Introduction

In recent times, more emphasis was given on gene expression studies including promoter level regulation. Several key pathways and mechanisms have regulatory checkpoints (e.g., cell cycle check points) wherein the gene expression is stimulated or suppressed at specific times or tissues. Steroids control important processes like metabolism, inflammation, immune functions, gonadal development, and maturation, and hence, regulation of steroidogenesis is extremely critical. Though few reports showed the promoter level regulation of steroidogenic enzyme genes, comprehensive analysis of all the enzyme genes in teleosts were not available, and hence, the present review attempted to highlight the transcriptional regulation of steroidogenesis in teleosts by emphasizing on the promoter level regulation. Owing to the wide variation in regulation of teleosts, more general and comprehensive view is given to provide a clear perspective.

Steroid hormones regulate embryonic development, gonadal differentiation, neuroprotection, stress response, and gametogenesis in teleosts as in other vertebrates (Mayer et al. 1990; Borg 1994; Baroiller et al. 1999; Devlin and Nagahama 2002; Handa et al. 2008; Tokarz et al. 2015). The major sites of steroidogenesis include gonadal and non-gonadal tissues like head kidney, liver, intestine, and adipose tissue (Nagahama 1994; Jiang et al. 1998; Rasheeda et al. 2010a, b; Swart et al. 2013). In teleosts, steroidogenesis occurs in specific cell types of gonads, wherein cholesterol is processed into pregnenolone and subsequently into different steroids such as estrogens, androgens, and progestins, while conversion to corticosteroids and mineralocorticoids occur at peripheral tissues (Borg 1994; Mommsen et al. 1999; Senthilkumaran et al. 2004; Blazquez and Somoza 2010; Scott et al. 2010). Owing to its diverse role in several biological processes, tissue specific expression, and their intrinsic regulation, it is essential to study the regulation of steroidogenic enzyme genes at promoter motif level. In addition, steroidogenesis is regulated by multiple means like substrate limitation, co-factor preference, allosteric mechanism, and expression of steroidogenic enzymes genes (Stocco 2000; Zhou et al. 2005; Guiguen et al. 2010; Rasheeda et al. 2010a; Raghuveer et al. 2011; Rajakumar and Senthilkumaran 2014a, b, 2015, 2016). This regulation is critical with respect to stage or reproductive cycle, tissue, season, and sex-specific biosynthesis of steroids.

Gonadal steroidogenesis is primarily regulated by hypothalamo-hypophysial axis, while autocrine and/or paracrine regulation and steroidogenic cell niche (microenvironment) are also critical for stage and sex-specific steroidogenesis (Peter et al. 1991; Nagahama 1994; Senthilkumaran and Joy 1996; Goos et al. 1999; Ge 2005; Scott et al. 2010). Another important aspect is their involvement in gonadal differentiation, development, and growth (Devlin and Nagahama 2002; Vizziano et al. 2007; Guiguen et al. 2010; Raghuveer et al. 2011; Sudhakumari and Senthilkumaran 2013). In the case of mammals, sex determination and gonadal development are strictly genetic (Wilhelm et al. 2007) and shows limited or no gonadal plasticity (Tanaka and Nishinakamura 2014). Conversely, teleost gonads show plasticity, often retaining the ability to change gonadal sex at different stages of development or even in adulthood in various species, which can be directed to undergo complete reversal from testis to ovary and vice-versa through hormonal treatments irrespective of genetic/chromosomal sex or through environmental cues (Francis 1992; Baroiller et al. 1999; Kobayashi et al. 2003; Sudhakumari and Senthilkumaran 2013; Kobayashi et al. 2013). Further, the levels of hormones during critical period of gonadal differentiation determine the development of undifferentiated gonad into either testis or ovary (Devlin and Nagahama 2002; Baroiller et al. 2009; Guiguen et al. 2010). Consequently, two hypotheses had been proposed, the first one considers the differential expression of aromatase (cytochrome P450, family 19, subfamily A, polypeptide 1a [Cyp19a1a/ ovarian aromatase] gene) for ovarian development and elevated temperature and/or cytochrome P450 family 11 subfamily B member 1 (cyp11b1/11β-hydroxylase)/ 11β-hydroxysteroid dehydrogenase (hsd11b) gene expression for testicular development (Baroiller et al. 2009; Blasco et al. 2010; Fernandino et al. 2012, 2013; Guiguen et al. 2010; Nakamura et al. 2010). Recent study using gene editing methodologies (TALEN and CRISPR) showed that the knockout of Cyp19a1a results in all-male offspring in zebrafish (Lau et al. 2016). While the second hypothesis perceives androgens do not participate in early testicular differentiation, active expression of cyp19a1a induces ovarian differentiation and its inhibition alone is sufficient for testicular differentiation (Fernandino et al. 2012, 2013; Guiguen et al. 2010; Hattori et al. 2009; Sudhakumari and Senthilkumaran 2013; Yamaguchi et al. 2010). In gonochoristic fishes like pejerrey or the Japanese flounder, elevated temperature and other environmental stressors result in increased cortisol levels and decreased aromatase, which leads to activation of androgen pathways, increased hsd11b expression, and gonadal masculinization (Fernandino et al. 2012, 2013; Hattori et al. 2009; Yamaguchi et al. 2010). Regulation of steroidogenic enzyme genes seems to play central role in gonadal differentiation. Hence, the role of sex steroids and regulation of steroidogenic enzyme genes by certain transcription factors during the critical stages of gonadal differentiation have a direct impact on gonadal fate and sex of the organism as well as the reproductive cycle.

