Swathi Tenugu, Akanksha Pranoty, Sajwan-Khatri Mamta,Balasubramanian Senthilkumaran

Department of Animal Biology, School of Life Sciences, University of Hyderabad, P.O. Central University, Hyderabad, 500046, Telangana, India

Keywords:

ABSTRACT

1. Introduction

Steroidogenesis is the synthesis of biologically active steroids that plays a crucial role during various physiological processes involved in gonadal development, growth and maturation. Fishes normally exhibit a wide range of reproductive strategies for sex determination which is followed by differentiation to express their sexuality (Devlin &Nagahama, 2002). Reproduction and gonadal steroidogenesis in teleost are also influenced by various environmental and dietary factors (Gupta,1975; Sumpter, 1997; Van Der Kraak & Chang, 1990). Depending upon the impact of sex steroids, fishes display diverse sexual pattern and are characterised by distinct gonadal differentiation types such as gonochoristic species that possess either testis or ovary and hermaphroditic species that changes the sex to become as protoandric (male to female)or protogynic (female to male) species (Devlin & Nagahama, 2002). Sex steroids that are required during all stages of the reproductive cycle in teleost which include sex differentiation, maturation, growth, and sexual behaviour (García-L´opez, S´anchez-Amaya, & Prat, 2011; Murata et al., 2011; Young, Kusakabe, Nakamura, Lokman, & Goetz, 2005). In most teleost, three major classes of steroids, progestins, androgens and estrogens play a vital role in orchestrating gonadal function (Kazeto,Tosaka, Matsubara, Ijiri, & Adachi, 2011). Interrenal tissue of the head kidney is a vital steroidogenic component in fishes wherein the synthesis of glucocorticoids and mineralocorticoids regulates ionic plus osmotic balance, stress response, immune function, body homeostasis and to some extent reproduction (Faught & Vijayan, 2018; Gallo & Civinini,2003; Goikoetxea, Todd, & Gemmell, 2017). Incidentally, after the initial development, growth of endocrine glands in particular gonads and interrenal, regulatory influence of brain-pituitary became inevitable to evoke the establishment of endocrine axis as well as feedback response (Yaron & Levavi-Sivan, 2011). In vertebrates, the regulation of steroidogenesis is through hypothalamo-hypophyseal-gonadal (HHG)axis that corresponds to gonadotropin-releasing hormone (GnRH)vis-`a-vis gonadotropins (GTHs) and sex steroids. Upon stimulation of GTHs, referring follicle stimulating hormone (FSH) and luteinizing hormone (LH), biologically active steroids such as 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-DP), estradiol-17β (E), testosterone (T)and 11-ketotestosterone (11-KT) are produced abundantly in gonads that are primarily derived from cholesterol (Miura, Yamauchi, Takahashi, & Nagahama, 1991; Nagahama, 1997; Nagahama & Yamashita,2008; Senthilkumaran, 2011; Rajakumar & Senthilkumaran, 2014a, c;Tokarz, M¨oller, Hrabˇede Angelis, & Adamski, 2015).

2. Involvement of mitochondria and endoplasmic reticulum in the development of steroidogenesis

Steroid biosynthesis in fishes occurs in gonads, interrenal tissue and central nervous system (CNS)/brain (Chai, Liu, & Chan, 2003) wherein gonadal and interrenal tissues are considered as major sites (Miller &Bose, 2011) and CNS accounts for neurosteroids production (Diotel et al., 2011). All steroid hormones are synthesized by the mobilization of cholesterol from the outer to inner membrane of mitochondria by steroidogenic acute regulatory protein (star). Subsequently, cholesterol to pregnenolone conversion is achieved by cytochrome P450 side-chain cleavage (P450scc or cyp11a1) enzyme which is a rate-limiting step(Simpson & Boyd, 1966; Wang et al., 1998; Stocco, 2001) and is the first event of steroidogenesis in vertebrates including mammals and pisces(Clark & Stocco, 1995; Stocco, 1996; Stocco & Clark, 1996; Bauer,Bridgham, Langenau, Johnson, & Goetz, 2000). Increased expression of star by human chorionic gonadotropin (hCG) induction has been observed in the zebrafish, Danio rerio and the African catfish, Clarias gariepinus during spawning phase of gonadal cycle and final oocyte maturation (Ings & Van Der Kraak, 2006; Sreenivasulu et al., 2009). In the Nile tilapia, Oreochromis niloticus two different isoforms of star,referred as star1 and star2, have been characterised in the gonads (Yu et al., 2014). However, the expression of star was rarely detectable in undifferentiated gonads of the catfish, C. gariepinus, which later increased gradually during gonadal differentiation (Raghuveer et al.,2011a). In the case of Japanese medaka, Oryzias latipes, expression of star was evident during 20 days post hatch (dph) in XX gonads and 25 dph in XY gonads. It was also observed that star co-expressed with hydroxysteroid 3β-dehydrogenase/Δ-Δisomerase (hsd3b), in the interstitial/Leydig cells to drive steroidogenesis. This process is transcriptionally regulated by adrenal-4-binding protein/steroidogenic factor 1 (ad4bp/sf-1) during development of testis but contradictorily its onset of expression was seen at later stages of ovarian development in medaka (Nakamoto et al., 2012). Knockout (KO) of star and luteinizing hormone β subunit (lhb) in zebrafish using transcription activator-like effector nucleases (TALENs), decreased the levels of maturation inducing hormone (MIH) in the oocytes of both mutants. Furthermore,expression of star was found to be downregulated in lhb mutants indicating its pivotal role in MIH synthesis and star might act as a downstream target to the luteinizing hormone (LH) signalling (Shang et al.,2019). Each step of steroid biosynthesis is catalyzed by various steroidogenic enzymes such as P450scc, (cyp11a), steroidogenic cytochrome P450 17-hydroxylase/lyase, (cyp17), hsd3b, hydroxysteroid 17β-dehydrogenase 1, (hsd17b1), cytochrome P450, family 11, subfamily b, polypeptide1, (cyp11b1), hydroxysteroid 11β-dehydrogenase(hsd11b) and cytochrome P450, family 19, subfamily a, polypeptide 1(cyp19a1). Various steps involved in steroid biosynthesis are mostly conserved between human and teleost with a difference in aldosterone synthesis in human, MIH synthesis in fishes and divergence in androgen synthesis pathway (Tokarz et al., 2015). Since most of the steps are conserved, especially the rate-limiting cholesterol mobilization step, it was presumed that initial steps occurring in mitochondria and endoplasmic reticulum might be conserved. Mobilization of cholesterol into mitochondria was driven by transduceosome and metabolon (Liu, Rone,& Papadopoulos, 2006; Rone et al., 2012) and which are multiprotein complexes containing cytosolic proteins including, protein kinase cAMP-dependent type I regulatory subunit α, Star protein,diazepam-binding inhibitor and acyl-CoA-binding domain containing 3 and mitochondrial proteins including voltage-dependent anion channel,translocator protein, AAA domain containing 3A, ATPase family,Cyp11a1 and ferredoxin reductase 3, 14-3-3γ adaptor protein (Rone et al., 2012; Aghazadeh et al., 2012). Cyclic adenosine monophosphate(cAMP) signal at outer mitochondrial membrane is amplified by transduceosome whereas unwanted cholesterol interaction with other pathways is prevented by metabolon making it available for cyp11a1 for the synthesis of pregnenolone, common precursor for most of the steroid hormones (Midzak & Papadopoulos, 2016). Teleostean steroidogenesis pathway is shown in Fig.1. Cholesterol for steroidogenesis is known to be derived from lipid droplets and the interactions between lipid droplet, endoplasmic reticulum and mitochondria suggesting a loop(Issop, Rone, & Papadopoulos, 2013).

Fig.1. Schematic representation of steroidogenesis in teleosts. Mobilization of cholesterol by star to inner mitochondrial membrane and major steroidogenic enzymes catalysing the reaction for the synthesis of steroids. Blue box indicates major sex steroids and pink box indicates maturation inducing hormones. Abbreviations are: cyp, cytochrome P450; DHEA, dehydroepiandrosterone; OH-, hydroxy-; hsd, hydroxysteroid dehydrogenase; star, steroidogenic acute regulatory protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3. Sexual development and steroidogenesis onset: role of transcription factors, steroidogenic enzymes and growth factors

3.1. Transcription factors

A duplicated copy of the autosomal doublesex and mab-3-related transcription factor (dmrt) dmrt1a, named as DMY/dmrt1bY, was first identified in the medaka, which had a prominent role as a male sex determining gene (Matsuda et al., 2002; Nanda et al., 2002). Furthermore, introduction of dmrt1bY transgene in genetic females (XX) of medaka promoted testis development (Otake et al., 2009). Dmrt family comprises several members which majorly contribute in sex determination and differentiation processes. The characteristic feature of dmrt is the presence of a conserved DNA-binding motif known as the doublesex and mab-3-related domain. This domain is a non-canonical cysteine-rich DNA binding motif with two intertwined finger structures chelating one zinc ion that binds to the minor groove of DNA (Herpin & Schartl, 2011;Zhu et al., 2000). In spite of the differences in gonadal structure among different phyla, dmrt, specifically, was found to be expressed in developing gonad of several vertebrates including teleost. Several forms of dmrts, dmrt1, dmrt2, dmrt3, dmrt4, dmrt5 and dmrt6 were found to be expressed in gonads of fishes (Cao, Chen, Wu, Gan, & Luo, 2009; Guo et al., 2004; Liu et al., 2008; Wen et al., 2008; Winkler et al., 2004;Zhang et al., 2014). dmrt1 has been identified in a wide variety of gonochoric, protandrous, protogynous and temperature-dependent sex determining fish species including catfish, where it was highly expressed in differentiating testis, spermatogonia, spermatocytes, spermatids,Sertoli and epithelial cells in the efferent duct (Herpin & Schartl, 2011;Raghuveer & Senthilkumaran, 2009) as well as in the germ cells of zebrafish and the Atlantic cod, Gadus morhua (Guo et al., 2005; Johnsen,Seppola, Torgersen, Delghandi, & Andersen, 2010). C. gariepinus undergo seasonal patterns of gonadal resting and recrudescence, wherein expression of dmrt1 was higher during testicular recrudescence, particularly in spermatogenesis (Raghuveer & Senthilkumaran, 2009). In O.latipes, testis differentiation is retained by dmrt1 after DMY induction which elicited differentiation pathway in male. However, in XY medaka lacking dmrt1, testis normally developed first that later differentiated into ovary which was rescued by transgenesis at dmrt1 genomic region(Masuyama et al., 2011). Mutation in dmrt1 by using TALENs in the Chinese tongue sole, Cynoglossusse milaevis caused intersex and ovary-like testis with disrupted spermatogenesis along with over expression of female related genes like foxl2 and cyp19a1a and declined expression of anti-Mϋllerian hormone (amh) and sox9a (Cui et al., 2017)suggesting dmrt1 as a male sex-related gene in this species. Reports from various groups of researchers established the expression of other members of dmrt family in different stages of gonadal maturation in teleost. A study using medaka indicated the expression of dmrt2/3/4 in differentiating gonad and mature testis (Winkler et al., 2004). Expression of dmrt5 was reported in developing germ cells of gonads while dmrt3 expression was evident in spermatocytes, spermatogonia and developing oocytes in zebrafish (Guo et al., 2004; Li et al., 2008). KO of dmrt6 in the Nile tilapia resulted in reduced expression of cyp11b2, fewer spermatocytes and low levels of serum 11-KT (Zhang et al., 2014).Incidentally, authors recorded recovery of these correlates from 150 to 180 dph fish. Germ cell development is directly and locally influenced by sex steroids which also affect cell and organs engaged in sex differentiation. Exposure to feminization inducing compounds downregulated dmrt1 indirectly impacting the transcriptional regulation in the rare minnow, Gobiocypris rarus (Zhang, Zha, & Wang, 2008), the pejerrey,Odontesthes bonariensis (Fernandino et al., 2008) and D. rerio (Schulz et al., 2007). Transcriptional upregulation of dmrt1 was also reported upon treatment with masculinization inducing compounds in several teleost (Herpin & Schartl, 2011). A report using zebrafish model suggested direct downregulation of dmrt1 by a member of SRY-like HMG-box (sox) gene, Sox5 during various developmental processes including spermatogenesis (Gao et al., 2005). Expression of dmrt1 and sox9 in the lambari, Astyanax altiparanae showed alterations during testicular cycle and incidentally, downregulation of those genes were evident after spermiation (Adolfi et al., 2015). A recent study using the Nile tilapia reported positive regulation of sox30 by Dmrt1 by binding to a putative cis-regulatory element within sox30 promoter (Tang et al.,2019). These reports established the interaction between dmrt1 and sox family genes.

