King et al. (2020) Sox9a is an early expressed, male sex-determining gene which plays a role in spermatogenesis and is predominantly expressed in the testis (Groh et al., 2011; Chen et al., 2017; Lin et al., 2017). A high expression of sox9a has been found in both zebrafish in males, juveniles, 28dpf and during earlier development (Jørgensen et al., 2008; King et al., 2020). Despite the fact that sox9a has strong potential as a candidate gene for early sex determination, previous studies involving long-term exposure to natural and synthetic P4 and norgestrel and other drugs affecting steroid levels have been shown to affect sox9a expression, thereby altering sex-determination of zebrafish. Tamoxifen is a selective oestrogen receptor modulator used to prevent and treat breast cancer. Exposure to tamoxifen during development induces abnormal upregulation of sox9 in sertoli cells of female mice which in turn causes testis differentiation (Yu et al., 2014). Therefore, its use as a sex-determining gene in pharmaceutical studies cannot be justified (van boxtel et al., 2010; Liang et al., 2015).

Cyp26b1 is expressed highly in male and juvenile zebrafish (King et al., 2020). Retinoic acid (RA) is essential for spermatogenesis in male zebrafish. The relative level of RA in the gonads is correlated with sex-differentiation. Cyp26b1 degrades RA and has a higher expression in the testis. Cyp26b1 is upregulated in individuals of male fate. The loss of RA prevents developing gonads from entering into meiosis to form the ovaries, the opposite is true for female fated individuals (Rodríguez-Marí et al., 2013; Pradhan and Olsson, 2015). In mice, male transcript factors, sf1 and sox9 upregulate and activate cyp26b1. In gonads fated to become ovaries, foxl2 is highly expressed and acts an antagonist to cyp26b1 (Kashimada et al., 2011).

Retinoids have been well studied for their chemo-preventive properties. Retinoids are activated by their conversion to RA (Sedjo et al., 2004). In all vertebrates and some invertebrates, RA is a potent teratogen that, at pharmacological concentrations, can induce defects in congenital processes. RA signalling can be blocked by pharmaceuticals, thereby altering cyp26b1 level and sexual fate (Mark et al., 2006).

Anti-convulsants are a broad group of pharmacological agents which stabilise the level of nerve cell impulses and are used to treat epileptic seizures, bipolar disorder as well as for the treatment of neuropathic pain. Retinoids and vitamin A control have a vital role in sex-determination while the embryo is developing. Pharmaceuticals with anti-convulsant agents can have a huge effect on the retinoid metabolism. Due to the effect of cyp26b1 on RA expression during juvenile development, the altered retinoid metabolism by these drugs may significantly affect sex-determination (Nau et al., 1995). These results taken together indicate that cyp26b1 is not suitable to indicate sex variations during pharmaceutical trials.

Atp1b1a is highly expressed in adult male, juvenile and developing zebrafish (Wang et al., 2008b; Hatzold et al., 2016; King et al., 2020). The oestrogen medication, Ethinylestradiol (EE) is commonly used in combination with progestins in birth control pills. EE has often been used to treat menopausal symptoms, gynaecological disorders, and certain hormone-sensitive cancers. Sewage effluent sometimes contains concentrations of EE2 which alters reproduction in fish. After zebrafish were exposed to EE2, atp1b1a was downregulated (Martyniuk et al., 2007).

In recent times, graphene oxide (GO) has been researched for application of drug delivery in order to discover its properties for multi-pharmaceutical action including those with photothermal therapy. GO has been found to be useful in cancer therapies. In vitro studies have shown that GO is a valuable nanomaterial for stem cell research in medicine as it has the potential to promote stem cell adhesion, growth and differentiation (Duran et al., 2015; Priyadarsini et al., 2018). After zebrafish were exposed to 100 μg/L GO, the expression of atp1b1a was vastly altered. GO exposure to zebrafish caused atp1b1a to be downregulated. With these points considered, atp1a1b is not suitable as a sex-determining marker due to the alteration in expression of this gene with application of oestrogen-based drugs or those containing GO (Zou et al., 2018).

Fkbp5 gene is an important target of androgen signalling in the prostate. Androgen-deprivation leads to a decrease in FKBP51 and androgen stimulation resulting in up to 5-fold upregulation of FKBP5 (Amler et al., 2000). Therefore, FKBP5 is termed androgen-responsive, is conserved among species and plays a role in zebrafish male development (Magee et al., 2006). The transcript data shown in this study indicates juvenile expression of fkbp5 and an almost a 3-fold increase of fkbp5 gene expression in adult male zebrafish compared to females. This is in correlation with previous studies; therefore, it can be concluded that fkbp5 can be used as an early indicator of sex in zebrafish.