In addition to sex steroids, glucocorticoids play an important role in gonadal differentiation in few teleosts like pejerrey and the Japanese flounder by downregulating cyp19a1a, thereby inducing testicular differentiation (Hattori et al. 2009; Yamaguchi et al. 2010). Differential expression of specific steroidogenic enzyme genes during gonadal differentiation/development, maturation, and seasonal cycle results in the production of pertinent sex steroids. However, very little is known about their promoter regulation and transcription factor interaction in teleosts. Any detailed analysis on these aspects will unravel their role in androgen and estrogen production vis-à-vis gonadal differentiation. On these perspectives, the current review focused on the transcriptional regulation of steroidogenesis in teleosts, based on scientific literature available till date.

Steroidogenic enzyme gene expression and their regulation

Steroidogenesis starts with the rate-limiting transport of cholesterol into the mitochondria mediated by steroidogenic acute regulatory protein (StAR), where it is converted into pregnenolone, a first precursor in the steroidogenic cascade and it is the rate-limiting step in steroidogenesis (Stocco 2000). However, existence of additional rate-limiting steps in steroidogenesis is not clear. Cyp11a1 is the only enzyme involved in the conversion of cholesterol to pregnenolone, which thereby initiates the whole process of steroidogenesis (Stocco 2000; Rajakumar and Senthilkumaran 2014a; Tokarz et al. 2015) after the initial stint from StAR. Sequential action of several steroidogenic enzymes results in the conversion of pregnenolone into active steroids like 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), testosterone (T), 11-Ketotestosterone (11-KT), and estradiol-17β (E2) as well as corticosteroids (Senthilkumaran 2011; Rajakumar and Senthilkumaran 2014a, b; Tokarz et al. 2015). Maturation-inducing steroids (MISs), 17α,20β-DP and 17α,20β,21-trihydroxy-4-pregnen-3-one, have been implicated in the final oocyte maturation and to some extent in sperm maturation of teleosts (Senthilkumaran et al. 2004; Sreenivasulu et al. 2012a; Trant and Thomas 1989). Interestingly, 11-hydroxytestosterone (11-OHT) and 11-KT (a potent androgen in fishes) are present in mammals, except that it was detected when induced with human chorionic gonadotropin (hCG; Yazawa et al. 2008; Senthilkumaran et al. 2009; Scott et al. 2010; Sreenivasulu et al. 2012a; Rajakumar and Senthilkumaran 2013; Rege et al. 2019). However, 5α-dihydrotestosterone (DHT), a potent androgen of mammals, was detected in plasma of fathead minnow and found to have androgenic potency (Margiotta-Casaluci and Sumpter 2011; Margiotta-Casaluci et al. 2013). Further studies are needed to understand the implications of 11-oxygenated androgens in mammals and DHT in fishes during testicular development. Studies using specific chemical blockers or targeted gene silencing provide reason for varied pattern of androgen metabolism to act more potentially than the natural potent androgen, T in reference to evolution. In the case of Japanese eel, immature testes have the ability to produce 11-KT, in the presence of 11-OHT, and it was proposed that synthesis of 11-KT is arrested in immature testis earlier in the steroidogenic pathway other than the step from 11-OHT to 11-KT (Ozaki et al. 2006). Hence, cohesive study of all other steroidogenic enzyme genes and their stringent control might provide novel leads in unraveling the critical steps of testicular steroidogenesis. The steroidogenic enzyme genes, cyp11a1, cyp17, and cyp19a1a, are best characterized in the pathway, because they constitute three important regulatory inputs in the steroidogenesis. Steroidogenesis is strictly regulated by gonadotropins (GTHs)/hCG/cAMP, wherein acute and chronic stimulation of Mouse Leydig cells (with 8-bromo cAMP in culture containing