Proteins encoded by sox family of genes have a characteristic Sry related high mobility group (HMG) domain has been reported in a wide range of species including fishes (Wei, Yang, Tao, & Wang, 2016).Among teleost, other than sox9, the role of several members of the family are less established. The involvement of sox9 in male gonadal development was evident among vertebrates. In several species of teleost, including zebrafish, medaka and stickleback, two isoforms of sox9, sox9a and sox9b have been reported (Klüver, Kondo, Herpin,Mitani, & Schartl, 2005). sox9a expression varied with species as it was expressed more in zebrafish testis (Chiang et al., 2001) in contrast to prominent expression in medaka ovary (Klüver et al., 2005). Expression of sox9a in C. gariepinus was important during spermatogenesis. Further,treatment of testicular slices with hCG and 11-KT resulted in upregulation of sox9a indicating GTH mediated stimulation of sox9 (Raghuveer &Senthilkumaran, 2010). Conversely, the expression of sox9b declined in ovary from preparatory to post-spawning phases (Raghuveer & Senthilkumaran, 2010). Spatial expression analysis of sox genes in the Nile tilapia confirmed the presence of 5 sox genes in ovary and 15 in testis wherein sox3 expression was the highest in ovary while sox30 in testis(Wei et al., 2016). In O. dancena, sox3was localized in the neural tube,neuromasts in XX embryo and in somatic cells that surrounds germ cells in XY gonad, however, no expression was seen in mature ovary or testis indicating a plausible role for sox3 in testis determination which was further confirmed by sex reversal in sox3transgenic fish (Takehana et al., 2014). Further, it was also demonstrated that sox3 activates its downstream gonadal soma derived factor (gsdf) which is essential for testicular differentiation. Study in C. batrachus indicated higher sox3 expression in testis and brain as compared to ovary. Furthermore, high expression of sox3 in immature testis and ovary confirmed the significance during gonadal development and maturation. In addition to this,high expression of sox3 was found in spawning testis which further elevated after hCG induction indicating its GTH dependency (Rajakumar& Senthilkumaran, 2014b). The existence of sox30 in a non-mammalian vertebrate was first reported accidently in the Nile tilapia while attempting to clone sox9b wherein four isoforms of sox30 have been reported, out of which, only three were expressed in the adult gonads.Expression of isoform-I was found to be higher in testis than other tissues whereas isoform-II was evident only in testis. Expression of isoform-IV was more in ovary as compared to testis while expression of isoform-III was restricted to earlier stages of development i.e., 10 dph fish larva (Han et al., 2010). Interestingly, amongst all the members of sox family of genes, sox19 was found only in fish however an ortholog of sox19 has been reported in mammals, named as, sox15 belonging to a different sox family subgroup (Navarro-Martín, Galay-Burgos, Piferrer,& Sweeney, 2012). In this context, in the European sea bass, Dicentrarchus labrax the expression of sox19 was evident in brain, skin as well as gonads. Furthermore, its expression during gonadal differentiation remained relatively constant in males whereas in females, a dramatic increase in expression was seen during ovarian differentiation (Navarro-Martín et al., 2012). Another member of the family, i.e., sox17 was characterised in very few species wherein genomic structural similarity was found between the rice field eel, Monopterus albus and mouse (Zhou et al., 2002). Furthermore, the expression of sox17 was detected in developing spermatogenic cells of testis, lamellae of ovaries and ovotestis in eel suggesting its role during sex reversal (Wang et al.,2003). However, the presence and significance of several other forms of sox remains unclear justifying further studies.

ad4bp/sf-1 is known to be a key modulator for steroid biosynthesis and transcriptional regulator for cyp19a1a gene expression in medaka and the Nile tilapia ovary (Watanabe et al., 1999; Yoshiura et al., 2003).Expression of cyp19a1a and ad4bp/sf-1 was found to be high during vitellogenic phase which declined during final oocyte maturation in the Nile tilapia (Yoshiura et al., 2003). Additionally, hCG was found to induce the expression of cyp19a1a and ad4bp/sf-1 in post-vitellogenic immature follicles when incubated in vitro (Yoshiura et al., 2003).ad4bp/sf-1 regulates the transcription of hsd3b in medaka, where co-expression of both correlates were observed in the interstitial/Leydig cells during testicular development while in developing ovary major expression was evident in the follicular cells (Nakamoto et al., 2012).Expression of ad4bp/sf-1 along with cyp19a1b was found to be higher in testis as compared to ovary in 3 and 10 months old carp which revealed its involvement in testicular development (Tang, Hu, Hao, & Zhu, 2010).Moreover, ad4bp/sf-1 regulated the expression of cyp19a1a in GTH dependent manner as evident from the report of hCG induction during oocyte development in the rohu, Labeo rohita (Moulik et al., 2016) and the Nile tilapia, O. niloticus (Yoshiura et al., 2003). Report from the banded gourami, Trichogaster facitata revealed regulation of ad4bp/sf-1 by FSH and LH and it was localized in both vitellogenic and post-vitellogenic stages of ovary (Guchhait, Chatterjee, Mukherjee, &Pramanick, 2018). KO of sf-1 through CRISPR/Cas9 technique in tilapia induced gonadal dysgenesis with decrease in the number of steroidogenic cells in gonads (Xie et al., 2016). Additionally, these authors recorded decreased expression of cyp19a1a, foxl2 and Elevels in XX fish, whereas cyp19a1a, foxl2, cyp11b2 and 11-KT levels in XY fish after KO indicating the regulatory influence of sf-1 on steroidogenesis and reproduction. It is important to note that ad4bp/sf-1 is also represented as sf-1 in some teleosts (Moulik et al., 2016; Xie et al., 2016).

In line with this, fushi tarazu factor-1 (ftz-f1), a subfamily member of nuclear receptors is also considered analogous to sf-1 and certain reports include sf-1 as a member of ftz-f1. Like ad4bp/sf-1, ftz-f1 is known to function as a transcriptional regulator and it regulates the expression of salmon gonadotropin IIβ subunit gene in zebrafish (Liu, Le Drean, Ekker,Xiong, & Hew, 1997). It is a potential regulator of steroidogenesis whose increase in expression was evident along with star and cyp11a during reproductive maturation of medaka showing its regulatory influence on cyp19a1a promoter in ovarian follicle (Watanabe et al., 1999). Increased expression of ftz-f1 was evident in the gonads of both juvenile and mature black rockfish, Sebastes schlegelii with abundance of transcripts in testis (Shafi et al., 2012). Similarly, in the Nile tilapia, O. niloticus the ftz-f1 transcript expression was evident only in gonad and liver which specified a role in gonadal development (Cao, Chen, Jiang, Luo, & Gan,2012). In the Arctic char, Salvelinus alpinus, expression of ftz-f1 reduced after Etreatment in males while 11-KT treated males showed decreased expression of hsd3b and cyp11a1 indicating the regulation of ftz-f1 by E(Von Hofsten, Karlsson, Jones, & Olsson, 2002). Four homologues of ftz-f1 referred as ff1a, ff1b, ff1c and ff1d were found in zebrafish wherein ff1a expression was evident in pronephric duct, liver, somites and hindbrain whereas ff1d co-expressed with amh in both gonads which in general indicated a role in reproduction, sex determination and differentiation (Von Hofsten, Karlsson, Jones, & Olsson, 2001). In C. gariepinus, ftz-f1 along with foxl2 cooperatively binds to cyp19a1b promoter and upregulate cyp19a1b expression, which might promote ovarian differentiation and recrudescence indirectly (Sridevi, Chaitanya,Dutta-Gupta, & Senthilkumaran, 2012; Sridevi, Dutta-Gupta, & Senthilkumaran, 2011).

foxl2 belonging to forkhead family of transcription factors plays a major role during ovarian differentiation along with ad4bp/sf-1 by regulating cyp19a1 expression in teleost. The forkhead domain of the foxl2 binds with ligand-binding domain of ad4bp/sf-1 as a heterodimer,thus, acting as a co-activator (Wang et al., 2007). Further, it also increased ad4bp/sf-1 dependent cyp19a1 expression in tilapia. Initiation of foxl2 expression was linked to differentiation of ovary as observed in teleost such as rainbow trout, medaka, tilapia, and southern catfish(Baron et al., 2004; Ijiri et al., 2008; Liu, Zhang, & Wang, 2008; Nakamoto, Matsuda, Wang, Nagahama, & Shibata, 2006). In C. gariepinus,foxl2 expression was evident in ovary particularly follicular layer of oocytes at 60 dph (Raghuveer et al., 2011a). Localization of Foxl2 protein was evident in the follicular layer of oocytes of medaka and the Nile tilapia, while in zebrafish it was observed in germ and somatic cells in the case of ovary whereas germ and Leydig cells in testis (Caulier et al., 2015; Nakamoto et al., 2006; Wang et al., 2007). TALENs based targeted KO approach on foxl2 in XX tilapia by Li et al. (2013) showed decreased cyp19a1a expression with varied degree of oocyte degeneration as well as low serum E. Additionally, evidence for sex reversal in foxl2-deficient fish with significant expression of dmrt1 and cyp11b2 was shown. Double KO of foxl2a and foxl2b in zebrafish caused female to male sex reversal accounting to mutual involvement of both genes to regulate ovarian development with dominant role of the former in preventing ovary from testis differentiation (Yang, Wang, Li, Zhou, &Gui, 2017).

In teleost, cAMP acts as an important second messenger for synthesis of both Eand 17α, 20β-DP and many GTH related processes. For example, in the coho salmon, O. kisutch testicular hormones were produced by the stimulation of GTH where elevated cAMP levels act as a mediator of spermatogenesis (Planas, Swanson, & Dickhoff, 1993).Further, induction of 17α, 20β-DP production during spermiation by dibutyryl cAMP indicated its regulatory influence to entrain the steroidogenic activity of GTH (Sakai, Suzuki, & Nagahama, 1996). Even in the brook trout, S. fontinalis, ovarian steroidogenesis was stimulated by GTH through cAMP/PKA signalling pathway with additional involvement of protein kinase C (PKC)/Ca(Planas, Goetz, & Swanson, 1997)as shown in Fig.2. Similarly GTH induced steroidogenesis through multiple signalling transduction pathways was found in common carp ovary (Paul, Kundu, Pramanick, Bandyopadhyay, & Mukherjee, 2010).cAMP signalling pathway is mediated by cAMP response element binding (Creb) protein and cAMP response element (CRE) interaction that is observed in the promoter region of CRE related genes. For example, CREs were appeared in hsd20b and cyp19a1a promoters that were transcriptionally regulated by crebs during shift in steroidogenesis in rainbow trout and the Nile tilapia (Senthilkumaran, Yoshikuni, &Nagahama, 2004; Sreenivasulu, Senthilkumaran, Sudhakumari, et al.,2012; Huang, Zhou, Li, & Gui, 2009). Multiple forms of creb such as creb1, creb2 and creb3 were identified in the gonad of the Nile tilapia while a single form of creb was observed in catfish (Senthilkumaran et al., 2015a). In addition, creb3 was found to be prominently expressed during spawning as well as hCG-induced oocyte maturation as compared to creb1 and creb2 in the Nile tilapia whereas in the catfish, C. gariepinus elevated expression of creb was evident during spawning and after hCG induction of ovary (Senthilkumaran et al., 2015a). This spatio-temporal expression of creb indicated a major role for this correlate during oocyte maturation as well as regulating oocyte growth in fish. creb1 partially regulated cyp19a1a expression whereas hsd20b was regulated by creb3 as well as creb2 which was evident in the Nile tilapia and in catfish a single form of creb is the regulator. Thus, multiple forms of creb might be involved in other fish species like that of rainbow trout or tilapia(Murugananthkumar & Senthilkumaran, 2013; Senthilkumaran, 2011;Senthilkumaran et al., 2015a; Sreenivasulu, Senthilkumaran, Sudhakumari, et al., 2012).