Fkbp5 and fkbp51 expression has been linked to stress response regulation and can be used in anti-depressant treatment. Fkbp5, the gene expressing the protein fkbp51 (FK506-binding protein 51), is a strong inhibitor of the glucocorticoid receptor (GR). Restoration of GR signalling has been found to be a treatment for depression. In a previous study, patients who responded to anti-depressant treatment had a significant reduction of fkbp5 gene and associated fkbp51 protein expression, whereas an increasing expression level of fkbp5 was observed in non-responders (Ising et al., 2019). Therefore, the use of fkbp5 as a gene for identifying sex related differences in drug trials cannot be justified.

In zebrafish, hbaa1 is expressed 19 organs including, eye; hematopoietic system; presumptive blood; and vasculature; with highest expression level in the spleen and 13th highest expression in the testis of zebrafish at fully formed stage. Zebrafish hbaa1 is orthologous to several human genes including HBZ (haemoglobin subunit zeta) (Zfn.org—ZFIN ID: ZDB-GENE-980526-79). Hbaa1 is expressed during zebrafish early development from zygote-1-cell-stage to adult-stage (Higano et al., 1997). Highest expression of hbaa1 in early development occurs at 35dpf (Tiedke et al., 2011). There are very few studies to our knowledge in which adult male and female zebrafish hbaa1 expression are compared. Our data indicates a higher expression of hbaa1 in adult male zebrafish compared with females as well as a high expression level in juveniles, therefore, hbaa1 should be considered as an early marker for male sex-determination in zebrafish but more research should be done to confirm this.

Again, few studies were found which show the effect of drugs on the expression of hbaa1 and its orthologs. Valproate (VPA) and its valproic acid, sodium valproate, and valproate semi-sodium forms are medications primarily used to treat epilepsy and bipolar disorder and prevent migraine headaches. A study found that VPA blocked the expression of human HBZ (Belrose et al., 2011; Gazon et al., 2016). More studies should now be carried out to investigate this pattern of expression in hbaa1 and its orthologs to discover for certain whether it can be discounted as a gene for uncovering sex-related differences during drug trials.

King et al. (2020) Ccnb1 has shown its potential to be an early expressed gene to indicate female sex (Yasuda et al., 2010; Horie and Kotani, 2016; King et al., 2020). The gene is expressed in the unfertilised egg, oocyte and mature ovarian follicle and involved in maturation and female sex determination in zebrafish (Kondo et al., 1997; Kondo et al., 2001; Knoll-Gellida et al., 2006; Wang et al., 2007; Nagahama and Yamashita, 2008; Kotani et al., 2013; Yasuda et al., 2013; Takahashi et al., 2014; Dingare et al., 2018; Takei et al., 2018; Yi et al., 2019).

Ccnb1 is significantly overexpressed in various cancer types including breast and prostate. A study showed that several tested drugs had an influence the expression of ccnb1; Doxorubicin, Cisplatin, Etoposide, Fluorouracil, Irinotecan, Ochratoxin A, Oxaliplatin and Tretinoin could decrease the expression of ccnb1, while Paclitaxel and Tamoxifen could increase the expression of ccnb1 (Ding et al., 2014). Additionally, resveratrol, a plant compound that acts like an antioxidant, has been used as an inhibitor of ccnb1 and can therefore regulate both the proliferation and apoptosis of pituitary tumour cells as well as altering the expression level of various EMT (epithelial-to-mesenchymal transition) markers (Li et al., 2019a). All things considered, the use of ccnb1 as an early sex determining gene in drug trials is not appropriate.

Rdh10b is more highly expressed in female and juveniles than in males (King et al., 2020). The gene is necessary for oxidisation of retinol to retinal and RA (Sandell et al., 2007). Retinoids are found in eggs and oocytes of marine and freshwater fish, teleosts, amphibians and mammals (Costaridis et al., 1996; Irie and Seki, 2002). RA is highly expressed in the gonad of female zebrafish and is necessary for sexual development in adult female but not adult male mice (Koubova et al., 2006; Belyaeva et al., 2020b). A decrease in RA results in a male phenotype (Rodríguez-Marí et al., 2013). In the ovary of zebrafish, genes which are necessary for the synthesis of retinal are upregulated (Levi et al., 2012). Insulin represses rdh10 expression by inactivating FoxO1 (Obrochta et al., 2015). The alteration rdh10b gene expression with insulin shows that it is not suitable as a gene to reveal the confounding variable of sex in FELS tests.