synthetic serum-free medium containing 0.1% BSA and insulin (500 μg/ml) for up to 15 days) is required for the steady-state expression of steroidogenic enzyme genes which in turn stimulate the production of sex steroids (Payne and Youngblood 1995). A recent study using FreeStyle 293-F cell lines producing recombinant Japanese eel Follicle-stimulating hormone (reFsh) and Luteinizing hormone (reLh) and gonadotropin receptors-expressing COS-7 cells indicated reFsh stimulated its cognate receptor; meanwhile, reLh activated both receptors. Both reFsh and reLh induced testicular 11-KT production in a dose- and time-dependent manner by upregulating expression of steroidogenic enzyme genes (Suzuki et al. 2019). Owing to the receptor sharing of luteinizing hormone (LH) and hCG (Bogerd et al. 2001; Choi and Smitz 2014; Vischer et al. 2003), and promoter regulation, several researchers used hCG for functional studies on hormonal profiles and on the expression of steroidogenic enzyme genes (Choi and Smitz 2014; Rajakumar and Senthilkumaran 2015). Vischer et al. (2003) used human follicle stimulating hormone, hCG, and human LH, in 25 μl of Hepes-modified Dulbecco’s modified Eagle’s medium containing 0.1% bovine serum albumin and 0.1 mM 3-isobutyl-1-methylxanthine in 96-well plate for ligand stimulation and receptor binding assays. Production of E2, T, and 11-KT was stimulated by hCG by binding to luteinizing hormone receptor (LHR) and both directly and indirectly through an increase in the expression of steroidogenic enzyme genes like cyp11a1 (Rajakumar and Senthilkumaran 2014a), 17β-hydroxysteroid dehydrogenase 1 (hsd17b1) and hsd17b12 (Rajakumar and Senthilkumaran 2014b), cyp19a1a and cyp19a1b (brain aromatase; Rasheeda et al. 2010b), cyp11b1 (Jiang et al. 1996; Rajakumar and Senthilkumaran 2015), and hsd11b (Jiang et al. 2003; Ozaki et al. 2006; Rasheeda et al. 2010a; Rajakumar and Senthilkumaran 2016). These reports provided detailed information on the regulation of all steroidogenic enzyme genes comprehensively with changes in the gene expression during gonadal differentiation, development, and gametogenesis in teleosts. In fact, it has been well established that hCG binds to LHR and complements LH action in gonads at different time points of active reproduction in teleosts including advancing the gonadal maturation (Kagawa et al. 2009; Murugananthkumar et al. 2017). Nevertheless, it will be ideal to check the effect of teleostean LH independently as hCG shows differential response in teleosts.

Cyp11a1

Cholesterol is converted by cholesterol side-chain cleavage enzyme, P450scc or cyp11a1, into pregnenolone, which is the slowest step, and thus, controls the rate of synthesis of steroid hormones. Hence, the regulation of cyp11a1 together with cholesterol mobilization is very important in controlling overall steroidogenesis (Rajakumar and Senthilkumaran 2014a). Though the expression changes during gonadal development, maturation, and gametogenesis were studied in teleosts (Hsu et al. 2002; Hu et al. 2004; Kazeto et al. 2006; Rajakumar and Senthilkumaran 2014a), promoter motif regulation seems to be less understood except for zebrafish. In zebrafish, Ff1b (homolog of steroidogenic factor 1 [Ad4BP/Sf-1 referred as SF-1]) binds to two conserved FF1 response elements (FRE) on the putative promoter of cyp11a1 and activates transcription (Quek and Chan 2009). Deletion and mutagenesis studies revealed that only the proximal FRE was essential for transcriptional activation, which critically regulates cyp11a1 expression (Quek and Chan 2009). Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSAs) further confirmed the importance of Ff1b in the transcriptional activation of cyp11a1 (Table 1; Fig. 1). Further, cyp11a1 promoter drives the EGFP expression specifically to the internal gland and genital ridge when transiently expressed in microinjected zebrafish embryos (Quek and Chan 2009).