Activator protein-1 (AP-1) act as the transcription factor that play a very important role during signalling cascades such as PKC pathway that is involved in the expression of Star protein during steroidogenesis(Manna & Stocco, 2005). In vertebrates, AP-1 factor usually formed by the combinational heterodimerization of JUN (junB, junD and cjun) and FOS (fosB, fra1, fra2 and cfos) members that are ubiquitously expressed(O’Shea, Rutkowski, & Kim, 1992). Transcriptional cofactors such as CREB-binding protein (CBP) and its functional homolog, referred as p300 (CBP/p300) interacts with other factors like creb along with cjun and cfos which usually binds to the CRE regions that are present on star promoter to regulate its transcription (Manna et al., 2002; Manna,Wang, & Stocco, 2003). Most of the transcription factors such as cjun and gata4 regulating the star promoter has been extensively studied in mouse as compared to lower vertebrates (Martin, Bergeron, Viger, & Tremblay,2011; Tremblay & Viger, 2001). These genes as well as cofactors involved in star promoter activation and regulation during steroidogenesis are yet to be studied in most teleost.

Fig.2. Postulated signalling pathway and major Δ4/ Δ5 pathways involved during gonadal steroidogenesis in teleosts. Binding of gonadotropins to the tropic receptor and activation of protein kinase A, protein kinase C/Ca2+ signalling pathways and transcription factors by phosphorylation and binding to the promoter region of star as well as steroidogenic enzymes and modulating steroid hormone biosynthesis with either Δ4/ Δ5 pathways. Abbreviations are: AP-1,activator protein-1; ATP, adenosine triphosphate; ATF, activating transcription factor; cAMP, cyclic Adenosine monophosphate; cAMP response element binding protein, Creb; cfos, fos proto-oncogene; cjun, jun proto-oncogene; cyp, cytochrome P450; DAG, diacylgycerol; DHEA, dehydroepiandrosterone; FSH, follicle stimulating hormone; OH-, hydroxy-; hsd, hydroxysteroid dehydrogenase; IP3, inositol 1,4,5-trisphosphate; junD, junD protooncogene; LH, luteinizing hormone; PKA,protein kinase A; PKC, protein kinase C; PLC, phospholipase C; star, steroidogenic acute regulatory protein.

wt1 is important for the interrenal development in most teleost (Hsu,Lin, & Chung, 2003). It was found to be involved in the expression of ffb in zebrafish that helps in interrenal differentiation as well as activation of cyp11a1 (Hsu et al., 2003). Two forms of wt1, wt1a and wt1b have been identified in medaka where both act as critical transcription factors for primordial germ cell maintenance during gonadal development in addition to expression in testis, ovary, liver, kidney and spleen of adult fish (Klüver, Herpin, Braasch, Drieˆale, & Schartl, 2009). The GATA family, zinc finger DNA binding protein, gata4 is ament as observed in tn important marker for gonadal differentiation and development as observed in tilapia (Li et al., 2011). The binding sites of Gata4 and Wt-1 have been identified on cyp19a1a gene promoter in zebrafish indicating their regulatory role (Tong & Chung, 2003). Increased expression of gata4, wt-1 along with ad4bp/sf-1 were evident in testis as compared to other tissues and these genes were found to be upregulated after hCG induction during prespawning and spawning phases in catfish. This interactive role of wt-1 in the catfish, C. batrachus was further proved by using wt1-esiRNA, which decreased both ad4bp/sf-1 and gata4 expression along with certain steroidogenic enzyme genes (Murugananthkumar & Senthilkumaran, 2016).

3.2. Steroidogenic enzymes

The steroidogenic enzyme cyp11a1, catalysing the rate limiting step,is involved in cholesterol to pregnenolone conversion during steroid hormone biosynthesis (Miller & Auchus, 2011). This step is highly crucial for the synthesis of all three major classes of steroid hormones such as mineralocorticoids, glucocorticoids, and sex steroids. In rainbow trout, increased expression of cyp11a1 was evident in post ovulatory follicles indicating a role in the final oocyte maturation by rapidly elevating 17α, 20β-DP production (Takahashi et al., 1993). In the case of medaka, dmrt1 activates cyp11a expression (Wang et al., 2010). In the Japanese eel, expression of cyp11a1 was evident in ovary and head kidney, which was induced in ovary by salmon pituitary homogenate indicating GTH dependency (Kazeto, Ijiri, Adachi, & Yamauchi, 2006).Expression of cyp11a was evident in interstitial cells at 15 dph till testicular development in male medaka while in females the expression was seen from 5 dph till ovarian development (Nakamoto et al., 2010).cyp11a1 expression was observed in gonads, interrenal glands and brain of zebrafish (Hsu, Hsiao, Kuo, & Chung, 2002). Additionally in zebrafish, two forms of cyp11a were found in which cyp11a1 was restricted to gonads during early development from 0 to 48 h post fertilization (hpf)while cyp11a2 was evident after 32 hpf which is required for both the initiation and maintenance of interrenal steroidogenesis (Parajes et al.,2013). In the catfish C. batrachus, cyp11a1 was highly expressed in mature testis especially during prespawning and spawning phases as well as after hCG induction (Rajakumar & Senthilkumaran, 2014a).Recent study using a mutant cell lines on cyp11a2 by CRISPR/Cas9 approach in zebrafish revealed downregulation of male related genes i.e.sox9a, dmrt1 and amh in cyp11a2 homologous group whereas in homozygous mutants, the spermatocyte marker such as sycp3 and spermatid marker odf3b were downregulated which specified a major role for this correlate in both interrenal and gonadal steroidogenesis (Li,Oakes, Storbeck, Cunliffe, & Krone, 2020). However, in zebrafish, Ff1b which is a homolog of ad4bp/sf-1 activated cyp11a1 expression by binding with two conserved Ff1 response elements identified in the cyp11a1 putative promoter (Quek & Chan, 2009).

cyp17a is essential for the production of 17α-hydroxyprogestins in gonads and cortisol in the interrenal tissue. This enzyme is required for Cto Csteroid conversion, the second step of steroidogenesis involved in the production of 17α-hydroxyprogesterone (17-OHP) and androstenedione. It is a single enzyme having both 17α-hydroxylase as well as 17, 20-lyase activity (Kazeto, Ijiri, Todo, Adachi, & Yamauchi,2000b). In medaka and the Nile tilapia, cyp17a1 had both the activities whereas cyp17a2 possess 17α-hydroxylase lacking 17, 20-lyase activity however in the Nile tilapia, expression of cyp17a1 and cyp17a2 was evident in ovary and head kidney which synthesize Ein ovary whereas Csteroids such as cortisol in the head kidney (Zhou et al., 2007a).Similarly, in medaka, expression of cyp17a1 was observed during oocyte growth for Eproduction while cyp17a2 contributes for cortisol production in the head kidney. Incidentally, the expression of both the genes is regulated by foxl2, dax1, lrh-1 and ad4bp/sf-1 during spawning stage in medaka (Zhou et al., 2007b). But for instance, in the Japanese eel only one form cyp17a is present which had both the activities wherein the elevated 17α-hydroxylase activity was seen from vitellogenic till the maturation stage while decreased Clyase activity was evident in the same stage (Kazeto et al., 2000b). In a natural sex reversal fish, rice field eel, cyp17 was predominantly expressed in testis, very less in ovary and negligible in ovotestis (Yu, Cheng, Guo, Xia, & Zhou, 2003).Predominant expression of cyp17 was observed in gonads and with weak signal of cyp17 in whole kidney signifying its role in gonadal and to some extent on interrenal steroidogenesis of zebrafish (Wang & Ge, 2004).Furthermore, in C. gariepinus, the expression of single form of cyp17 was found in follicular layer of ovary as well as in spermatocytes and interstitial cells of testis. Elevated expression of cyp17 was observed during preparatory and prespawning stage of follicular development whereas testis showed maximum expression during preparatory and spawning phases (Sreenivasulu & Senthilkumaran, 2009a). Zhai et al.(2017) showed KO of cyp17a1 in zebrafish caused reduction in serum levels of T and 11-KT with decreased expression of sox9a and elevated expression of amh indicating the synergistic mechanism of androgen signalling with sox9a in regulating transcription of amh. Lack of cyp17a1 hindered ovarian differentiation at 32 dph which was rescued after the treatment of sex steroids (Zhai et al., 2018). cyp17a1 KO males, upon treatment with 11-KT and T rescued male secondary sexual characters in zebrafish indicating androgen dependency (Shu, Zhai, Pradhan, Olsson,& Yin, 2020).

hsd17b is the steroidogenic enzyme required for production of T and Ewhich are involved during sex differentiation and gametogenesis processes (Adamski & Jakob, 2001). hsd17b exists as isoforms wherein hsd17b3 was involved in the conversion of androstenedione to T in most of the fishes whereas hsd17b1 converts an inactive form of estrone to an active receptor binding Eduring steroidogenesis (Mindnich, Deluca, &Adamski, 2004). hsd17b3 has been well characterised in medaka and zebrafish while hsd17b1 is partially characterised in the Japanese eel,zebrafish, the Nile tilapia and the Atlantic cod (Kazeto, Ijiri, Matsubara,Adachi, & Yamauchi, 2000a; Mindnich et al., 2005; Zhou et al., 2005;Breton & Berlinsky, 2014). In zebrafish, expression of hsd17b3 was evident at sphere stage to 84 hpf during embryogenesis whereas in adults it exhibited sexual dimorphism by showing elevated expression of hsd17b3 in ovary and comparatively very less detection in testis(Mindnich et al., 2005). Elevated expression of hsd17b3 at vitellogenesis and hsd17b1 in pre-vitellogenesis was observed in zebrafish during ovarian follicular development (Ings & Van Der Kraak, 2006). On the other hand, in the Atlantic cod, expression of hsd17b1 was evident during mid vitellogenesis (Breton & Berlinsky, 2014). Dominant expression of hsd17b1 was observed in ovary compared to testis where hsd17b12 expressed exclusively in testis, however hsd17b8 is ubiquitous in both sexes of the Nile tilapia (Zhou et al., 2005). Expression of hsd17b1 was observed during early vitellogenesis in the Japanese eel and after artificial maturation by salmon pituitary homogenate (Kazeto et al., 2000a). In C. batrachus, the expression of hsd17b1 was found to be high in developing as well as mature ovary while hsd17b12 was high in testis. Expression of both enzyme genes elevated after hCG induction indicating their dependency to GTHs (Rajakumar & Senthilkumaran,2014c).