Pdia is expressed during zebrafish development. It is found highly expressed in the female gonad at juvenile and adult stages and been suggested as an early female sex-determining gene in zebrafish (Zheng et al., 2018; King et al., 2020). It has been observed that the expression of pdia decreases in the presence of tunicamycin, a mixture of homologous nucleoside antibiotics (Nair et al., 2007). Additionally, application of dithiothreitol (DTT), a reducing agent that causes the formation of unfolded proteins, causes the upregulation of pdia2 (Al-Sheikh et al., 2004). Due to the changing expression of pdia with drug influence its use as an early sex-determining gene for the discovery of drug related differences cannot be applied.

Ctsla was previous selected as a female sex-determining gene (King et al., 2020). This maternally inherited gene is known to be involved in oogenesis and is highly expressed in juvenile zebrafish and the ovary and oocytes of adult zebrafish (Tingaud-Sequeira and Cerdà, 2007; Tingaud-Sequeira et al., 2011; King et al., 2020).

The mammalian ortholog of ctsla is ctsl. Nifedipine, a calcium antagonist, was shown to suppress ctsl activity and mRNA expression in mice (Nishimura et al., 2002). Previous studies have demonstrated that the expression of ctsl was upregulated in several cancers; therefore, supporting its use as a therapeutic target for cancer (Rudzińska et al., 2019). There is evidence that ctsl may be central in drug resistance development (Zheng et al., 2004). Further research has shown that by inhibiting ctsl, development of resistance to cancer drugs such as doxorubicin, a chemotherapy medication used to treat cancer, can be reversed, and prevented. No matter what level of resistance cancer cells have reached, their reaction to drugs is restored after ctsl inhibition (Zheng et al., 2009). Additionally, some anti-tuberculous and anti-leprotic drugs inhibit the lysosomal cysteine proteinases such as ctsl; therefore, changing the expression of the gene (Kamboj et al., 2003). These results taken together imply that ctsla is not suitable to indicate sex variations during drug trials.

Vtg1 exhibits antioxidant activity and is involved in cellular response to oestrogen, oestradiol, and xenobiotic stimulus. It is expressed in several structures, including the unfertilised egg and at various stages through early development; in zygote:1-cell-stage at 0hpf to 0.75hpf, at larval-stage, 5dpf and all the way to breeding adult-stage at 90dpf to 730dpf (Zfn.org—ZFIN ID: ZDB-GENE-001201-1). During vitellogenesis, vtg1 is secreted and transported to the ovaries in the plasma. NGS data in this study as well as previous research showed that vtg1 has a higher overall expression in females compared to males (Levi et al., 2012). Increased expression of vtg1 in females is essential for the maintenance of the female sex and the prevention of testis differentiation in zebrafish.

Observations from previous studies indicate that oestrogen, E2, stimulates gene expression of vtg1 (King et al., 2020). When juvenile zebrafish are exposed to the oestrogen 17alpha-ethynyl oestradiol, the sex ratio is significantly female-biased; in one case an entirely female population was produced after exposure (Örn et al., 2003). Therefore, the use of vtg1 would be unsuitable as a marker for identifying female zebrafish in FET with endocrine disrupting substances, since the gene is upregulated by oestrogenic substances (Leet et al., 2011). However, the evidence of juvenile expression of vtg1 from previous literature as well as the significant female transcript expression from NGS data displayed in this study, indicates the usefulness of vtg1 as an early precursor for sex-determination. For these reasons, vtg1 can be used as a molecular marker for the indication of feminisation but not for uncovering sex-related differences in drug discovery (Jin et al., 2009).

Cyp17a1 is involved in androst-4-ene-3,17-dione biosynthetic and p4 metabolic process. Human orthologs of this gene are implicated in several diseases, including prostate cancer. In the zygote of zebrafish, cyp17a1 is already expressed during the 1-cell stage. Additionally, its expression is seen in the zygote at larval-stage, 5dpf; to breeding adult, 90dpf to 730dpf (Zfn.org—ZFIN ID: ZDB-GENE-040213-2). Elevated expression of cyp17a1 in female zebrafish can be seen in previous research; whereas cyp17a1a deficient zebrafish had male phenotypes (Zhai et al., 2018). A significantly higher expression of cyp17a1 can be seen in adult female zebrafish compared to males based on transcript data in this study and this is supported by previous research (Lin et al., 2017; Yan et al., 2017). These observations confirm that cyp17a1 is essential for female sexual development. It can be concluded from these findings that cyp17a1 could be suitable as a marker of female sex-specific expression in juvenile zebrafish.