Table 1 Summary of regulation of steroidogenic enzyme genes in teleosts
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Fig. 1
figure 1

A schematic pathway of steroidogenesis and regulation of steroidogenic enzyme genes with its transcription factors in the gonads of teleosts. + indicates stimulation; − indicates suppression. White arrows indicate the proposed androgen synthesis pathway. Gray arrows indicate progestogen synthesis pathway (Adapted from Young et al. 2004)

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Cyp17

The conventional type of cyp17 (cyp17a1) and a novel type of cyp17 (cyp17a2) were identified in tilapia (Zhou et al. 2007a), medaka (Zhou et al. 2007b), and Japanese eel (Kazeto et al. 2000; Su et al. 2015) and these two types of Cyp17 are encoded by two different genes (Zhou et al. 2007a). The cyp17a1 showed both 17α-hydroxylase and 17,20-lyase activities (Fig. 1) while cyp17a2 possesses unique 17α-hydroxylase activity, without any 17,20-lyase activity which involve in 17α,20β-DP production (Fig. 1). Cyp17a2 was expressed not only in the gonads, but also in the head kidney, while cyp17a1 was exclusively expressed in the gonads (Zhou et al. 2007a, b). It is well known that T and E2 are involved in gametogenesis and decreased during gamete maturation during which progestin levels might get elevated (Scott et al. 2010; Sreenivasulu et al. 2012a; Rajakumar and Senthilkumaran 2013). The specific molecular mechanisms are not clearly understood. One possibility is the differential expression of cyp17a2 in few teleosts, yet specific promoter regulation of cyp17 gene is not studied in any teleosts to validate the contention. In other teleosts, cyp17a1 through its 17α-hydroxylase and 17, 20 lyase activities synthesizes androgen from progestins (Scott et al. 2010; Sreenivasulu and Senthilkumaran 2009). Comprehensive studies on gene expression and enzyme activity analysis in catfish warrant the cyp17 is indeed involved in androgen production, and the presence of second isoform has been ruled out (Sreenivasulu and Senthilkumaran 2009). The expression of cyp17a2 was significantly decreased in the testis of the GTH receptor knockout zebrafish (Chu et al. 2015). Incidentally, in the commercially important Japanese eel, Anguilla japonica, the MIS, 17α,20β-DP is generated from its precursor by cyp17 which has both 17α-hydroxylase and 17, 20 lyase activities. In order to elucidate the regulatory mechanism underlying the steroidogenic shift from E2 to 17α,20β-DP, and the mechanistic basis for the failure of this shift in natural versus artificially induced eel will be rewarding. Taken together, specific activity of cyp17a1 and cyp17a2 as well as targeted gene silencing might provide relevant information to establish the specific role of these enzymes in MIS and androgen production as well as shift in steroidogenesis.

3β-hydroxysteroid dehydrogenase (Hsd3b)

Regulation of hsd3b (Δ5-Δ4 isomerase) gene at the promoter level is not studied in any teleost. In mouse Leydig cell culture, cAMP induction of gonadotropin regulation was observed in Hsd3b (Payne and Youngblood 1995). In the case of Nile tilapia, two forms of hsd3b were reported to be similar to human (Senthilkumaran et al. 2009). GTHs regulate hsd3b expression in Protogynous Orange-Spotted Grouper, Epinephelus coioides (Huang et al. 2019) and several other fish species (Levavi-Sivan et al. 2009). The expression of hsd3b was significantly decreased in the testis of the GTH receptor knockout zebrafish (Chu et al. 2015).

In the case of human, gene regulation of hsd3b is complex and involves several different factors including a number of endocrine and paracrine regulatory mechanisms. The enzyme, hsd3b, is involved in the synthesis of several natural steroid hormones like progesterone and T and the hepatic degradation of the pheromone androstenone (Rasmussen et al. 2013). Transcriptional activity of hsd3b was influenced by several signaling and regulatory pathways like JAK-STAT, LH/hCG, estrogen receptor alpha, androgen receptor, Ad4BP/Sf-1, and peroxisome proliferator-activated receptor alpha in mammals (Rasmussen et al. 2013). Whether similar phenomenon exists in the case of teleosts remains to be elucidated.