One of the most conserved enzymes, cyp19a1 encoding aromatase which converts androgens to estrogens is common to all teleost species in controlling ovarian differentiation, development and growth. Unlike mammals, teleost exhibit intense aromatase activity that are encoded by two genes, referred as cyp19a1a and cyp19a1b were localized in ovary and brain, respectively. These two genes were identified in variety of species such as O. mykiss, C. auratus, D. rerio, O. niloticus, D. labrax, O.latipes, C. carpio and C. gariepinus (Barney, Patil, Gunasekera, & Carter,2008; Bl´azquez & Piferrer, 2004; Callard & Tchoudakova, 1997; Chiang,Yan, Guiguen, Postlethwait, & Chung, 2001; Kuhl, Manning, & Brouwer,2005; Kwon, McAndrew, & Penman, 2001; Rasheeda, Sridevi, & Senthilkumaran, 2010; Tanaka et al., 1992; Valle, Ramina, Vianello,Belvedere, & Colombo, 2002). cyp19a1a is known for its pivotal role in gonadal sex differentiation in fishes (Guiguen, Fostier, Piferrer, &Chang, 2010). Upregulation of cyp19a1a is crucial for ovarian differentiation while downregulation is needed for testicular differentiation(Wang et al., 2007). Previous study reported that cyp19a1a activity is mainly regulated at transcription level in ovarian follicles rather than during ovarian development in the Japanese eel (Ijiri, Kazeto, Mark Lokman, Adachi, & Yamauchi, 2003). In the Nile tilapia, cyp19a1a expression was found to be evident at 15 to 90 dph in females along with synchronous expression of ad4bp/sf-1 in contrast to males where it showed only after 90 dph as well as elevated expression during vitellogenic stage of ovary indicating its role during sex differentiation and ovarian development (Chang et al., 2005; Sudhakumari et al., 2005).Expression of cyp19a1a showed a declining trend when post-vitellogenic follicles of the Nile tilapia were incubated with hCG, in vitro (Yoshiura et al., 2003). Promoter analysis of cyp19a1a gene suggested that ad4bp/sf-1 acts as a primary regulator with co-activating function of foxl2 as well as nr5a1 in the rainbow trout, medaka, zebrafish, gilthead bream, the Nile tilapia and the Japanese founder (Baron et al., 2005;Kanda et al., 2006; Yamaguchi, Yamaguchi, Hirai, & Kitano, 2007;Yoshiura et al., 2003). In the Nile tilapia, dmrt1 binds to the upstream of the cyp19a1a promoter and represses its expression and estrogen production in gonads during early testicular differentiation (Wang et al.,2010). Few reports demonstrated cyp19a1a expression in interstitial as well as follicular cells during ovarian development of medaka (Suzuki,Tanaka, & Shibata, 2004) which is critical during various developmental and maturational stages (Nakamoto et al., 2010). cyp19a1a mutants developed into males whereas cyp19a1b mutants showed no defects indicating cyp19a1a is indispensable for ovarian development (Lau,Zhang, Qin, & Ge, 2016; Yin et al., 2017). Double KO of dmrt and cyp19a1a in zebrafish showed normal development of follicles even in the absence of cyp19a1a or estrogens till pre-vitellogenic stage (Wu,Song, Zhang, & Ge, 2020). Further studies are required to evaluate this contention using other teleost fish models.

11b-hydroxylase (11b-h) is encoded by cyp11b which is required for the synthesis of precursor for 11-KT production (Jiang et al., 1996),conversion of respective precursors to corticosterone and cortisol (Jiang,Young, Kobayashi, & Nagahama, 1998). Increased expression of cyp11b/11b-h was evident during ovary to testis transformation in zebrafish (Wang & Orban, 2007). Available reports suggested that sex steroids can alter the expression of cyp11b in the testis of rainbow trout(Baron, Fostier, Breton, & Guiguen, 2005; Govoroun, McMeel,Mecherouki, Smith, & Guiguen, 2001) and in the gonad of Japanese flounder (Meng et al., 2019). Both gonadotropin receptors and cyp11b1 mRNA showed positive correlation in the European sea bass males(Rocha, Zanuy, Carrillo, & G´omez, 2009). Interestingly, male-specific expression was observed in the midshipman fish, Porichthys notatus where territorial type I males exhibited higher level of 11b-h than sneaker type II males which can be correlated with high 11-KT levels(Arterbery, Deitcher, & Bass, 2010). Four forms of 11b-h, variant 1,variant 2, variant 3 and a regular type were identified in the testis of C.batrachus wherein the expression of variants were high in testis (Rajakumar & Senthilkumaran, 2015). Same study also revealed higher expression of regular type 11b-h during the prespawning phase of testicular cycle and after hCG treatment in vitro, whereas all types of 11b-h were upregulated during hCG treatment in vivo. In the Japanese flounder 11b-h was localized in oocytes, spermatocytes and Sertoli cells,however high expression was observed in testis as compared to other tissues including ovary (Meng et al., 2019). Mutation of cyp11c1(cyp11c1) in zebrafish revealed its importance in juvenile ovary to testis transformation, development of Leydig cells and spermatogenesis by 11-KT in males while oocyte maturation and ovulation by cortisol in females (Zhang et al., 2020).

hsd3b/Δisomerase, hsd20b and hsd11b are other key enzymes crucial for steroid synthesis. hsd3b has been identified in several species including fishes such as in rainbow trout (Sakai et al., 1994), zebrafish(Lai, Hsiao, Guiguen, & Chung, 1998), the Japanese eel (Kazeto, Ijiri,Matsubara, Adachi, & Yamauchi, 2003), the Nile tilapia (Senthilkumaran et al., 2009), catfish (Raghuveer & Senthilkumaran, 2012) and C.batrachus (Bhat et al., 2018). In fact, novel multiple forms of hsd3b was identified in the Nile tilapia for the first time which showed prominent changes during gonadogenesis (Senthilkumaran et al., 2009). In zebrafish, two hsd3b genes were identified, first the adult form, hsd3b1 expressed in head kidney and adult gonad whereas the embryonic form,hsd3b2 expressed as maternal transcripts in early embryos till 1 daf (Lin et al., 2015). In the orange-spotted grouper, Epinephelus coioides, a protogynous hermaphrodite species, high expression of hsd3b1 was found to be observed in gonad during female to male sex reversal whereas expression of hsd11b2 was high in brain of grouper (Xiao et al.,2020). Interestingly in midshipman fish, similar to 11b-h, expression of hsd11b was high in territorial type I males which again can be correlated with higher 11-KT level (Arterbery et al., 2010). In the pejerrey, O.bonariensis larvae at male producing temperature, cortisol modulated hsd11b2 expression for the production of 11-KT (Fernandino, Hattori,Kishii, Strüssmann, & Somoza, 2012). Ontogeny studies in catfish revealed sex specific expression of hsd11b from 50 dph onwards and ubiquitous expression was found to be observed with prominent in testis as well as during spawning phase of reproductive cycle (Rajakumar &Senthilkumaran, 2016). Promoter analysis study using luciferase reporter assay revealed binding of Sox3 to hsd11b promoter and regulates male reproduction. hsd20b is required for conversion of 17α-OHP to MIH required for shift in steroidogenesis during oocyte maturation (Nagahama, 1997). Carbonyl reductase-like hsd20b in the Nile tilapia was expressed in various tissues including gonad with distinct expression in ovarian follicles on the day of spawning whereas no expression was observed in immature testis of 30 dph fish (Senthilkumaran et al., 2002).High expression of hsd20b in post-vitellogenic immature follicles during final oocyte maturation and within 1-2 h after hCG treatment suggested its important role during final gamete maturation. hCG induced hsd20b expression and oocyte maturation in the Nile tilapia was inhibited by actinomycin D provided clue for involvement of transcription factors in regulating hsd20b and MIH production (Senthilkumaran et al., 2002). A recent study using the same species suggested the involvement of hsd17b12 in MIH production instead of hsd20b (Aranyakanont, Ijiri,Hasegawa, & Adachi, 2020). Though the recombinant protein of Hsd17b12 producing MIH is shown, the authors did not demonstrate enzyme activity or western blot analysis of hsd17b12 from ovarian tissue source to correlate the expression analysis during ovarian cycle or spawning as done in catfish (Sreenivasulu & Senthilkumaran, 2009b).Considering the work done on hsd-20b including some reports with enzyme activity analysis in various fish species like rainbow trout, ayu and catfish it seems both hsd17b12 and hsd20b might involve in MIH production in teleost (Nagahama, 1997; Guan et al., 1999; Tanaka et al.,2002; Sreenivasulu & Senthilkumaran, 2009b). Further, a report using the snakehead murrel, Channa striatus revealed lower expression of hsd20b in pre-vitellogenic whilst higher expression in full-grown immature follicles which further elevated after 2 h of hCG treatment(Sreenivasulu et al., 2005). Promoter motif regulation of hsd20b was performed in catfish and rainbow trout where potential consensus motifs of creb and ad4bp/sf-1 had been dealt clearly (Sreenivasulu, Senthilkumaran, Sudhakumari, et al., 2012). In this context, it is important to perform promoter motif analysis for hsd17b12 to categorize its function in tilapia. Promoter motif regulation of several steroidogenic enzymes and other factors have been studied in several species of teleost which is listed in Table 1. Apart from these, studies on transcriptional regulation of hsd3b and hsd17b needs to be elucidated (Rajakumar &Senthilkumaran, 2020).

3.3. Growth factors

Sub family gsdf, belongs to the superfamily of transforming growth factor-β (tgfβ) whose other members include several sub families of tgfβ,bone morphogenetic proteins (bmp), growth and differentiation factor(gdf), activin/inhibin as well as amh in vertebrates including fishes (Lal,2013). Gsdf is conserved in gasterosteiformes (stickleback), salmoniformes (trout, salmon), cypriniformes (roach, zebrafish, fathead minnow), gadiformes (morhua), tetraodontiformes (spotted green pufferfish, takifugu) and beloniformes like medaka (Gautier, Le Gac, &Lareyre, 2011). In the medaka, O. luzonensis, gsdfis the master sex-determining gene instead of DMY (Myosho et al., 2012). In rainbow trout testicular cell culture, media enriched with recombinant Gsdf promoted proliferation of type-A spermatogonia and the effect was inhibited by antiserum against Gsdf suggesting its role in the spermatogonial proliferation (Sawatari, Shikina, Takeuchi, & Yoshizaki,2007). Expression of gsdf in medaka was found to be high in testis as compared to ovary which could be localized in the Sertoli and somatic cells around the efferent ducts of adult testis and in granulosa cells surrounding the pre-vitellogenic oocytes (Shibata et al., 2010). Role of gsdf as an inducer of testicular development has been reported in medaka (Imai, Saino, & Matsuda, 2015), which acts downstream of dmy to maintain the male sex (Zhang et al., 2016). KO of gsdf using CRISPR/Cas9 in tilapia revealed the initiation of male pathway at 10 dph in F2 generation XY gsdffish but subsequently developed as functional ovaries at 36 dph, which was rescued by the use of aromatase inhibitor, resulting in XY gsdfwith normal testis. These results suggested the requirement of gsdf for maintenance of the male pathway(Jiang et al., 2016). Promoter analysis in the same study suggested that in the presence of ad4bp/sf-1 and dmrt1, activation of gsdf was seen in a dose-dependent manner. Significance of gsdf in ovary cannot be denied as it was specifically upregulated in granulosa cells of the coho salmon,O. kisutch at cortical alveolus stage during pre-vitellogenesis (Luckenbach, Iliev, Goetz, & Swanson, 2008). KO of gsdf in medaka resulted in females with low fertility rate having few mature oocytes while infertile females having no mature oocytes suggesting its role in oocyte growth(Guan et al., 2017). Other members of the tgfβ family are not well studied in bony fishes. Activin and tgfβ1 modulates steroid production by acting as local regulators in the ovary of goldfish, Carassius auratus(Kathryn Calp, Matsumoto, & Van Der Kraak, 2003). In zebrafish,epidermal growth factor/TGFα regulates oocyte maturation in zebrafish wherein modulation of intraovarian activin has been documented (Pang& Ge, 2002). In zebrafish, it was reported that tgfβ1 inhibited GTH and 17α, 20β-DP induced oocyte maturation (Kohli et al., 2003). Inhibitory effect on oocyte maturation is through the action at multiple sites that include membrane progestin receptor β, LH receptor (lhr) and hsd20b(Kohli, Clelland, & Peng, 2005). High expression of bmp4, bmp7 and gdf9 was observed during pre-vitellogenesis which declined at laterdevelopmental stages suggesting an important role of these genes during early stages of ovarian development (Lankford & Weber, 2010). In the patagonian pejerrey, O. hatcheri male specific duplicated copy of amh named as amhy was expressed prior to autosomal amh expression(Hattori et al., 2012). Furthermore, knockdown of amhy in XY embryo resulted in ovarian development and upregulation of cyp19a1a and foxl2 suggesting amhy might be involved in sex determination. Similarly, male to female sex reversal in tilapia after KO of amhy authenticated the dominant role in male sex determination (Li et al., 2015). Further,CRISPR/Cas9 technology in zebrafish revealed amh is necessary for the development of male germ cells where both male and female mutants developed hypertrophic gonads and female-biased sex ratio (Lin et al.,2017).

