Cytochrome P450 aromatase (CYP19) is the enzyme responsible for the conversion of C19 androgens to C18 estrogens in brain and gonadal tissues of vertebrates (Castro et al.,2005; Hong et al.,2009 )^52,70. During early developmental stages, the hormonal balance between estrogens and androgens is essential particularly durring the sexual differentiation period and this balance is in turn dependent on the availability and activity of steroid synthesizing enzymes such as aromatase (Smirnov & Trukhina, 2019)⁶⁰. For a bipotential gonad to differentiate into testis, an increase in the level of androgens is required to trigger the male differentiation pathway, while ovary differentiation requires increasing levels of estrogens (DeFalco 2019; Nef & Parada, 2000) ^{17, 69}. As aromatase inhibitors block the synthesis of estrogens (by inhibiting the conversion of androgens to estrogens), the level of androgens in the developing organism increases, inducing testis differentiation and male maturation (Muth-Kohne et al., 2016)⁷.

Biological Plausibility

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Inhibition of cytochrome P450 aromatase (CYP19) during the critical period of sexual differentiation of non-mammalian vertebrates can induce a male differentiation pathway due to an increasing imbalance in the androgen-t­o-estrogen ratio. Androgens have a critical physiological role in reproductive biology and sexual differentiation, particularly in the development of male first and secondary sex characteristics(DeFalco 2019) ¹⁷. After sex has been determined, the increasing levels of androgens during the critical period of sexual differentiation will allow the morphological development of the testis, for which the early presence of three main differentiating cell types is fundamental; the gamete forming cells (spermatogonia), support cells (sertoli cells) and hormone secreting cells (leydig or interstitial cells) (Cotton & Wedekind, 2009)⁴⁴. As gonads continue to differentiate into testes, the secretion of testicular hormones will be sufficient to promote the complete masculinization of the embryo (Nef & Parada, 2000) ⁶⁹.

Empirical Evidence

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Fish

• Well known aromatase inhibitor, fadrozole, has shown to cause concentration-dependent inhibition of aromatase activity in Zebrafish during the critical period of differentiation leading to a complete shift towards male development. ^{3,4, 6,7,9}
• Generation of cyp19a1a and cyp19a1b gene mutant lines and a cyp19a1a;cyp19a1b double knockout line in zebrafish using transcription activator like effector nucleases (TALENs) has shown that cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants result in all male phenotypes ^8,13 . This was characterized by high number of apoptotic cells and stromal cells by 29 days post fertilization and by 40 days post fertilization the typical testicular structure had appeared showing cystic spermatogeic cells¹³.
• All nile tilapia fish treated with aromatase inhibitor Exemestane during the critical period of sexual differentiation (from 9 dah through 35 days after hatch) had well developed testes by 120 days after hatch.⁵
• Studies with Tilapia (Oreochromis niloticus), a species with genetic and environmental sex determination, have shown that aromatase repression in the gonad is required to drive sexual differentiation to testis. ^61,71,72

Birds

• Studies with chicken (Gallus g. domesticus) embryos using aromatase inhibitor (AI) letrozole on the first day of embryonic development has shown that the gonad of genetic females exposed to the AI had poorly developed seminiferous tubules suggesting that they had undergone testicular sexual differentiation pathway (Trukhina et al., 2016) ⁵⁹
• Female chicken (Gallus g. domesticus) gonads treated at embryonic day 3.5 with an aromatase inhibitor were masculinized by the embryonic day 9.5, and MIR202 expression was increased. Increased MIR202 expression correlated with up-regulation of DMRT1 and SOX9 which are required for proper testis development (Bannister et al., 2011)⁶²

Reptiles

• Administration of aromatase inhibitors to red-eared slider turtle (Trachemys scripta) eggs incubated at female producing temperatures has shown to produce all male offspring (Crews & Bergeron, 1994)⁵⁶

Amphibians

• Studies with in vitro Xenopus laevis gonads treated with aromatase inhibitor showed histological characteristics of the male phenotype. ⁵⁸

Uncertainties and Inconsistencies

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Response-response Relationship

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Time-scale

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Known modulating factors

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Known Feedforward/Feedback loops influencing this KER

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