Hsd17b

The enzyme hsd17b isoforms are involved in the interconversion between 17β-hydroxy- (active) and 17-keto- (inactive) steroids, which thereby regulate the level of specific substrates required for sex steroid biosynthesis (Adamski and Jakob 2001; Moeller and Adamski 2009; Rajakumar and Senthilkumaran 2014b). Various forms of hsd17bs were reported in teleosts (14 forms were reported in mammals), which are involved in several processes leading to the production of sex steroids. The enzyme hsd17b is required for the production of essential sex steroids like T and E2, and thus, is indirectly involved in the process of sex differentiation and gametogenesis in fish (Guiguen 2000; Mindnich et al. 2004; Zhou et al. 2005).

Incidentally, hsd17b1 is a key enzyme involved in E2 synthesis together with cyp19a1a. It is predominantly involved in interconversion of estrone (E1) to E2 in teleosts (Zhou et al. 2005). The enzyme hsd17b2 is involved in the inactivation of estrogens and androgens while hsd17b3 is androgenic which is essential for testicular production of T (Adamski and Jakob 2001; Moeller and Adamski 2009). Hsd17b12 is able to reduce E1 to E2 (Mindnich et al. 2004). The enzyme is the most recent addition to this family which shares close relationship to hsd17b3 and was shown to be an ancestor of hsd17b3 by phylogenetic analysis (Mindnich et al. 2004). In zebrafish, hsd17b12-like was able to convert cortisone to 20β-hydroxycortisone, and hence, it was named as hsd20b2 (Tokarz et al. 2012). Transcriptional regulation of hsd17b was not studied for any of the isoforms identified in teleosts. Owing to the presence of different isoforms and substrate specificity and its role in T production, it is relevant to study the gene regulation of all the hsd17b subtypes, which might provide interesting data on promoter level control of tissue and stage-specific expression in teleosts.

Cyp19a1a

The enzyme cyp19a1a is responsible for the formation of C18 steroids and is thus the most important enzyme with reference to hormonal control of sexual development in teleosts (Rashid et al. 2007; Rasheeda et al. 2010b; Mills et al. 2014). Though the importance of cyp19a1a in female sexual differentiation is known, regulation of expression of cyp19a1a within the developing gonads remains to be elucidated (Ijiri et al. 2008). In fish, as well as in other vertebrates, T acts as essential substrate for cyp19a1a to produce E2 in granulosa cells (Tanaka et al. 1992; Nagahama et al. 1995; Senthilkumaran et al. 2004). Steroidogenic shift that occurs at the completion of vitellogenesis in female involves loss of stimulatory effects of FSH and Igfs on cyp19a1a expression and inhibition of cyp19a1a transcription by LH (Nakamura et al. 2016).

Promoter characteristics of cyp19a1a were described in few species (Kazeto et al. 2001; Tchoudakova et al. 2001; Valle et al. 2002). In the Nile tilapia and the Japanese medaka, an orphan nuclear receptor protein, fushi tarazu-factor I (FTZ-F1) named as Ad4BP/Sf-1, plays an important role in the transcriptional activation of cyp19a1a expression (Table 1; Fig. 1) and enzyme activity (Watanabe et al. 1999; Yoshiura et al. 2003). Forkhead family of transcription factor, forkhead box l2 (Foxl2), is shown to be involved in the transcriptional regulation of cyp19a1a expression as a co-regulator (Govoroun et al. 2004; Wang et al. 2007). Though the expression pattern and levels of their transcripts vary between the transcription factors, foxl2 and nuclear receptor subfamily 5 group A member 1 (nr5a1) are both thought to be involved in the regulation of steroidogenesis. These factors, FTZ-F1 and foxl2, are also involved in the transcriptional activation of cyp19a1b expression which were authenticated using ChIP and EMSA methodologies (Sridevi et al. 2012). Transcription of cyp19a1b is positively regulated by E2 in zebrafish (Cheshenko et al. 2007; Diotel et al. 2010).

Cyp11b

Cyp11b is involved in the biosynthesis of 11-hydroxyandrostenedione (11-OHA) and 11-OHT, precursors for 11-KT production (Borg 1994; Lokman et al. 2002; Rajakumar and Senthilkumaran 2015). It is also involved in the production of corticosterone and cortisol in interrenal cells of kidney of several teleosts (Jiang et al. 1998). Various splice variants of cyp11b were reported in catfish showing its pivotal role in androgenesis (Rajakumar and Senthilkumaran 2015). Transcriptional regulation of cyp11b has not been studied in any teleost till date except for a preliminary study in catfish, Clarias batrachus (unpublished data), which revealed the regulation of cyp11b1 expression by Ad4BP/Sf-1 (Table 1; Fig. 1).