Cyp17a1 has been found to be highly expressed in half about of human prostate carcinomas. Intracellular androgen is synthesised by these cancer cells. Abiraterone, a drug used to treat prostate cancer, can block nuclear accumulation of androgen receptors (ARs) which consequently suppresses expression of cyp17a1 (Giatromanolaki et al., 2019). The alteration of cyp17a1 gene expression with drug application means that it should not be selected to reveal sex-related differences with drug usage.

In zebrafish, Igf3 is expressed in several structures, including gill; gonad; head; kidney; and skeletal muscle; with fifth highest expression level in the female gonad at fully-formed stage (Zfn.org—ZFIN ID: ZDB-GENE-080611-1). Igf3 is expressed in early ovary development (Wang et al., 2008a; McMenamin et al., 2013) and its expression increases throughout the course of ovary differentiation (Li et al., 2011). Igf3 has been found to be expressed from zygote 1-cell-stage, 0.0 h–0.75h, to adult, 90days to 730 days (Zfn.org—ZFIN ID: ZDB-GENE-080611-1). Igf3 is generally thought of as a female biasing gene (Xia et al., 2018). The increased expression of igf3 in adult female zebrafish can be explained by its role in oocyte maturation (Li et al., 2011). Using transcript data from this study, an increased expression of igf3 can be seen in adult female zebrafish compared to males. Since the whole zebrafish was used for the analysis of gene expression and no tissue-specific expression analysis of the ovaries was performed, the eggs present in the females in our study possibly contributed to the increased expression of igf3. Despite this, analysis of igf3 reveals it as a potentially suitable gene for assigning a sex-specific expression profile in developing zebrafish.

Insulin-like growth factor 1 receptor (IGF-1R) has already been used as a therapeutic target (Dolgin, 2020). Studies have shown that the stable overexpression of igfbp3 could suppress the growth of tumour cells by inducing apoptosis and suppressing survival and growth of cells (Dar et al., 2010). Despite its use as an early genetic marker of sex-determination, due to the change in the expression of igf3 with drug application it cannot be used as a gene to reveal sex-related differences during drug trials.

Nr0b1 is predicted to have transcription corepressor activity and transcription factor binding activity. It is involved in negative regulation of male gonad development and water homeostasis. Nr0b1 is predicted to localise to the cytoplasm and nucleus and is expressed in several structures, including central nervous system; digestive system; fin; inter-renal primordium; and reproductive system, with highest levels of expression in the liver (Zhao et al., 2006). The gene is also expressed in earlier zebrafish development from gastrula-50%-epiboly-stage, 5.25hpf to 5.66hpf until adult-stage, 90dpf to 730dpf (Zfn.org—ZFIN ID: ZDB-GENE-070130-1). In zebrafish, nr0b1 mutants developed mostly as males (Chen et al., 2016). Mutations of the gene nr0b1 in zebrafish larvae, 13dpf, caused a decreased expression of the female-specific aromatase-encoding gene, cyp19a1a. This suggests that nr0b1 works during the early bipotential stage to promote female development (Chen et al., 2016).

Long-term oestradiol (E2) administration to the protandrous black porgy fish resulted in high levels of nr0b1 (Wu et al., 2008). Additionally, RA has been widely used to treat photoaged skin cancer and psoriasis (Lalli, 2014). Vitamin A is also crucial in activating Retinoic Acid Receptors (RAR). Both tretinoin and isotretinoin, are two synthetic forms of vitamin A. RA treatment has been found to increase the expression of nr0b1 (Nagl et al., 2009). These previous findings highlight changes in expression of nr0b1 after application of steroid-based and retinoid drugs, therefore, nr0b1 should not be used to indicate confounding sex identity with drug exposure (Li et al., 2016b).

