sex-determining system, such as sturgeons. Moreover, the direct effects of the SDG on genes involved in ovarian or testicular programs are not well understood. The male program in fish is characterized by early sexually-dimorphic expression of transcription factors such as doublesex- and mab-3-related transcription factor 1 (dmrt1), sry-box containing gene 9 a1 (sox9a1), sry-box containing gene 9 a2 (sox9a2), nuclear receptor subfamily 0 group B member 1 (nr0b1 or dax1), and T-box transcription factor 1 (tbx1) (Baron et al., 2005, Marchand et al., 2000, Vizziano et al., 2007, Yano et al., 2011), as well as (Baron et al., 2005, Marchand et al., 2000, Vizziano et al., 2007, Yano et al., 2011) type beta transforming growth factors such as anti-Müllerian hormone (amh), gonadal soma-derived factor 2 (gsdf2), and gonadal soma-derived factor (gsdf Baron et al., 2005, Hattori et al., 2012, Lareyre et al., 2008, Myosho et al., 2012, Shibata et al., 2010, Vizziano et al., 2007, Yamamoto et al., 2014) (Baron et al., 2005, Fernandino et al., 2008, Hattori et al., 2012, Lareyre et al., 2008, Myosho et al., 2012, Shibata et al., 2010, Takehana et al., 2014, Vizziano et al., 2007, Yamamoto et al., 2014). However, few functional studies have been performed to confirm the participation of these genes in testicular differentiation. gsdf deletion induces XY gonads to undergo ovarian development in Oryzias latipes (Imai et al., 2015), and the insertion of a fragment containing gonadal soma-derived growth factor on the Y chromosome (gsdfY) —which has supplanted the Y-specific DM-domain gene or dmY as the SDG in Oryzias luzonensis— converts XX individuals into fertile XX males (Myosho et al., 2012). In Oryzias dancena, the SDG SRY-box containing transcription factor 3 on the Y chromosome (sox3-Y) initiates testicular differentiation by upregulating expression of gsdf (Takehana et al., 2014). Furthermore, insertion of the feminine gene R-spondin1 (rspo1) (Zhou et al., 2012) suppresses male-biased genes (dmy, gsdf, sox9a2, and dmrt1) and inhibits testicular differentiation in O. latipes (Zhou et al., 2016). These results show that gsdf is essential for inducing testicular development in Oryzias species, along with sox9a2 and dmrt1. In the pejerrey Odontesthes hatcheri, inhibition of the SDG Y-linked anti-Müllerian hormone (amh-Y) induces a male-to-female sex reversal through activation of feminine genes (Hattori et al., 2012). In future male fish, amh-Y is expressed during very early development, later supplanted by amh after the onset of testicular differentiation. In trout, deletion of the SDG sexually dimorphic on the Y chromosome (sdy) induces sex reversal, and its activation triggers a masculine phenotype in XX females (Yano et al., 2012). However, there are no studies on the effect of sdy inhibition on the masculine or feminine programs. Previous works showed that aromatase inhibitors induce female-to-male sex reversal, mediated by early up regulation of masculine genes such as dax1, platelet-derived growth factor receptor alpha (pdgfra), and dmrt1 (Vizziano et al., 2008); in contrast, estrogen treatment induces male-to-female sex reversal, mediated by strong inhibition of masculine genes such as amh and sox9a2. Recently, it was discovered that cyp19a1a -/- and foxl2 -/- fish from mutant lines of Nile tilapia may undergo female-to-male sex reversal if male-specific factors are induced, such as nuclear receptor subfamily 5, group a, member 1b (nr5a1b or sf1), dmrt1, and gsdf (Zhang et al., 2017). In sum, gsdf, amh, dax1, pdgfra, dmrt1, sox9a2, and sf1 have been proposed as factors that control the male sex differentiation pathway in fish. Gonadal expression of testicular genes such as dmrt1, amh, and sox9 have been studied in morphologically-undifferentiated Siberian sturgeon (Acipenser baerii) (Berbejillo et al., 2012, Berbejillo et al., 2013, Vizziano-Cantonnet et al., 2016), but the lack of available sex markers has prevented any conclusion regarding activation of these genes during the testicular program.