Hsd11b

The enzyme hsd11b2 plays a key role in the synthesis of 11-oxygenated androgens and glucocorticoids (Borg 1994; Lokman et al. 2002; Jiang et al. 2003; Rasheeda et al. 2010a; Rajakumar and Senthilkumaran 2016). The other form, hsd11b1 (reductive), was not well characterized in teleosts.

Hsd11b2 is involved in the conversion of cortisol to cortisone as well as 11-oxygenated androgen synthesis pathway (Borg 1994; Lokman et al. 2002). Hence, it was suggested to protect the testicular tissue from circulating cortisol in addition to their role in 11-KT production (Kusakabe et al. 2003). A strong correlation exists between androgen and glucocorticoid pathways (Fernandino et al. 2012); however, the specific mechanism and regulation during different circumstances are unclear. In the next section, the relationship of glucocorticoid with androgen metabolism was described in detail based on the available information in teleosts. In spawning fishes, 11-KT is produced and released in higher levels, which shows that testicular expression of the enzyme is responsible for the conversion of 11-OHT to 11-KT; hsd11b2 would increase specifically during the spawning season. Similarly, in catfish, higher levels of hsd11b2 expression were evident during spawning followed by pre-spawning phase (Rajakumar and Senthilkumaran 2016). There is a correlation between hsd11b2 and sox3 in catfish (Rajakumar and Senthilkumaran 2016). Detailed analyses using site-directed mutagenesis (SDM), luciferase assay, EMSA, and ChIP revealed that sox3 binds to hsd11b2 gene promoter (Fig. 1; Table 1) and transactivates its transcription by binding to its specific promoter motifs in catfish (Rajakumar and Senthilkumaran 2016). Partly, hsd11b2 is also moderately regulated by Wt-1 (Rajakumar and Senthilkumaran 2016; Murugananthkumar and Senthilkumaran 2016). Further, in a previous work, cortisol was shown to induce hsd11b2 expression (Fernandino et al. 2012).

Hsd20b

Hsd20b is involved in the production of MIH, i.e., 17α,20β-DP, which has been explicitly shown in few teleosts using recombinant protein in combination with radiometric assays using ovarian tissues, implicating their pivotal role in final oocyte maturation (Senthilkumaran et al. 2002, 2004; Sreenivasulu et al. 2012a). Incidentally, the work has also been extended with gene promoter motif analysis in teleosts (Sreenivasulu et al. 2012a). On the other hand, based on the work on masu salmon and medaka, the production of MIH is different in relation to steroidogenic enzymes in other teleosts (Zhou et al. 2007a, b). Recent detailed study in masu salmon showed that hsd 17b12-like (not hsd20b) is responsible for MIH synthesis by granulosa cells during final oocyte maturation (Ijiri et al. 2017). The enzyme hsd20b2 (hsd17b12-like) was able to convert cortisone to 20β-hydroxycortisone in zebrafish (Tokarz et al. 2012). In view of these, the production of MIH seems to operate differently in teleosts with differential gene regulation mechanism. 17α,20β-DP is essential for the initiation of meiosis in the spermatocyte, milt production, and sperm mobility and acts as a pheromone in cyprinids (Miura et al. 1992; see review by Scott et al. 2010; Schulz et al. 2010; Sreenivasulu et al. 2012a; Rajakumar and Senthilkumaran 2013). Promoter motif regulation of hsd20b was explored in detail in both catfish and rainbow trout (Sreenivasulu et al. 2012b) wherein the importance of cAMP-responsive element binding protein (CREB) and Ad4BP/Sf-1 was shown explicitly (Table 1; Fig. 1). Incidentally, differential expression of CREBs was shown in tilapia and catfish gonads wherein their pivotal role in gonadal growth and maturation was demonstrated with special emphasis on final oocyte or meiotic maturation (Senthilkumaran et al. 2015). Taken together, hsd20b and CREB seem to be essential for MIH production in few teleosts.

Important aspects of steroidogenic enzymes are the existence of isoforms, and it is essential to probe this phenomenon to delineate specific function. The next section of the review will highlight these with reference to functional perspectives and genome duplication.