Table 1 Steroidogenic enzyme gene/factors and their promoter binding protein in teleosts.

Other growth factors like insulin-like growth factors (igf) and fibroblast growth factors (fgf) have also been reported in fish gonads however, studies pertaining to these are limited, yet expression of igfs was reported. In rainbow trout, the expression of igf1 was observed in primary spermatocytes and Sertoli cells and regulation of growth hormone (GH) by igf1 was also postulated (Le Gac, Loir, Le Bail, & Ollitrault,1996). Incidentally, in C. batrachus, during early recrudescence phase,chronological influence of GH mediated regulation of ovarian growth and its development was proposed by analysing plasma T and E(Singh& Lal, 2008). However, this report is substantiated with igf1 as suggested earlier, justifying further studies. Cell culture studies using the testis of Japanese eel, Anguilla japonica confirmed the interaction of igf1 with 11-KT wherein igf1 acts as a local regulator of spermatogenesis (Nader,Miura, Ando, Miura, & Yamauchi, 1999). Incidentally, the role of igfs in the regulation of the ovarian system has been well studied. Expression of igf2 was found to be evident in rainbow trout ovaries in vitro as compared to igf1 at the time of acquiring follicular maturational competence, indicating the role of igf2 during folliculogenesis (Bobe,Maugars, Nguyen, Rime, & Jalabert, 2003). Furthermore, igf1 increased germinal vesicle breakdown (GVBD) in oocyte of the red seabream,Pagrus major (Kagawa, Kobayashi, Hasegawa, & Aida, 1994), the common carp, C. carpio (Mukherjee, Mukherjee, Sen, Paul, & Bhattacharyya,2006) and the stringing catfish, Heteropneustes fossilis (Chourasia & Joy,2008). In the ovary of mummichog, Fundulus heteroclitus igf1 stimulated oocyte maturation much more quickly than 17α, 20β-DP and igf2 was also reported to induce GVBD similar to igf1 (Negatu, Hsiao, & Wallace,1998). In the white perch, Morone americana, T and Eproduction by ovarian fragments was increased by igf1, however, 17α, 20β-DP was below the detection level (Weber, Moore, & Sullivan, 2007). In the previous report of coho salmon, O. kisutch it was revealed inhibition of basal and GTH-II stimulated T and 17α-OHP production by igf1 during pre-ovulatory phase of ovarian cycle by theca-interstitial cells whereas both Eand 17α, 20β-DP production was stimulated in granulosa cells before GVBD and only 17α, 20β-DP after GVBD, suggested involvement of igf1 in regulation of steroidogenesis during preovulatory phase(Maestro et al., 1997). In red sea bream, igf1 increased LH dependent cyp19a1a gene expression in ovarian follicles (Kagawa, Gen, Okuzawa,& Tanaka, 2003) whereas in theca layer of trout, igf1 modulated cAMP-dependent protein kinase activity which in turn affected the activity of LH on steroid production which might be via MAPK signalling pathway (M´endez, Montserrat, & Planas, 2005). Expression of igf3 in tilapia testis showed less changes as compared to ovary, further age-related decrease was evident during ovarian development (Berishvili, Baroiller, Eppler, & Reinecke, 2010). Igf3 mediated oocyte maturation was demonstrated in zebrafish (Li, Liu, Wang, & Cheng, 2011).Involvement of igf3 in fish gonadal steroidogenesis was further confirmed by the fact that it regulated the expression of transcription factors like foxl2, dmrt1, and nr5a1 as well as certain steroidogenic enzymes (Li et al., 2012).

Roles of fgfs and fgf receptors (fgfr) are well characterised during various biological processes of vertebrates. Fgfs and Fgfrs have been localized in gonad of fishes but the studies elucidating their role during gonadal development, growth and maturation which are limited. In medaka, fgf2 was found to play an important role in initiating oocyte development through follicular cells (Watanabe, Kobayashi, Ogawa, &Onitake, 1998). In tilapia, expression of fgf16 was detected in both testis and ovary, fgf20b was detected only in ovary while fgf20a was not found either in testis or ovary as compared to tissue analysed (Sun et al., 2012).Sex reversal studies in the same report revealed decreased expression of fgf16 and fgf20b upon treatment with aromatase inhibitor or estrogen receptor (ER) antagonist whereas upregulation was seen after the treatment with Esuggesting that these two genes might play a role in ovarian development and growth. Study using fgf24 mutant zebrafish revealed its requirement for proliferation, differentiation, and morphogenesis of the early somatic gonad where most of the adult mutant fish were sterile (Leerberg, Sano, & Draper, 2017). It was also confirmed that Fgf24 signalling is essential for differentiation of inner population of mesenchymal cells that are directly in contact of germ cells to functional cell type in the larval as well as early juvenile-stage gonad (Leerberg et al., 2017). Although several members of the growth factor family have been localized in gonads of several fish species, studies pertaining to their roles during gonadal development,growth and recrudescence is not well established, which warrants further investigation.

4. Impact of steroid hormones on sex differentiation in teleost

Steroid hormones regulate various biological processes such as embryonic development, reproduction, metabolism, stress response, sex differentiation, immune responses and circadian rhythms in most of the vertebrates. The major steroid hormones involved in sexual differentiation in teleost are E, T and 11-KT which are synthesized by the common precursor cholesterol upon stimulation of GTHs. The other minor hormones are MIH such as 17α, 20β-DP and 17, 20β, 21-trihydroxy-4-pregnen-3-one (20β-S) are involved majorly in gamate maturation(Nagahama, 1997; Pati˜no, Thomas, & Yoshizaki, 2003; Senthilkumaran et al., 2004; Sreenivasulu, Senthilkumaran, Sridevi, Rajakumar, &Rasheeda, 2012). Apart from these, oxygenated androgens that are involved in steroidogenic enzymes production were also found to be expressed during crucial period of sex determination and differentiation in many teleost (Fernandino et al., 2012; Rajakumar and Senthilkumaran 2014a, 2015, 2016). Additionally, glucocorticoid such as cortisol is also involved in downregulation of cyp19a1a in teleost like the Japanese founder and the European seabass (Navarro-Martin et al., 2011;Yamaguchi, Yoshinaga, Yazawa, Gen, & Kitano, 2010). Among estrogens, Eis crucial for ovarian differentiation and cyp19a1a is the key enzyme for its synthesis in most of the species. Teleost fish normally express cyp11b and hsd11b activities in testis for the synthesis of 11-KT which indicated dominance of 11-KT over T in many species (Billard,Fostier, Weil, & Breton, 1982; Borg, 1994; Idler, Bitners, & Schmidt,1961; Idler, Schmidt, & Ronald, 1960; Kusakabe, Nakamura, Evans,Swanson, & Young, 2006; Schmidt & Idler, 1962; Wang & Orban, 2007).Among androgens, 11-KT is involved in spermiation along with the expression of male secondary sexual characters in goldfish, salmonids and medaka (Hishida & Kawamoto, 1970; Idler et al., 1961; Yamazaki &Donaldson, 1969). The androgens normally bind to the androgen receptor (ar) which belong to nuclear receptor protein family that act as an inducible transcription factor in regulating the gene expression(Wadosky & Koochekpour, 2017). In targeted disruption of ar through CRISPR/Cas9 technique in zebrafish increased the number of females and offspring weight whereas ar-null mutant males developed female secondary sex characteristics (Yu et al., 2018). In an another study of ar KO in zebrafish resulted in very few stage III/IV with more number of stage I/II follicles causing premature ovarian failure. On the other hand,males exhibited structurally disorganised testes with improper sperm production (Crowder, Lassiter, & Gorelick, 2018). Interestingly, in the same study, majority of the ar mutant embryos had ovaries with female secondary sexual characteristics with differential upregulation of cyp19a1a, foxl2a in ar mutant testes and amh, dmrt1 genes in ar mutant ovaries warranting ar regulation in sexual dimorphic gene expression.Levels of 11-KT were considerably higher in male compared to female teleost such as the rainbow trout, O. mykiss, the sockeye salmon, O.nerka, brown trout, S. trutta, the Atlantic salmon, S. salar, goldfish, C.auratus, coho salmon, O. kisutch, winter flounder, Pseudopleuronectes americanus Walbaum (Campbell, Walsh, & Idler, 1976; Kime & Manning, 1982; Leatherland, Copeland, Sumpter, & Sonstegard, 1982; Rosenblum, Yamada, Callard, & Callard, 1985; Simpson & Wright, 1977;Stuart-Kregor, Sumpter, & Dodd, 1981; Truscott, Idler, So, & Walsh,1986). Elevated plasma levels of 11-KT was evident during spermiation in brown trout and concentration of 11-KT were high in coho salmon compared to T during final maturation in males (Fitzpatrick, Van Der Kraak, & Schreck, 1986; Kime & Manning, 1982). In the juvenile catfish,C. gariepinus, T accelerated pituitary gonadotroph development and 11-KT stimulated spermatogenesis (Cavaco et al., 2001). Interestingly,during pubertal development of male catfish, co-treatment of 11-KT and T abolished the stimulatory effect of 11-KT on testis growth and spermatogenesis due to T, indicated the requirement of balanced production of androgens with critical regulation during puberty (Cavaco, Bogerd,Goos, & Schulz 2001). In testis, under the stimulation of the pituitary hormone FSH, 11-KT synthesis gets triggered, which in turn induced spermatogenesis in teleost (Borg, 1994; Rinchard, Dabrowski, & Ottobre, 2001; Schulz et al., 2010; Zhang et al., 2010). In male common carp,11-KT levels were twice as high as T during maturation when incubated with hypophyseal homogenate (Koldras, Bieniarz, & Kime, 2006). In relation to sex differentiation, sex reversing teleost were found to be sensitive to androgens, when exposed or induced, as they have functionally changed from female to male sex either at juvenile or adulthood stage (Baroiller & Guiguen, 2001; Kobayashi, Furukawa, Kim, & Aida,1997). Incubation of spermatogenic cells, in vitro with 11-KT in the Japanese eel, stimulated whole process of spermatogenesis along with activin B, produced by Sertoli cells (Miura & Miura, 2001; Miura,Yamauchi, Takahashi, & Nagahama, 1991). Normally, the levels of 11-KT and Eusually increase during gametogenesis but at the end of gamete maturation, upon stimulation of the pituitary hormone LH both the hormones were found to be decreased with concurrent elevation of MIH such as 17, 20β-DP and 20β-S catalyzed by hsd20b in teleost(Lubzens, Young, Bobe, & Cerd`a, 2010; Ma˜nan´os, Duncan, & Mylonas,2008; Rajakumar & Senthilkumaran, 2020; Scott, Sumpter, & Stacey,2010; Senthilkumaran, 2011; Sreenivasulu et al., 2012a).