Gonadal steroids are crucial for sex differentiation in fish (Baroiller et al., 1999, Baroiller and D'cotta, H., , 2001, Devlin and Nagahama, 2002, Piferrer and Guiguen, 2008). However, the role of androgens as endogenous testicular inducers is not universal (Blasco et al., 2013, Ijiri et al., 2008, Vizziano et al., 2007). The participation of androgens in the male sturgeon program has not been studied, but it is known that various factors involved in steroid synthesis and receptivity, such as cytochrome P450, family 17, subfamily A, polypeptide 1 (cyp17a); steroidogenic acute regulatory protein (star); and androgen receptor (ar), are expressed prior to sex differentiation (Berbejillo et al., 2012, Vizziano-Cantonnet et al., 2016).
The female program in fish is characterized by early sexually-dimorphic expression of cytochrome P450, family 19, subfamily A, polypeptide 1a (cyp19a1a); forkhead box L2 (foxl2); and follistatin (fst) (Ijiri et al., 2008, Vizziano et al., 2007). Estrogen production is mediated by the product of the aromatase gene (cyp19a1a). The key role of estrogens in the sex differentiation of fish is well established (Baroiller et al., 1999, Devlin and Nagahama, 2002, Guiguen et al., 2010, Piferrer and Guiguen, 2008). Estrogens stimulate genes essential for ovarian differentiation, such as foxl2, fst, and rspo1 (Baron et al., 2004, Nicol et al., 2013, Vizziano-Cantonnet et al., 2008, Zhou et al., 2016). Disruption of cyp19a1a inhibits foxl2 expression in all-female trout populations during androgen-induced ovary-to-testis sex reversal (Vizziano et al., 2008), and deletion of cyp19a1a and foxl2 induces female-to-male sex reversal in medaka (Zhang et al., 2017), supporting the idea that foxl2 plays a key role in female sex differentiation. In medaka, rspo1 shows female-specific activation during gonadal sex differentiation (Zhou et al., 2012) under estrogen control (Zhou et al., 2016). Moreover, rspo1 activation induces femininity in XY fish by enhancing the feminine genes wingless-type MMTV integration site family, member 4b (Wnt4b), and catenin beta-1 (ctnnb1) and suppressing masculine genes (Zhou et al., 2016), suggesting that this gene participates in ovarian differentiation. Briefly, the ovarian program in fish is controlled by estrogens that regulate feminine genes such as foxl2, fst, and rspo1, or by genes that regulate estrogens such as foxl2. In sturgeons, cyp19a1a and foxl2 are activated in some fish and repressed in others prior to sex differentiation (Vizziano-Cantonnet et al., 2018), strongly suggesting that these genes participate in the ovarian program.
Few studies have attempted to identify the genes that regulate sexual differentiation in non-model fish such as sturgeons (Berbejillo et al., 2012, Berbejillo et al., 2013, Vizziano-Cantonnet et al., 2016, Vizziano-Cantonnet et al., 2018). The gonadal transcriptome provides a basic tool for advancing knowledge of genes that control differentiation in non-model organisms. Gonadal transcriptome databases are emerging for several sturgeon species, including the lake sturgeon (Acipenser fulvescens, Hale et al., 2010), Adriatic sturgeon (Acipenser naccarii, Vidotto et al., 2013), and Chinese sturgeon (Acipenser sinensis, Yue et al., 2015). However, to date, the sturgeon genes involved in sex differentiation have not been studied in depth, with efforts limited to analyses using tissues from a single ovary and testis. In the Siberian sturgeon, transcriptomes have been built using samples from undifferentiated embryos and larvae (Song et al., 2015) with undeveloped gonads (Rzepkowska and Ostaszewska, 2013), but there are no published data on the Siberian sturgeon gonadal transcriptome.