Isoforms and variants role in regulation of steroidogenesis

Teleost-specific genome duplication has occurred around 350 million years ago, which provides several different evolutionary processes and adaptive mechanisms in teleosts (Meyer and Schartl 1999; Meyer and Van De Peer 2005). After genome duplication, the duplicated gene copies had been attributed to different or modified functions in different species or no function at all (Meyer and Schartl 1999). Several isoforms or variants of steroidogenic enzyme genes are detected in several teleosts: cyp11a1 (Parajes et al. 2013), cyp17 (Zhou et al. 2007a, b), hsd3b (Senthilkumaran et al. 2009), and cyp11b1 (Zhang et al. 2010; Rajakumar and Senthilkumaran 2015). Specific role for each isoform has been shown in some teleosts, yet the role of different variant forms was least understood for example hsd3b variants in tilapia (Senthilkumaran et al. 2009). The gene duplication in teleost fishes is different from the human and hence, several duplicated genes were reported in teleosts. This opens the possibility for diverse regulatory processes in teleosts like tissue-specific and/or developmental stage-specific effects.

Germ and Sertoli cell steroidogenesis

Leydig cells of testes and thecal and granulosa cells of ovaries contribute largely for steroidogenesis. Androstenedione (A) and T are converted into E1 and E2, respectively, by the cyp19a1a localized in the granulosa cells. However, recent studies in several fish species have revealed the presence of different steroidogenic enzymes in the germ (Spermatogonia/Spermatocyte) and Sertoli cells of testis (Hinfray et al. 2013; Rajakumar and Senthilkumaran 2015). In chondrichthyes, Sertoli cells producing steroids were reported (Sourdaine and Garnier 1993; Prisco et al. 2008). In the spotted ray testis, hsd3b and hsd17b were localized in Sertoli and Leydig cells and these cells were indirectly involved in the hormonal control of spermatogenesis (Prisco et al. 2008). Likewise, expression of steroidogenic enzyme genes is well characterized in zebrafish and cyp19a1a mRNA was detected in presumptive granulosa cells surrounding oocytes (Wang and Orban 2007).

Glucocorticoids and androgens relationship

Stress hormones can interfere with the reproductive signaling by accelerating, delaying, and/or inhibiting reproduction (Nematollahi et al. 2009; Schreck et al. 2001; Schreck 2010). Inversely, sex steroids can influence the stress response (Fuzzen et al. 2011) thereby maintaining reproductive cycle. Incidentally, the role of androgen and cortisol crosstalk involved in the male pathway is not clearly understood. In the case of pejerrey and the Japanese flounder, cortisol acts directly by downregulating cyp19a1a expression (Hattori et al. 2009; Yamaguchi et al. 2010). Further, in the Japanese flounder cortisol binds to glucocorticoid receptor, which acts as a transcription factor by binding to glucocorticoid responsive element in the upstream of cyp19a1a promoter and thereby suppresses its transcription (Yamaguchi et al. 2010). In the European sea bass, hyper-methylation of cyp19a1a promoter was reported, which leads to the downregulation of cyp19a1a gene expression (Navarro-Martin et al. 2011). Androgens are involved in testicular differentiation, which was shown by the treatment of androgen or androgenic analogues (Devlin and Nagahama 2002; Raghuveer and Senthilkumaran 2009). Involvement of 11-oxygenated androgens and the expression of steroidogenic enzyme genes were reported during the critical period of sex determination/differentiation in several teleosts (Liu et al. 2000; Blazquez et al. 2001; Hattori et al. 2009; Fernandino et al. 2012; Rajakumar and Senthilkumaran 2014a, 2015, 2016). The main enzymes involved in the pathways are cyp11b1 and hsd11b2. The enzyme cyp11b2 converts A and T to 11-OHA and 11-OHT, respectively (Borg 1994; Lokman et al. 2002). Interestingly, in pejerrey, it was reported that hsd11b2 is regulated by cortisol thereby promoting the synthesis of 11-KT in temperature-dependent sex determination (Fernandino et al. 2012).

In fish, 11-OHA is considered as the principal steroid produced by the gonads whereas 11-KT is the predominant androgen (Borg 1994; Cavaco et al. 1997). Nonetheless, the expression of gene that codes for cyp11b showed an increase at later stages of morphological gonadal differentiation in males only (Socorro et al. 2007; Blasco et al. 2010; Raghuveer et al. 2011).