Human orthologs of the gene, lhcgr, are implicated in leydig cell tumour; breast cancer; and gonadal disease. It is expressed in digestive system; gonad; and hypophysis from juvenile-stage; 45dpf to 89dpf through to adult-stage; 90dpf to 730dpf (Zfn.org—ZFIN ID: ZDB-GENE-040806-3). Recent studies have shown that lhcgr is more important in controlling female reproduction compared to its role in male development (Zhang et al., 2015; Song et al., 2020). In female, lhcgr plays a vital role in triggering final oocyte maturation and ovulation. Lhcgr, a follicle cell-specific marker is expressed in follicles and isolated follicle layers, interacts with female-specific, gdf9, and is thought to be essential for ovulation (Wang et al., 2005; Li et al., 2016a). Lhcgr encodes luteinizing hormone subunit beta, lhβ; the transcription levels of the pituitary glycoprotein hormone, lhβ, were upregulated in cyp17a1-deficient fish pituitary (Zhai et al., 2018).

Oestradiol (E2)-based drugs have been found to alter the expression of lhcgr. Use of 17β-oestradiol (E2), but not testosterone, changed the regulated the expression of lhcgr (Liu et al., 2011). Recent studies have shown that lhcgr is expressed at high levels in various gynaecological cancers and so it was predicted be a potentially important biomarker for cancer diagnosis and therapy, in particular for ovarian cancer (Xiong et al., 2019; Zhong et al., 2019). FSH receptor binding inhibitor (FRBI) suppresses the expression of lhcgr mRNA and protein in sheep cumulus-oocyte complexes (COCs) (Wei et al., 2018). The mechanism of FRBI as an antagonist of lhcgr also has potential in aiding ovarian diseases and ovarian and follicular functioning in order to promote fertility in humans and animals (Wei et al., 2017; Lai et al., 2018). Although lhcgr has been identified as a potential drug target for cancer therapy and methods to apply this have been refined, there have so far been very few studies showing how these anti-cancer drugs affect the expression of lhcgr (Cavalli et al., 2016; Gao et al., 2018). Further studies of lhcgr expression levels in relation to drug application are needed to reveal if it can be used as an early expressed gene for unravelling confounding sex variables during anti-cancer drug development.

Cyp19a1a is expressed in brain; eye; female organism; the ovary, ovarian granulosa cells; and the reproductive system; with highest expression level in female gonad (Chiang et al., 2001). The expression of the gene begins at zygote-1-cell-stage, through to adulthood (Zfn.org—ZFIN ID: ZDB-GENE-990415-43). Cyp19a1a is expressed at the highest level in the female gonad at juvenile stage, 30 to 44 days. It is also expressed in the immature gonad, at larval stage, 14 to 20dpf; in the gonad and female organism at larval stage, 21 to 44dpf; and in the mature ovarian follicle at fully-formed stage (Santos et al., 2007; Yu et al., 2014; Valdivieso et al., 2019). Cyp19a1a is a female promoting gene; the importance of cyp19a1a in the process of female sex differentiation was demonstrated when the silencing of the gene produced only male offspring (Lau et al., 2016; Yin et al., 2017). Other studies complement these findings whereby cyp19a1a is stated to be a female sex-determining gene (Santos et al., 2007; Jørgensen et al., 2008; Tong et al., 2010a; Rodríguez-Marí et al., 2010; Chen et al., 2017; Crowder et al., 2018b; Valdivieso et al., 2019) The NGS transcript data described here also complies with these findings. Taken together this implies that cyp19a1a could be used as an earlier genetic marker for indicating female sex-determination.

The expression of cyp19a1a in the gonad of labeo rohita fry changed after treated with fadrozole, an aromatase inhibitor. Equally treatment with 17β-oestradiol (E2) had no effect on the expression of cyp19a1a However, other studies have shown that two endocrine disrupting chemicals (EDCs) - an exogenous oestrogen 17β-oestradiol (E2) and the oestrogen mimic 4-n-nonylphenol (NP) cause a significant reduction in the expression of cyp19a1a in ovarian tissues with complete inhibition at the higher concentrations; therefore, implying that drugs with these actions can affect the expression of cyp19a1a (Shanthanagouda et al., 2013). More studies should be carried out to clarify the effect of different drugs on the expression of cyp19a1a, so that its use as an early sex-determining gene for uncovering sex-related difference with drug application can be definitively stated.