Eplays a vital role in ovarian differentiation which is synthesized by the steroidogenic enzyme, cyp19a1a by aromatisation of androgens, a key step in fish steroidogenesis (Guiguen et al., 1999). In teleost, Eis the major estrogen that stimulates the production of vitellogenin which is the major constituent of egg proteins (Mommsen & Walsh, 1988).cyp19a1a expressing cells were usually present in the primordial cells of gonads in tilapia particularly at 7 dph (Sakai, Kobayashi, Matsuda, &Nagahama, 2008). Testicular differentiation was observed when cyp19a1a was blocked in XX tilapia fry which inhibited the Eproduction that demonstrated Eacting as a natural inducer for ovarian differentiation (Kawamura, Omura, Sakai, & Yamashita, 2003). In amago salmon, partially purified chinook salmon GTH stimulated Eproduction only when both theca and granulosa cells were present in intact compared to isolated theca cells indicated the requirement of both the layers for Eproduction (Kagawa, Young, Adachi, & Nagahama, 1982).Elevated levels of Ewere observed during prespawning and spawning phases in protandrous female black porgy, Acanthopagrus schlegeli.Additionally, Einduced sex reversal in one-year-old protandrous black porgy by inhibition of both gonadal growth and spermiation when it was fed with Etreated diet for 5 months indicating its role in sex reversal(Chang, Lee, & Chen, 1994). Spermatogonial stem cell division was observed in testis when induced by Einduction, in vivo in the Japanese eel, which indicated estrogen acts as a male hormone during early spermatogenic cycle (Miura et al., 1999). Furthermore, rainbow trout showed elevated expression of cyp19a1a and foxl2 when administered with 17α-ethinylestradiol for first two months during gonadal development (Guiguen et al., 2010; Vizziano-Cantonnet et al., 2008).Zebrafish also showed abundant expression of cyp19a1a in those gonads which were transformed into ovaries (Jørgensen, Morthorst, Andersen,Rasmussen, & Bjerregaard, 2008). Interestingly, forskolin in the presence of cyanoketone, stimulated the conversion of exogenous T to Ein a dose-dependent manner by involving adenyl-cyclase system in the induction of cyp19a1a activation by vitellogenic follicles of goldfish in response of hCG (Tan, Adachi, & Nagahama, 1986). Expression of three types of ERs was evident in gonads of the Chinese rare minnow, rainbow trout, zebrafish, tilapia and medaka as ERα, ERβ1 and ERβ2 and two forms ERα and ERβ in the case of Atlantic salmon and channel catfish whereas only one form ERα was observed in the Japanese eel and goldfish (Bardet, Horard, Robinson-Rechavi, Laudet, & Vanacker, 2002;Chakraborty et al., 2011; Ijiri et al., 2008; Nagler, Cavileer, Sullivan,Cyr, & Rexroad, 2007; Pati˜no et al., 2000; Rogers, Llewellyn, Wigham, &Sweeney, 2000; Tchoudakova, Pathak, & Callard, 1999; Todo, Adachi, &Yamauchi, 1996; Wang et al., 2011). A unique third type classical ERγ was found in the Atlantic croaker, Micropogonias undulatus along with ERα, ERβ wherein ERγ phylogenetically derived through gene duplication and later studies in the Nile tilapia denoted that ERγ seems to be the isoform of ERβ(Hawkins et al., 2000; Wang et al., 2005; Zhu, Rice,Pang, Pace, & Thomas, 2003). Among all ER forms, ERβ binds to Ewith higher affinity as compared to ERα in several species such as zebrafish,the Atlantic croaker and channel catfish (Hawkins & Thomas, 2004;Menuet et al., 2002; Xia et al., 2000). Furthermore, ovarian differentiation occurred in genetic males of medaka when exposed to Edue to the presence of ERα in XY gonads which showed susceptibility of males to feminization (Kawahara & Yamashita, 2000). Reports on double or triple KOs of the three nuclear estrogen receptors, ERα, ERβ1 and ERβ2 in zebrafish showed folliculogenic arrest at pre-vitellogenic stage II as well as during female to male sex reversal which indicated their essential role in female reproduction, particularly, ERβ1 and ERβ2 (Lu, Cui, Jiang, &Ge., 2017). Interestingly, normal development and reproductive function were evident in both sexes in single nuclear ER mutants indicating the requirement of all the three ERs in zebrafish. No significant changes in sexual characteristics as well as reproduction were observed in both males and females when ERα KO was performed in medaka and tilapia(Tohyama et al., 2017; Yan et al., 2019). Homozygous mutants of both ERβ1 and ERβ2 in tilapia displayed abnormal testes and ovarian development which eventually resulted in infertility showing the dependency of ERβ over ERα in teleost (Yan et al., 2019). Upregulation of ERs when exposed to Estimulated vitellogenin production revealing the presence of estrogen response element (ERE) in the promoter region of ERα in rainbow trout (Le Drean et al., 1995). In tilapia, both XX and XY fishes showed sex reversal to males and females when treated with 17α-methyltestosterone and estrogens, respectively and these fishes also showed normal serum levels of sex steroids (Chen et al., 2016). Sun et al.(2014) showed that in the female Nile tilapia blockade of cyp19a1a caused various phenotypic as well as physiological alterations together with secondary sex reversal, declined Elevels, and reduction of ovary-specific with simultaneous triggering of testis-specific gene pathways. However, blockade of androgen followed by simultaneous Eadministration resulted in differentiated testis transdifferentiating to ovary in tilapia (Shi et al., 2017). On the other hand, in protogynous orange-spotted grouper, masculinization of gonads was observed after exogenous androgen treatment even in the presence of estrogen (Huang et al., 2018).

5. Regulation of gonadal steroidogenesis by environmental and dietary factors

5.1. Environmental factors

Environmental factors such as temperature and photoperiod are known to influence sex determination, differentiation as well as gonadal steroidogenesis in several species of the fishes (Gupta, 1975; Billard &Breton, 1978; Korpelainen, 1990; Bromage et al., 1993; Pandian &Koteeswaran, 1999; Baroiller & D’Cotta, 2001; Goto-Kazeto et al., 2006;Pankhurst & King, 2010). In red seabream, exposure to high water temperature suppressed oocyte development in the ovarian portion of hermaphroditic gonads and decreased the expression of both cyp19a1a and 11b-h (Lim, Kagawa, Gen, & Okuzawa, 2003). In the pikeperch,Sander lucioperca, temperature seems to be the primary factor to induce puberty. This was revealed by endocrine parameters and histological analysis, where moderate temperature i.e. 12-15C was required minimally for 3 months to attain initial maturation whereas gonadal maturation was prevented by high, 23C or low, 6C temperature(Hermelink et al., 2011). Apart from temperature, photoperiod, hypoxia, pH and density were also reported to affect reproductive activity of fishes (Carlson & Hale, 1973; Baroiller, D’Cotta, & Saillant, 2009).Exposure of hypoxia condition to zebrafish downregulated cyp19 and altered the ratio of T to Eduring early sexual development which resulted in male-biased F1 generation (Wu, 2009). In brook trout,exposure to long photoperiod (16L: 08D) followed by short photoperiod(08L: 16D) had resulted in precocious maturation thereby regulating gonadal function (Carlson & Hale, 1973). Short photoperiod (06L:18D)along with the ambient temperature (21C) was found to be effective in stimulating vitellogenesis in grey mullet, Mugil cephalus thus triggering ovarian recrudescence independent of season (Kuo & Nash, 1975). In the female catfish, H. fossilis, it was observed that the photothermal treatment (14L: 10D or 09L: 15D and 30C) along with LH administration induced early spawning and also four times within the prespawning period of the same year (Anand & Sundararaj, 1974; Sundararaj & Vasal,1976). Short photoperiod (8L: 16D) along with 16C temperature exposure induced spermatogenesis and spermiation in rainbow trout as compared to continuous illumination wherein gonadal recrudescence was not induced (Billard & Breton, 1978). C. batrachus upon growth hormone treatment (1 and 10 μg/fish) along with the ambient warm temperature (28C ±2C) and photoperiod (12.40L: 11.20D) exposure during early recrudescence, was found to induce IGF-I production vis-`a-vis steroidogenic activity in testis in a dose dependent manner with increased levels of T and Eby stimulating spermatogenesis. However,at lower water temperature (15.2C ±1C) and photoperiod (11L: 13D)such activity was not evident during the late quiescence phase (Gopal,Kumar, & Lal, 2014). In fact, the impact of climate change and its related factors such as pH/pCO2, water temperature, salinity and oxygen content on brain-pituitary-gonad axis have been well reviewed by Servili,Canario, Mouchel, and Mu˜noz-Cueto (2020). Effect of pH on reproduction was observed in some species of fishes belonging to families Cichidae and Poecilidae where low (6.2) and high pH (7.9) favoured male and female populations, respectively (R¨omer & Beisenherz, 1996; Rubin,1985). Environmental factors undoubtedly play significant roles in fishes, factors which arose due to human activity such as pollution particularly effluent from both domestic and industrial sources contains endocrine-disrupting chemicals and anthropogenic activities byproducts like mercury also influence various reproductive activities and HHG axis(Crump & Trudeau, 2009; Kar, Sangem, Anusha, & Senthilkumaran,2020; Sumpter, 1997). Although various environmental and related factors directly or indirectly influence sex determination, gonadal differentiation, development and recrudescence, temperature and photoperiod were reported to be most influencing factors in various teleost(Abdollahpour, Falahatkar, & Lawrence, 2020; Alix, Kjesbu, & Anderson, 2020; El-Sayed & Kawanna, 2007; Lundova et al., 2019).

5.2. Dietary factors

Nutritional i.e. dietary factors influence reproduction in vertebrates including fishes (Volkoff & London, 2018). Dietary fatty acids such as arachidonic acid (ARA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) have been reported to play an important role in regulating steroidogenesis in fishes (Van Der Kraak & Chang, 1990; Izquierdo,2005; Turchini, Torstensen, & Ng, 2009; Norberg et al., 2017). Preovulatory ovarian follicles of goldfish when incubated with ARA stimulated T production in a dose- and time-dependent manner and the magnitude of stimulation was found to be similar when incubated with hCG. Furthermore, effect of ARA on T production was found to be inhibited by cyclooxyenase inhibitors, ibuprofen or indomethacin whereas reduction by 50% was observed by the use of the lipoxygenase inhibitor, nordihydroguaiaretic acid suggesting the role of ARA in the regulation of steroidogenesis (Van Der Kraak & Chang, 1990). ARA gets converted to prostaglandin and stimulated T production in the testis of European seabass (Asturiano et al., 2001). Dietary ARA feeding elevated the production of T in mature tongue sole testes while it reduced Esynthesis in both immature and mature ovaries. Incidentally, the treatment also differentially regulated the expression of steroidogenic enzyme genes based on fish gender and maturation stage (Xu et al.,2017). Furthermore, different combinational ratio of dietary DHA and EPA had revealed apparent regulation on steroid hormone synthesis indicated DHA is required more than EPA in males for gonadal steroidogenesis as compared to females suggesting sex specific nutritive dependent strategy in teleost (Xu, Cao, Wei, Zhang, & Liang, 2017).Earlier reports revealed the impact of dietary significance on hepatic and circulating IGF-I level and its correlation with growth rate in teleost(Larsen, Beckman, & Dickoff, 2001; Uchida et al., 2003). Earlier report using red seabream showed that IGF-1 can induce final oocyte maturation directly without any mediation of MIH, 17α, 20β-DP (Kagawa et al.,1994). In fact, common and disparate action of IGF-1 on teleostean ovarian steroidogenesis has been reviewed (Reinecke, 2010). More in-depth studies are required to relate IGF family and its specific role on ovarian function especially steroidogenesis exerted through dietary factor. In the yellow catfish, Pelteobagrus fulvidraco feeding of different dietary ratio of linolenic acid (LNA)/linoleic acid (LA) revealed its influence on steroid hormone biosynthesis during ovarian development.Further, high levels of LNA/LA ratio feeding favors highly unsaturated fatty acid-rich gonadal development and steroid biosynthesis (Fei et al.,2020). Taken together, it seems that the dietary component, lipid plays a role in the regulation of gonadal steroidogenesis, either directly or indirectly. This may perhap warrants to analyze the influence of other essential dietary factors on gonadal steroidogenesis.