As already stated, hsd11b plays a key role in the synthesis of 11-oxygenated androgens and also known to be involved in the metabolism of glucocorticoids (Oppermann et al. 1997). Recently, it was proposed that hsd11b is the key enzyme involved in the warm temperature-induced masculinization in pejerrey and was also suggested that the enzymatic machinery necessary for the local production of 11-oxygenated steroids was already active in the undifferentiated gonads during this critical period (Fernandino et al. 2012). Enzymes hsd11b2 and hsd20b2 were suggested to consecutively metabolize cortisol to 20β-hydroxycortisone, which subsequently might be glucuronidated or sulfated and excreted from fish (Tokarz et al. 2012). Environmental stress-induced testicular differentiation was reviewed in depth with other intrinsic factors by Fernandino et al. (2013).

An elaborate view on steroidogenesis (steroidogenic enzyme genes expression and enzymatic activity) and the steroid hormone receptors, impact of duplicated genome on these processes, and influence of anthropogenic endocrine-disrupting compounds on steroid hormone were described in detail by Tokarz et al. (2015). An overview on the impact of pesticides on the reproduction, endocrine signaling and the resulting adverse physiological effects on teleost fishes was illustrated by Senthilkumaran (2015).

Approaches for studying regulation of gene expression at the promoter level

Function of every genome involves regulatory sequences, such as enhancers, co-regulators, and promoters. Instructions for when, where, and to what level each gene should be expressed in an organism are encoded by these correlates. Transcription factors binding to its target promoter/enhancers is a tightly controlled process, which governs the connectivity of gene networks which coordinately regulates complex spatiotemporal and gender-specific gene expression with modulating effects from co-regulators (Geertz and Maerkl 2010; Rajakumar and Senthilkumaran 2016). In principle, experimental methods can be divided into either in vitro or in vivo approaches. In vitro methods like EMSA and in vivo methods like ChIP provide evidences for DNA-protein interaction. However, each method has their own advantages and limitations in studying different aspects of transcription factor binding to their target motifs which can be an enhancer or regulator or promoter. Combining selective methods for understanding research problem can provide robust results. ChIP being an in vivo technique provides more reliable data in native condition on DNA-protein interaction than EMSA (Yan et al. 2004; Gade and Kalvakolanu 2012), but specific bp region cannot be ascertained using basic ChIP. EMSA is a rapid and sensitive method which provides evidence for specific DNA binding site for transcription factors on the designated oligos to ascertain functional promoter motifs (Hellman and Fried 2007). The main drawback of EMSA is that it is an in vitro technique, wherein the samples are not in chemical equilibrium during EMSA gel run and also many complexes are significantly more stable in the gel than at free solution (Gade and Kalvakolanu 2012). Single bp resolution can be achieved using Chip-Seq and other related methodologies, which cannot be done in general using basic ChIP methodology. ChIP is a versatile technique wherein analysis of proteins interacting within a native chromatin environment can be ascertained, and it also provides unbiased observations into the chromatin changes occurring in response to extracellular signals and/or hormone stimuli (Yan et al. 2004; Gade and Kalvakolanu 2012) and hence it is a better method for hormone interaction studies. Moreover, ChIP has been used to determine the allele-specific transcription factor-binding patterns (Heckman and Boxer 2002). Therefore, it is very essential to use all the available methods to obtain more credible data on DNA-protein interaction with respect to the analysis on steroidogenic enzyme gene regulation.

Future prospects

There is still limited knowledge on the neuroendocrine mechanisms, dietary, and environmental conditions that control the regulation of steroidogenesis to modulate gonadal development and maturation in teleosts. Identification of new or novel factors in relation to gametogeneis as well as steroidogenesis broadens the cross talk and interaction. Hence, understanding these aspects through molecular level studies integrated with systemic approach might delineate the importance of their cohesive regulation on teleost reproduction.

Though steroidogenesis is relatively well understood in teleosts, species-specific variation together with functions of all the reported isoforms/variants of genes is poorly deciphered. Further studies are needed to understand the role of miRNA interaction and epigenetic regulation of essential genes related to steroidogenesis. In addition, identification of new genes from evolutionarily/mechanistically diverse teleost species might provide novel leads on their functional relevance and overall survival of the organism. These understandings together with information on regulation of steroidogenic enzyme genes are crucial to unravel the evolutionary significance of several reproductive modes such as gonochorism, protandry, protogyny, true hermaphroditism, gynogenesis, androgenesis, and basic biology of this group of organisms. Current and the future research should address the lacunae in the information on altered endocrine conditions either naturally through genetic means or through anthropogenic sources as in the case of endocrine disruption for the sustainable environment leading to better aquaculture practice and seed production.