Foxl2a is expressed in female zebrafish; in the gonad; ovarian follicle and ovary at larval-stage, 14dpf to 20dpf until breeding adult, 90dpf to 730dpf (Cocquet et al., 2002; Uchida et al., 2002; Tong et al., 2010a). Our data also shows adult female and juvenile, 28dpf, expression of foxl2a in zebrafish. Foxl2a in combination with other genes is essential to regulate zebrafish hormone producing ovarian cells and is necessary for ovary development, growth, maintenance and folliculogenesis; its loss significantly affects female development (Selman et al., 1993; Uchida et al., 2002; Cocquet et al., 2003; Tong et al., 2010b; Herpin and Schartl, 2015; Kossack and Draper, 2016). This indicates the importance of foxl2a in preventing female-to-male sex reversal by repressing testis determining genes, dmrt1 and amh (Uchida et al., 2002; Uhlenhaut et al., 2009; Matson et al., 2011; Schlessinger et al., 2011; Huang et al., 2017; Yang et al., 2017; Crowder et al., 2018b). Based on transcript data in this study, a 1.8-fold increase of foxl2a gene expression was seen in adult female zebrafish compared to males. This provides valid evidence for its use as an early female gene marker in zebrafish.

Mutations in foxl2a have been found to cause several diseases; blepharophimosis syndrome (BPES), a complex eyelid malformation associated with premature ovarian failure, ovarian dysfunction–polycystic ovary syndrome (PCOS) and granulosa cell tumours and keloid scars (Cocquet et al., 2002; Fleming et al., 2010; Verdin and De Baere, 2012). Regulation of expression of foxl2 or its downstream targets with drugs may provide a potential target for therapy of these diseases but these must first be tested for clarification of this (Verdin and De Baere, 2012). Studies have shown that after treatment with oestradiol and 2-methyl-testosterone, foxl2 was upregulated and downregulated, respectively (Jiang et al., 2011). This suggests that foxl2 would be affected by steroid-based drug application and therefore, not suitable for uncovering sex-related variables to steroid-based drug treatment (Wu et al., 2019).

King et al. (2020) The gapdhs gene encodes a protein and the testis-specific enzyme, glyceraldehyde-3-phosphate dehydrogenase spermatogenic, gapdhs. They are vital for energy production, sperm motility to ensure male fertility (Welch et al., 1995; Kuravsky et al., 2011). Gapdhs is expressed in adult male zebrafish as well as during development and in juveniles (Liu et al., 2013; King et al., 2020). This potential target is known to belong to a well-known druggable protein family which implies that gapdhs can be used as an early sex determining gene without its expression level being affected by drug application (Gottwald et al., 2006). More research is needed to clarify this finding.

Apobb1 is expressed in intestine; liver; and yolk syncytial layer. Expression occurs in early development from gastrula 50% epiboly-stage and continues to adulthood (Otis et al., 2015). Apobb1 is expressed at lower levels with vitellogenin and not expressed in the ovary (Levi et al., 2012). There is a lack of studies regarding expression levels of apobb1 comparing adult male and female zebrafish. Our data shows on average more than a two-fold increase in transcripts of male zebrafish compared to female zebrafish as well as expression in juvenile. These findings give reason for the selection of apobb1 as a potential early male sex-determining gene. No previous literature indicates that drug activity influences apobb1 expression levels, further studies should also be carried out to prove this in order to highlight apobb1 as an early male sex-determining gene for uncovering sex-related variables with drug application.

In mice and zebrafish, spata6 is predicted to localise to the sperm connecting piece during spermiogenesis. The sperm connecting piece is essential for linking the developing flagellum to the head in late spermiogenesis. When the expression of spata6 is disrupted males are sterile; this is due to the disruption to the formation of the sperm connecting piece which leads to acephalic spermatozoa in both the epididymis and ejaculates (Yuan et al., 2015; Sujit et al., 2020). In zebrafish, spata6 is expressed in neural tube; spinal cord; and ventral mesoderm, spermatids and mature spermatozoa (Yuan et al., 2015). Spata6 is expressed from 20dpf and increases to adulthood (Oh et al., 2003). Spata6 is a known pro-male gene and was upregulated in the ovaries of fish living in high-population density groups, showing stress from environmental factors causes masculinisation of zebrafish populations (Valdivieso et al., 2019). Previous literature taken with our NGS results show spata6 to be an early male sex-determining gene.

Spata6 is thought to play a key role in testicular germ cell tumours (TGCTs). Downregulation of spata6 results in apoptosis of TGCTs (Huo et al., 2015). This indicates that spata6 could be used as a potential gene target for drug therapy. However, more studies are needed to prove or disprove this and therefore indicate whether it can be used when reveal confounding sex variables during drug trials.