6. Δ4 or Δ5 pathway: variation among teleost

Synthesis of sex steroids in fishes follows Δor Δpathways that have been reported in several fishes including guppy, Poecilia reticular(Venkatesh, Tan, Kime, Loy, & Lam, 1992) and medaka (Zhou et al.,2007b). In males, conversion of pregnenolone to testosterone follows two pathways, Δpathway via androstenedione and Δpathway through dehydroepiandrosterone (Fan et al., 2014). Fig.2 depicts the Δand Δpathway operating in teleost. In the ovary of Tilapia aurea, both the Δand Δpathway operates, similar to the mammalian ovary(Eckstein, 1970). Same study also proposed the existence of a third pathway where progesterone was converted to T without intermediate androstenedione. Presence of Δpathway has been reported in immature ovary of the one-sided livebearer, Jenynsia lineata (Charreau &Tesone, 1974), the ovary of female guppy, Poecilia reticular (Lambert &Pot, 1975) and at silver stage ovary of the European eel A. anguilla(Colombo & Belvedere, 1976). Interestingly, in the zebrafish, Brachydanio rerio, Δpathway, was active just after oviposition whereas the Δpathway was active before next oviposition (Lambert, 1978). During pre vitellogenesis, Δsteroids were abundant in the ovary of rainbow trout S. gairdneri whereas Δsteroids were evident at the end of exogenous vitellogenesis (Lambert & Van Bohemen, 1979). Further, it was also assumed that steroidogenesis in fish testis follows Δpathway predominantly (Fostier, Le Gac, & Loir, 1987). In medaka, Δpathway was preferred over Δpathway (Zhou et al., 2007b). These reports tentatively suggested the operation of both Δand Δpathways, which varied based on species and reproductive cycle. Incidentally, present status of knowledge favours operation of Δover Δpathway in teleost. Implicating 17α, 20β-DP as MIH (Nagahama, 1997) and Eas vitellogenic hormone (Hara, Hiramatsu, & Fujita, 2016; Wallace, 1985) in addition to T and 11-KT as potent androgens for spermatogenesis and sperm maturation (Rajakumar & Senthilkumaran, 2020; Schulz et al., 2010) in majority of teleost confirms the occurrence of Δpathway mostly.More-in depth studies might compliment to understand steroidal pathway differences based on species and reproductive cycle pattern.

7. Regulatory influence of hypothalamo-hypophyseal-gonadal(HHG) axis on gonadal steroidogenesis

Hormones from HHG axis regulates reproductive events leading to fertility at all stages by modulating steroidogenesis via feedback modulation at the level of brain and pituitary (Zohar, Mu˜noz-Cueto, Elizur, &Kah, 2010). In fact, sex steroids trigger the positive and negative reproductive track in the brain as well as kisspeptin neurons, by regulating HHG axis in the European sea bass during puberty (Alvarado et al., 2016). The key decapeptide neurohormone, GnRH regulates hypophyseal-gonadal axis by regulating the release of GTHs vis-`a-vis gametogenesis and steroidogenesis (Zohar et al., 2010; Maruska &Fernald, 2011; Gomes, Costa, & Borella, 2013). In this context,species-specific form of GnRH assumes major importance for GTH regulation when multiple forms exist (Powell et al., 1994; Senthilkumaran, Okuzawa, Gen, Ookura, & Kagawa, 1999; Zohar et al., 2010).Besides the hypophysiotropic function, the neuromodulatory action of GnRH has a prominent influence on reproductive behaviour (White,Kasten, Bond, Adelman, & Fernald, 1995; Soga, Ogawa, Millar, Sakuma,& Parhar, 2005). The presence of three forms of GnRH was first demonstrated in a teleost (Powell et al., 1994; Gothilf et al., 1996) and later reports authenticated the distribution and identification of GnRH forms to distinguish their independent role in neuroendocrine control of reproduction (White et al., 1995; Gomes et al., 2013; Levavi-Sivan,Safarian, Rosenfeld, Elizur, & Avitan, 2004). In earlier studies, it was shown that fish had one GTH, and later the existence of two forms,GTH-I (FSH) and GTH-II (LH) was well demonstrated in chum salmon(Suzuki, Kawauchi, & Nagahama, 1988). Presently, the dual GTHs (FSH and LH) are well documented in variety of teleosts like carp (Van Der Kraak, Suzuki, Peter, Itoh, & Kawauchi, 1992), killifish (Lin, Rupnow,Price, Greenberg, & Wallace, 1992) and coho salmon (Swanson, Suzuki,Kawauchi, & Dick-hoff, 1991). Involvement of GTH-I (FSH) is recognized to entrain early gonadal development, puberty and gonadal steroidogenesis while GTH-II (LH) plays a vital role in regulating the gamete maturation and spawning in salmon and other teleosts (Swanson et al., 1991; Senthilkumaran et al., 2004; Maugars & Schmitz, 2007).Significant role of GTH receptors, FSH receptor (fshr) and lhr in the gonadal development of teleost had been explicitly shown (Levavi-Sivan, Bogerd, Ma˜nan´os, G´omez, & Lareyre, 2010; Burow et al., 2020).Incidentally, in zebrafish and medaka single and double KO of GTH receptors mutants lines have been established (Chu, Li, Liu, & Cheng,2015; Chu, Li, Liu, Hu, & Cheng, 2014). Subsequently, single KO mutant lead to sub-fertility in female but infertility in male, then again double KO mutants triggered infertility in all male offspring of zebrafish and medaka (Chu et al., 2015; Takahashi, Kanda, Abe, & Oka, 2016).

Though implication of GnRH-GTH in the regulation of gametogenesis is well demonstrated, their role in instigating gonadal differentiation is controversial because of the late appearance of GnRH protein amid sex dimorphism besides inconsistency in GTH detection during early stages of sexual development (Sakai et al., 2005; Swapna et al.,2008; Yan et al., 2012; Senthilkumaran, Sudhakumari, et al., 2015).Recent report revealed that GnRH KO did not inhibit sex of zebrafish,although the mutant excluded ovulation in female but did not affect male medaka (Takahashi et al., 2016). Furthermore, GnRH antisense expression delayed the puberty without affecting reproduction in common carp (Trudeau, 2018). Neuropeptide Y (NPY) plays a regulatory role in sexual development through its influence on the HHG axis which is closely associated with the GTH cells of pituitary and GnRH neurons in teleost (Breton, Mikolajczyk, Weill, Danger, & Vaudry, 1990; Peng,Gallin, Peter, Blomqvist, & Larhammar et al., 1994; Cerd´a-Reverter &Larhammar, 2000). In fact, stimulation of GnRH release in preoptic anterior hypothalamus and pituitary, in vitro by NPY was clearly demonstrated in red seabream (Senthilkumaran, Okuzawa, Gen, &Kagawa, 2001). Expression of NPY in brain elevated concomitantly with gonadogenesis in catfish (Sudhakumari et al., 2017) which is also true in blunt snout bream (Ping et al., 2013). The impact of NPY was evaluated via transient gene silencing in catfish which revealed interplay of brain and gonadal development as well as recrudescence (Sudhakumari et al.,2017). Additionally, presence of NPY seems critical for the regulation of steroidogenesis which stimulates androgen production at testicular level(Priyadarshini & Lal, 2018). Sex steroids and reproduction are controlled by HHG axis, which modulates various biological processes such as steroidogenesis, gonadal growth and maturation rather than sex determination/differentiation (Gothilf et al., 1996; Prat, Zanuy, Carrillo,De Mones, & Fostier, 1990). In black porgy, administration of GnRH antagonist, in vivo prevented Estimulation of LH release signifying the phenomenon is essentially mediated by GnRH (Yen et al., 2002). Despite the fact that in pituitary cell culture, treatment of sex steroids, T, 11-KT and E, in vitro showed no impact on basal LH release while GnRH stimulated it (Yen et al., 2002). Emerging evidence indicated that steroid hormones i.e. androgens, estrogens, progestins produced from gonads and various other neurosteroids from brain in teleost fish tend to regulate reproduction (Young et al., 2005). In this context, neurosteroids synthesized in the brain might have a direct influence on the brain-gonadal axis to modulate gonadal function (Diotel et al., 2011). In teleost, gonadal sex related to the sexual function and behaviour at the level of brain had been strongly influenced by sex steroids and cortisol(Guiguen et al., 2010; Fernandino et al., 2013; Saoshiro, Kawaguchi,Hayakawa, & Kobayashi, 2013). Another report had shown that exogenous sex steroid Estimulated neurogenesis as well as steroidogenesis whereas aromatase inhibitor reduced proliferation of neuronal cells during gonadal differentiation in the black porgy (Lin, Fan-Chiang,Dufour, & Chang, 2015). The cyp19a1b transcript, encoding brain aromatase converts aromatizable androgens to estrogens and it is often seen in abundance in brain. High levels of E, aromatase activity (cyp19a1a and cyp19a1b) contributed by as well as cyp19a1b transcript expression were evident at the time of gonadal ontogeny in male brain when compared to female pejerrey (Strobl-Mazzulla et al., 2008) and rainbow trout (Vizziano-Cantonnet et al., 2011). On the other hand, in catfish brain, expression of cyp19a1b in female was considerably higher than males in early development indicating precise sex dimorphism(Rasheeda et al., 2010; Sridevi et al., 2012). Brain aromatase activity associate with sex steroid levels in circulation and in fact, cyp19a1b transcript abundance in female brain is a modulator loop by which sex steroids elevate the expression and incidentally this phenomenon entails ER binding on ERE motif located in cyp19a1b promoter to estrogenize rather than androgenizing the brain (Diotel et al., 2010). Moreover, the crucial steroidogenic enzymes, hsd3b, cyp17, cyp11a1 and cyp19a1b exhibited a comparable pattern mainly in the neuroendocrine regulatory areas in brain as well as cyp11a1, hsd3b and cyp17 transcripts were found to be expressed in aromatase containing radial glial cells of adult zebrafish (Diotel et al., 2010). Furthermore, in adult zebrafish brain cyp11a1 transcripts were expressed during gonadal differentiation (Hsu et al., 2002; Hu, Hsu, Guo, & Chung, 2004). Upregulated steroidogenesis related genes such as star, cyp11a1, cyp19a1b in the brain as well as ERs indicated steroidal action in addition to feedback regulation from gonads (Tomy, Wu, Huang, & Chang, 2009). Based on our research in catfish, regulation mechanism of various factors provided novel inputs to understand the neuroendocrine control in gonadal development in turn, reproduction. Overexpression of tryptophan hydroxylase (tph) and high serotonin (5-HT) levels modulate aromatase activity in the male brain leading to brain sex differentiation, while blockade of 5-HT biosynthesis by using para-chlorophenylalanine during sexual determination or differentiation leads to a greater number of female populations by initiating ovarian differentiation in the Nile tilapia and catfish(Raghuveer et al., 2011b; Tsai, Wang, Chang, & Kao, 2001). Additionally, tph was predominantly expressed in preoptic area of hypothalamus in male brain during gonadal development indicating sexual dimorphism in tilapia and catfish (Raghuveer et al., 2011b; Sudhakumari et al., 2010). Dominant transcript expression of tph only in the male brain but not in the female brain had been explicitly shown during early development in the Nile tilapia by Sudhakumari et al. (2010). On the other hand, tyrosine hydroxylase (th) which is the rate-limiting enzyme for catecholamine (CA) biosynthesis, showed high copy numbers and expression in the female brain as compared to males during early gonadal development. Moreover, elevated brain CA levels in developing females authenticated the importance of the CA-ergic system (Mamta et al., 2014). Concomitantly, glial cell line-derived neurotrophic factor family receptor α-1 (gfrα-1) expressed predominantly in female brain during early development contributes to regulate dopaminergic neurons. Furthermore, in vitro, and in vivo transient silencing of gfrα-1 as well as by neurotoxin, 1-methyl-1, 2, 3, 6-tetrahydropyridine treatment triggered neurodegeneration signifying the critical role for gfrα-1 together with dopaminergic activity. Subsequently, diminished expression of brain related genes after transient silencing of gfrα-1 showed regulatory influence (Mamta & Senthilkumaran, 2018). Nevertheless,the regulation of gonadal steroidogenesis by HHG axis gets activated only during gonadal differentiation or growth and the events followed thereafter intensely (Diotel et al., 2010; Senthilkumaran, Sudhakumari,et al., 2015). Overall, sexually dimorphic expression of certain brain-related genes including cyp19a1b, tph and th may possibly have an essential role in orchestrating “brain sex differentiation” which in turn alter gonadal development, growth, and recrudescence (Senthilkumaran, Sudhakumari, et al., 2015). Overexpression of tph vis-`a-vis 5-HT perhaps bring in causative response to induce testicular development which was evident from the studies using the Nile tilapia and catfish as models (Raghuveer et al., 2011b; Sudhakumari et al., 2010; Tsai et al.,2001). However, overexpression of cyp19a1b and th in females might require more regulatory inputs from gonads, especially ovary (Mamta et al., 2014; Rasheeda et al., 2010). More recently in catfish, alteration of expression of several genes related to brain-pituitary axis and CAs after the controlled release of sex steroids gender differently via osmotic pumps has been overtly shown indicating the feedback influence of sex steroids (Mamta, Sudhakumari, Kagawa, Dutta-Gupta, & Senthilkumaran, 2020). Therefore, it seems that the establishment of HHG axis occurs at gonadal differentiation, which then settles during gonadal growth. In other words, it is possible to suggest that the HHG axis plays an intense role after gonadal differentiation, precisely during gonadal growth and recrudescence.