To obtain a broader view of candidate genes involved in sex differentiation in the Siberian sturgeon, we built a gonadal transcriptome using tissue samples collected from sexually-undifferentiated fish during the molecular sex-differentiation period. In previous studies, we found that this period occurs at 2.5–6 months of age (Vizziano-Cantonnet et al., 2016). Gonads of recently differentiated fish and of immature male and female were also sequenced as a reference for key genes involved in sex differentiation, as expression of various steroid-related genes such as foxl2, fst, gsdf, dmrt1, amh, is sustained during early gametogenesis in teleosts.
The lack of known sex markers makes it challenging to recognize future females and males at undifferentiated stages. To circumvent this problem, we studied expression profiles of various pro-ovarian, pro-testicular, and steroid-related genes described as sexually dimorphic in the vertebrate literature (Brennan and Capel, 2004, Cutting et al., 2012, Lareyre et al., 2008, Lin and Capel, 2015, Nef et al., 2003, Piferrer and Guiguen, 2008, Russell and Wilson, 1994, Vizziano et al., 2007). We found that cyp19a1a and foxl2 were very good markers of ovarian differentiation. After sexing fish at undifferentiated stages, the gonadal transcriptomes of putative females were compared to those of the putative males to identify genes that modulate sex differentiation in the commercially-valuable Siberian sturgeon.
Section snippets
Experimental animals and rearing procedures
Research procedures involving animal experimentation complied with international principles on the use and care of laboratory animals and Uruguayan regulations on animal welfare (Comisión Honoraria de Experimentación Animal: CHEA). The protocol was approved by the “Comisión de Etica en el Uso de Animales” from the Comisión Honoraria de Experimentación Animal CHEA of Uruguay (Authorization Number 006-11). Siberian sturgeon individuals were obtained from a fish farm (Estuario del Plata, Uruguay)
Assessment of transcriptome quality
Quality assessment for the de novo transcriptome was performed in two ways. First, the reads for each condition were aligned against the contigs to assess the mapping rate. Second, ORF coverage and identity were checked against the database for L. oculatus, which was the species with the best annotation for our transcriptome (Fig. 1). The results of this second step are presented in the following section. The mapping rate of the reads on the contigs ranged from 93.17% to 97.90%. The rarefaction
Discussion
The SexSturg assembly represents a major contribution to the publicly-available sturgeon transcriptome reference data. There are two sturgeon transcriptomes, developed in the lake sturgeon and the Adriatic sturgeon (http://datadryad.org/handle/10255/dryad.37812; Vidotto et al., 2013). These transcriptomes were built from two sex-differentiated individuals, one male and one female, using the Roche 454 platform. Several gonad-relevant genes are missing from these databases, such as aromatase (
Conclusion
Our data confirm that ovarian development in the Siberian sturgeon involves estrogen-related genes with well-established roles in female sex differentiation, such as cyp19a1a and foxl2. However, we also found that genes related to the stem-cell niche, and genes associated with sex-specific nerve development, may play a role in this process. The potential participation of these genes in sex differentiation is a novel finding. The functions of genes associated with the stem-cell niche include
Acknowledgements
This work is dedicated to Dr. Florence Le Gac (INRA, France) who was Professor and great friend of the first author. We are grateful to the Chief Executive Officer of Estuario del Plata, Facundo Márquez; Dr. Andres Ryncowski; Eduardo Olivera, and the entire staff of Estuario del Plata (Uruguay) for their ample support and kindness during this project. Many thanks are due to our colleague Dr. Walter Norbis (Facultad de Ciencias, UdelaR) for his valuable help in selecting the statistical tests.
Funding
The work was funded by the Comisión Sectorial de Investigación Científica (CSIC), Universidad de la República Oriental del Uruguay, Grant C225-348-Uruguay; and by the Agencia Nacional de Investigación e Innovación – Dirección Nacional de Recursos Acuáticos ANII-DINARA – FPA 9975-Uruguay.
Conflict of interest declaration
None.
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