Dmrt1 is involved in gamete generation; male gonad development; and male sex. Dmrt1, which acts downstream of male SYR, has previously been shown to be more strongly expressed in males than in females (Jørgensen et al., 2008; Chen et al., 2017). It is known to maintain the male fate and control gonadal differentiation in vertebrate and non-vertebrate species (Matson et al., 2011; King et al., 2020). This is supported by evidence of higher expression of dmrt1 in male when compared with female zebrafish in NGS data from this study. It is expressed in 14 organs with highest expression level in testis (fully formed stage) of zebrafish. The expression of dmrt1 was also found to be expressed in juveniles throughout development; it is highly expressed in blastula stage of zebrafish (3.33h–3.66 h) through to breeding adult stage (90d-730 d) (Jørgensen et al., 2008).

Dmrt1 is thought to have an influence on sex-specific patterns of childhood asthma, and its expression in testis tissue suggests a potential involvement in hormone regulation (Schieck et al., 2016). It may therefore act as a potential drug target gene for asthma suffers and steroid-based genes may influence its expression. If drugs are found which change the expression of dmrt1 for therapeutic reasons, its use in human drug development as an early sex-determining gene for identification of sex-related difference is not appropriate.

King et al. (2020) Bmp15 is expressed in the gonads of juvenile and adult female zebrafish as well as in the ovaries of humans (Fitzpatrick et al., 1998; Paulini and Melo, 2011; King et al., 2020). It negatively regulates oocyte development, function and maturation through its own activity as well as by increasing expression of other genes which convert androgens to oestrogens, namely, cyp19a1a or decreasing the expression of the male determining gene, dmrt1 (Otsuka et al., 2000; Clelland et al., 2006; Matson et al., 2011; Yan et al., 2017; Hosseini et al., 2019). Loss of bmp15 in adult zebrafish results in a female-to-male sex reversal (Dranow et al., 2016; Kossack and Draper, 2016; Crowder et al., 2018a). In Humans, loss of bmp15 leads to disruption in ovary function (Galloway et al., 2000; Di Pasquale et al., 2004). Drug application has not been previously shown to affect bmp15 expression and so its use as a female sex-identifying gene in drug discovery could be warranted. More tests are needed to prove that bmp15 expression is not affected by drug application.

Chr4 expression has previously been linked to female sex determination in zebrafish and is expressed in juvenile stages (Charlesworth et al., 2005; Anderson et al., 2012; Howe et al., 2013; King et al., 2020). After research was carried out to discover changes in expression of chr4 with drug application, it was found that there were no previous studies which indicate that expression of chr4 is affected by drug application. Further studies should be carried out to clarify this result and its potential use for uncovering sex-related variables during drug trials.

Zpb3 gene expression is partly influenced by folliculogenesis-specific basic helix-loop-helix, Fig alpha, Fig α, which is needed in female development (Zfn.org—ZFIN ID: ZDB-GENE-031121-1, King et al., 2020). The germ cell-specific transcription factor, Fig α has been found in mammals and zebrafish where it is necessary for ovary differentiation and preservation (Soyal et al., 2000; Kanamori et al., 2006; Jørgensen et al., 2008; Schlessinger et al., 2011). As previously shown zpb3 is more highly expressed in oocytes and can be used as an early marker for female (Onichtchouk et al., 2003). No previous literature indicates that drug activity influences zpb3 expression levels. Further studies are needed to indicate the use of zpb3 to reveal sex differences with drug exposure during pharmaceutical trials.

Gyg1a is highly expressed in developing, juvenile and female adult zebrafish (Maanen et al., 1999; Wen et al., 2005; King et al., 2020). No previous study has provided evidence that drugs can influence the expression of gyg1a, more research is needed to clarify this finding.

Kpna2 is highly expressed in developing and adult female zebrafish gonads and its use as an early female sex-determining gene has been previously discussed (Major et al., 2011; Cui et al., 2020; King et al., 2020). Kpna2 overexpression has been linked to various human cancers, including, breast, ovarian endodermal sinus and ovarian primitive germ cell tumours (Huang et al., 2018; Christiansen and Dyrskjøt, 2020). An inhibitor of kpna2 could in theory act as a prospective drug to reduce growth of cancer cells. The genes, IRF1 has been shown to negatively regulate the expression of kpna2. Therefore, drugs which target IRF1 gene could disrupt normal kpna2 expression. Additionally, some studies have shown that kpna2 may be dependent on p53 nuclear translocation to support autophagy in order to allow chemoresistance and metastasis of certain cancerous cells, OSCC (Oral squamous cell carcinoma) (Lin et al., 2018). Although the use of kpna2 as a therapeutic target has been previously identified, studies regarding the relative gene expression levels with drug application are lacking and are required before kpna2 can be used as an early sex-determining gene for uncovering sex differences during drug trials.

