To induce sex conversion in fish, researchers have attempted to use third-generation irreversible AI, which is used to treat breast cancer in menopausal women, and to induce virilization in fish (Geisler et al., 1998; Miller et al., 2008). The exe of aromatase inhibitor lowers the concentration of E2 in the blood within a short time after administration by inhibiting the synthesis of E2. Furthermore, tam, a type of AE, is used to treat breast cancer. As a selective er modulator that competes with E2 for ers, tam acts as an agonist of E2 in some tissues, but as an antagonist in others (Cosman and Lindsay, 1999).In addition, AE inhibits the synthesis of vtg mRNA within a short time by inhibiting the transcription step of vtg conjugation through its antagonism with E2. In fish, AI and AE have been used to investigate sex conversion, gonadal gene expression, plasma vitellogenin, and female virilization.
In the in vivo experiments of this study, exe and tam were intraperitoneally injected into immature olive flounders, and the changes in gene expression of er and vtg as well as histological changes due to the hormone inhibitors were compared. Through a process called suicide inhibition of steroid hormones, exe, an AI, inhibits the function of aromatase that induces enzyme inactivation (Tiboni and Ponzano, 2016). Moreover, exe inhibits the production of E2 in the brain and peripheral tissues or inhibits the action of the already produced E2 on the receptor. The results of this study suggest that this AI may directly affect the inhibition of estrogen receptor expression itself in the liver (Fig. 2). In a 2010 study that fed exe to tilapia (Oreochromis niloticus), the comparison with E2 demonstrated that exemestane blocks the conversion of testosterone to E2 in the gonads, thus transforming the undifferentiated gonad into testes (Ruksana et al., 2010). Furthermore, in a 2018 study in which black sea bass (Centropristis striata) were exposed to exe, the early sex change and gonadal gene expression in the gonads, the CYP19A1A gene, which has the female gonad development function, decreased in females, though there was no significant difference from the control (Miller, K. A, 2018). In addition, the ESR2B gene, which has the E2 response function, and the ZPC2 gene, which has the egg envelope component function, also decreased, however, they were not significantly different from those of the control (Breton et al., 2019). In this study, when the AI was injected after the intraperitoneal injection of E2, the er was more inhibited than when the AI was injected alone; however, considering the sharp decrease, it is likely that the inhibition of the er directly in the liver can induce the inhibition of female sexual maturation (Figs. 2 and 6A). In addition to exemestane, which is a steroid, fadrozole, which is a non-steroid, has exhibited a wide range of sexual maturation inhibitory effects in various fish species (Hinfray et al., 2013). Thus, the technology for effectively controlling sexual maturation using AIs requires the use of various AI substances and additional experimentation. However, the use of exemestane alone, as in this experiment, showed the possibility of inhibiting female sexual maturation. The histological analysis showed intersex tissue at 5 days with injections of exemestane alone and mixed with E2 in the gonads. Thus, in addition to genotypic changes (Fig. 2), more obvious phenotypic changes were also observed (Fig. 5A).
Because of estrogen’s high lipophilicity when introduced in vivo, it enters directly into the cell and binds to the specific er in the nucleus; this er then binds to cis-acting elements (enhancer, ERE : estrogen responsive element) upstream of certain genes that are responsive to E2 (Savouret et al., 1991). However, by intraperitoneally injecting AI and anti-estrogen, the mRNA of both the estrogen receptor and vitellogenin reduced in the liver and gonads. E2 was not produced in the liver, but in the gonads (follicle cells), and likely inhibited expression itself by blocking the estrogen receptor. It appears that injection with exemestane reduced E2 synthesis. It was thought that besides directing the inhibition of androgens to E2s, it could have an effect elsewhere in the hypothalamus pituitary gonadal axis. However, there are no reports yet on the direct investigations of the estrogen receptor and level of direct expression in the liver of olive flounder. Future studies should include in-depth studies on the cascade of er blocking and the intraperitoneal injection of exe, which is expected to be able to inhibit sexual maturation in female fishes.
Tam is a non-steroidal anti-estrogen that blocks the binding of er and is known to bind and block the function of E2 (Massarweh et al., 2008). Furthermore, Tam has exhibited effectiveness as an antagonist of ER in fish, as well as in the inhibition of vtg production in the presence of E2 in fish (Leanos-Castaneda and Van Der Kraak., 2007). By intraperitoneally injecting AE into immature olive flounders, as expected, tam inhibited the induction of vtg receptors in immature flounder females by competing with the endogenous E2 for the estrogen receptors. These results are similar to those of the AI. In a study on chickens, after treatment with Tam, the female gonads exhibited degeneration and apparent testis morphology, although no changes were found during histological observation (Koo et al.,1985). In the experimental groups in which the immature olive flounders were injected with AE alone and mixed E2, genetic changes in mRNA were observed, although no changes were found during histological observation. In bony fish (Osteichthyes), research results have demonstrated the efficacy of tam in rainbow trout and tilapia, although the results also showed no effect on gonadal sexual differentiation (Guiguen et al., 1999). Furthermore, in a 2007 study, in which olive flounders were treated with tam, the er alpha and beta expression studies revealed anti-estrogenic activity (Kitano et al., 2007). Thus, tam not only exhibited differences among species, but also showed different effects on er and vtg receptors according to the time difference before and after sexual maturation. However, as mentioned above, tam has a function that binds it to the er and this binding may produce a reaction that reduces the amount of er. This study confirms that the expression of er and vtg is reduced (Fig. 2, 3). Therefore, tam appears to be sufficiently effective for the investigation of anti-estrogenic effects on the sex differentiation in olive flounder. Although these results can be used to predict the possibility of negative feedback across the brain-pituitary-gonad axis (BPG axis) in the synthesis of E2 by tam, this mechanism has not yet been fully investigated. As such, further examinations are needed to confirm if tam is directly involved.