8. Role of certain factors, wnt signalling, and nitric oxide in regulating steroidogenesis during gonadal growth and recrudescence

Apart from several steroidogenic enzymes, transcription factors and growth factors, signalling molecules like wnt and nitric oxide (NO), as well as few other factors like member of paired box family, pax2, pentraxin (ptx) and THO complex (thoc) play an important role during gonadal development, growth, recrudescence and steroidogenesis.Transient gene silencing of thoc3, decreased serum level of E, 17α, 20β-DP, along with several steroidogenic enzyme and transcription factor genes, with upregulation of igf1 and gdf9 in the common carp ovary(Gupta & Senthilkumaran, 2020b). This report suggested the regulatory role of Thoc3 on the ovarian system either directly or indirectly. Role of ptx in relation to fish reproduction is least studied. Report from the swamp eel, Monopterus albus revealed high expression of ptx in ovary whereas decreased expression during sex reversal suggested it might be regulating ovarian development (Sheng et al., 2015). In line with this report, a recent study from our laboratory in common carp using transient gene silencing and overexpression after sex reversal demonstrated the pivotal role of ptx in ovarian function, specifically in steroidogenesis(Gupta & Senthilkumaran, 2020a). Signalling cascade molecules like NO and wnt are another pivotal for sex differentiation and gonadal maturation. NO is a gaseous signalling molecule produced by nitric oxide synthases (NOS) enzymatic activity on arginine (Dixit & Parvizi, 2001).Two isoforms of NOS have been identified in gonads of C. batrachus wherein the role of NO has been demonstrated in folliculogenesis,vitellogenesis and steroidogenesis by modulating steroidogenic enzymes, hormones and vitellogenin (nee Pathak & Lal, 2010; Lal &Dubey, 2012; Singh & Lal, 2017).

wnt/β-catenin pathway is crucial for ovarian differentiation in which wnt4 regulates this process in mammals that is least studied in teleost.Signalling molecules Wnt4/5 are usually involved in ovarian growth in mammals, although wnt4 was identified in few teleost like zebrafish(Ungar, Kelly, & Moon, 1995), medaka (Yokoi, Nishimatsu, Ozato,&Yoda, 2003), black porgy (Wu & Chang, 2009), rainbow trout (Nicol,Guerin, Fostier, & Guiguen, 2011), half-smooth tongue sole (Hu, Zhu,Liu, Wang, & Chen, 2014), and orange-spotted grouper (Chen et al.,2015). In protandrous black porgy, increased expression of wnt4 along with cyp19a1a and foxl2 was observed in all fries that first developed as males which changed to females after three years indicating the importance of these genes in the initiation of gonadal development as well as sex reversal (Wu & Chang, 2009; Wu et al., 2010). Two wnt4 genes, wnt4a and wnt4b were identified in zebrafish whereas in rainbow trout wnt4a gene showed isoforms, wnt4a1/a2 and those were predominantly expressed in gonads while expression of wnt4b seems restricted to nervous system (Nicol et al., 2011). In catfish, expression of both wnt4 and wnt5 was found in ovary with high levels of wnt4 in prespawning and wnt5 in spawning phase of the ovarian cycle. Additionally, increased expression of wnt4 was observed after in vivo induction of hCG in a time dependent manner while wnt5 showed little difference (Prathibha & Senthilkumaran, 2017). Promoter analysis of wnt5 gene in catfish revealed the presence of binding sites of pax2, foxo1,sox9b, gata1/6, and CAAT/enhancer binding protein, among which pax2 was found to be dominant. Further studies including gene silencing demonstrated the potential role of pax2 during ovarian development and recrudescence either directly or indirectly by modulating steroidogenesis through wnt signalling in catfish (Prathibha & Senthilkumaran,2016). KO of wnt4a gene in zebrafish predominantly developed into males indicating its role in promoting female sex determination (Kossack et al., 2019).

9. Does shift in steroidogenesis occurs in males like females?

Shift in steroidogenesis that occurs in full grown immature follicles is an important phenomenon to attain maturational competence which has been well demonstrated in teleost. Shift in steroidogenesis occurs in granulosa cells where shift from Eto 17α, 20β-DP concurrently with the steroidogenic enzymes from cyp19a1a to hsd20b occurs before oocyte maturation under the control of LH (Nagahama, 1997; Senthilkumaran,2011; Senthilkumaran et al., 2004). 17α, 20β-DP act as a MIH (Matsuyama et al., 2005) for final oocyte maturation as identified in several bony fishes (Nagahama, 1997) including the Chub Mackerel, Scomber japonicas (Matsuyama et al., 2005).

This kind of shift, if any, in testis requires comprehensive analysis.Rapid shift from androgen to progestogen synthesis was observed in testis of common carp and during prespawning phase GTH surge was evident where C-17, 20-lyase get saturated with its substrate 17-OHP,diffused out from somatic cells, metabolized to 17α, 20β-DP and 17α,20α-dihydroxy-4-pregnen-3-one (17, 20α-P) in spermatozoa (Barry,Aida, Okumura, & Hanyu, 1990). These progestogens form an inhibitory feedback on the somatic cells for the activity of androgen biosynthesis enzymes including C-17, 20-lyase (Barry et al., 1990). This inhibition initiated a short positive loop resulting in increased production of 17α,20β-DP and 17, 20α-P which further suppressed the C-17, 20-lyase activity (Barry et al., 1990). Apparently, rapid fall in androgen and rise in progestogens controls the spermiation process. In the European sea bass,17α, 20β-DP and 20β-S have a pivotal role as MIH in both sexes(Asturiano et al., 2002; Nagahama, 1997). In catfish, role of hsd20b has been shown explicitly in males wherein elevated expression during spawning phase and overexpression after hCG induction during prespawaning phase of testicular cycle leading to sperm maturation was demonstrated with immunolocalization of the enzyme protein chiefly in interstitial cells and spermatogonia/spermatocyte (Sreenivasulu,Senthilkumaran et al., 2012a). In spite of these findings, in-depth analysis studies are needed to reveal the occurrence of shift in steroidogenesis in male fish as both androgens and progestins play a crucial role in sperm maturation (Rajakumar & Senthilkumaran, 2020; Senthilkumaran, 2011; Sreenivasulu et al., 2012a).

In conclusion, development and organisation of steroidogenesis in teleost are regulated by several steroidogenic enzymes, transcription factors, signalling molecules, environmental and dietary factors. Role of certain enzymes like cyp11a1, cyp19a1, cyp11b, cyp17, hsd20b, hsd3b,transcription factors such as dmy/dmrt1bY, sox, AP-1 and steroid hormones are indispensable. Role of growth factors and signalling molecules cannot be disregarded as they regulate gonadal development,growth and steroidogenesis differentially. Variations in steroid synthesis pathway i.e. Δor Δdo exist among teleost, yet in-depth analysis is required. Present status of knowledge favours predominance of Δpathway. HHG axis plays an important role during gonadal growth and recrudescence. Shift in steroidogenesis, a major phenomenon during final oocyte maturation, while in males it warrants further investigation as progestins and androgens are required for sperm maturation. The role of gonadal steroids as well as specific enzymes involved in sex-change warrants specific function in gametogenesis.

10. Future research directions

Knowledge underlying teleost sex determination, development,growth and steroidogenic regulation is still limited. Several factors and molecules modulating such processes have been least elucidated.Ontogeny studies are prerequisite to understand the expression of various genes and factors responsible for early sex differentiation and gonadal development in relation to steroidogenesis. Teleost animal models will help us to study the steroidogenic onset and regulation during maturation and especially during recrudescence which is less studied in higher vertebrates. Cholesterol mobilization is the most crucial step for onset of steroidogenesis in teleost but the molecular mechanism behind mobilization, role of two important organelles mitochondria and endoplasmic reticulum is least analysed in teleost.Such studies will provide evidence whether the processes behind mobilization are conserved among vertebrates like the action of star.Further, studies are required to identify the crucial transcription factors that can bind to the star promoter and trigger the mobilization of cholesterol during steroidogenesis. Most of the steroidogenic enzymes are conserved between human and fishes, elucidating their regulation may provide detailed knowledge of several diseases caused due to malfunction of steroidogenic enzymes. Shift in steroidogenesis is an important phenomenon well studied in females than males indicating the need for more work in males. Further, the mechanisms involved to maintain sex steroid levels during steroidogenic onset and recrudescence in seasonal breeders are yet to be studied. Several growth factors and signalling molecules are reported to play a pivotal role in reproduction among vertebrates, yet their exact role and regulation is still unclear. As many fish species exhibit sexual plasticity and diversity, sex reversal studies can be a useful to investigate the factors as well as mechanisms controlling sex determination/differentiation. More indepth studies on teleostean steroidogenesis are required to comprehensively understand the cascade of sex steroid biosynthesis in relation to gonadal and interrenal functions. These kinds of comprehensive analyses will pave way to decipher molecular mechanisms of steroidogenic onset, which in turn provide novel inputs to understand the process but also to develop novel biomarkers in perceiving endocrine disruptors targeting gonadal steroidogenesis (Kar et al., 2020). In this context,Zohar (2020) has categorically pointed out the significance of future research from basic to translational with powerful integrative physiological genomics approach with variety of teleost models including small fishes for better understanding of fish reproductive biology.

Declaration of competing interest

The authors declare that there is no conflicts of interest.

Acknowledgements

ST is thankful to Junior Research Fellowship support by Council of Scientific and Industrial Research (CSIR) (09/414(1150)/2017-EMR-I),India. AP is grateful to the University of Hyderabad for Non-NET fellowship. SKM is thankful to Women Scientist Program (SR/WOS-A/LS-303/2017) from the Department of Science and Technology (DST),India. BS is also a recipient of TATA innovation fellowship (BT/HRD/35/01/02/2013) from Department of Biotechnology, India (during the years: 2014-2019) and grant-in-aid from Science and Engineering Research Board (EMR/2017/000718), India and Council of Scientific and Industrial Research (37(1708)/18/EMR-II), India which is acknowledged.