Ftz-f1 is expressed in several structures, including digestive system; endocrine system; head; nervous system; and slow muscle cell. It is expressed from zygote:1-cell-stage, 0.0h–0.75 h until breeding-adult-stage, 90days to 730 days (Tong et al., 2010b) (Zfn.org—ZFIN ID: ZDB-GENE-990415-79). At around 12dpf, when female germ cells start to rapidly grow, the initial sign of sexual dimorphism occurs. At this time somatic cells start to increase expression of ftz-f1 transcription factors (Tong et al., 2010b). Ftz-f1 has been previously associated with female sex-determination in zebrafish (Cao et al., 2012). The zebrafish transcript data which is presented here showed an increase of 1.6-fold in females compared to males. Ftz-f1 can therefore, be considered for identification of female zebrafish early in development.

Overexpression of ftz-f1 enhances activation, implicating ftz-f1 as a critical component of the juvenile hormone response (Dubrovsky et al., 2011). Additionally, amino acid sequencing of ftz-f1 gene revealed that the gene protein is a member of the nuclear hormone receptor superfamily (Lavorgna et al., 1991). This research could therefore imply that the expression of ftz-f1 can be altered by hormone-based drugs. However, no previous studies revealed that ftz-f1 expression was altered by drug application and more research should be carried into this before it can be classified as an earlier sex-determining gene for use in revealing sex-related variables occurring from use of drugs.

Gdf9 is an oocyte specific factor encoding proteins which are secreted by oocytes (Clelland et al., 2006; Paulini and Melo, 2011). It is involved in juvenile and adult female sex-determination in zebrafish and is essential for ovarian follicle development in mammals (Silva et al., 2005; Clelland et al., 2006; Gilchrist et al., 2008; Oron et al., 2010; Chen et al., 2017; Strauss and Williams, 2019). It is expressed in fertilised egg; gonad; and immature gonad (Fitzpatrick et al., 1998). The expression of gdf9 starts in the gonads before sex differentiation, and its expression dramatically increases in the differentiated ovary, with a similar expression pattern to that of cyp19a1a. Gdf9 significantly suppresses the expression of male amh while increasing that of activin beta subunits, inhbaa and inhbb. Knock-down expression of gdf9 creates a male-biased sex ratio. Gdf9 promotes follicle-stimulating hormone (FSH)-induced P4 production and STAR expression (Leitão et al., 2014; Li et al., 2019b). P4 is an endogenous steroid and progestogen sex hormone involved in the menstrual cycle, pregnancy, and embryogenesis of humans and other species (Chen et al., 2017). Our transcript data along with previous knowledge supplies evidence for gdf9 as an early gene marker in zebrafish sex-determination.

In humans, gdf9 was recently shown to be a regulator of the development and spread of certain cancer cells including those of the breast and kidney. Gdf9 expression is decreased in clear-cell renal cell carcinoma (CCRCC) which is linked to long-term survival of patients. Although this indicates that gdf9 is a potential tumour suppressor in CCRCC which can be used a potential target for tumour drug development, very few studies have looked at drugs which target the gene for therapeutic reasons nor have they indicated the relative level of gdf9 expression (Hanavadi et al., 2007; Du et al., 2012; Du et al., 2014).

Ipo4 has a role in protein import into nucleus. It is expressed in 33 zebrafish organs with forth highest expression in the female gonad and 14th highest expression in the mature ovarian follicle [bgee. org—gene: ipo4— ENSDARG00000041493—Danio rerio (zebrafish)]. Ipo4 is expressed during development from 0dpf until 56dpf, peaking in expression level at 30dpf (Major et al., 2011). In this study, adult female zebrafish transcript data for ipo4 is around four times higher in adult females when compared to the adult males indicating it as a potential marker for early female sex-determination.

Ipo4 was found to be downregulated in both SARS-Cov-2 infection and aging (Belyaeva et al., 2020a). It could, therefore, provide a target for drug therapy for these. Ipo4 is thought to be a key gene contributing to the progression and development of gastric and other cancers (Xu et al., 2019; Zhou et al., 2020). The drugs, guanosine triphosphate and mkc-1 both have both been linked to the gene ipo4. Mkc-1 has an antineoplastic agent, meaning it inhibits the growth and spread of tumours or malignant cells (https://www.genecards.org/cgi-bin/carddisp.pl?gene=IPO4). This also highlights ipo4 as a gene target for cancer drug therapy. However, more studies are also needed for clarification of the action of these drugs on the expression of ipo4.