Opposite-directional sex change in functional female protandrous anemonefish, Amphiprion clarkii: effect of aromatase inhibitor on the ovarian tissue

It is known that the gonads in functional female anemonefishes consist of only ovarian
tissue and no testicular tissue 6], 9], 10]. In the present study, we confirmed that the females in the initial control group
were indeed functional and that their gonads were comprised of mature ovarian tissue,
completely devoid of testicular tissue. However, following treatment with the aromatase
inhibitor, Fadrozole, three out of five functional female fish presented with ambisexual
gonads, which possessed active spermatogenic tissues including a large amount of sperm
similar to what is seen in functional males, while the gonads from untreated females
retained developed ovaries with no testicular tissue. We also observed that the E2
levels in fish treated with AI were significantly lower than that in the control group.
These results strongly suggest that inhibition of E2 synthesis by AI treatment induces
mature testicular tissue formation in functional ovaries, a finding that has never
been reported in the protandrous anemonefish.

The exact origin of the spermatogenic cells and testicular somatic cells in the female
gonads remains unknown because there is no apparent testicular tissue in the ovary
22]. Previously, we induced female to male sex changes by AI treatment in some protogynous
fish, namely, three-spot wrasse (Halichoeres trimaculatus) 18], and grouper (Epinephelus merra) 23], 24]. It was suggested that some types of ovarian somatic cells survived and that the
gonial germ cells spreading across the ovary may have differentiated into sperm during
the gonadal sex changes in three-spot wrasse 22]. This study implied that the origin of testicular cells during sex changes is the
ovarian cells forming the ovarian tissue 22]. In addition, we induced sex change from adult females to mature males in gonochoristic
Nile-tilapia (Oreochromis niloticus), Medaka (Oryzias latipes), and zebrafish (Danio rerio) 12], 13]. These results revealed that following sex differentiation, germ cells and somatic
cells in the ovary retain bipotency for long time in the ovary of gonochoristic fish.
In addition, previous reports involving rainbow trout (Oncorhynchus mykiss) show that testicular germ cells possess a high level of developmental plasticity
and sexual bipotency, even after the animal reaches maturity 25]. Based on these findings, we conclude that some germ and somatic cells in the ovaries
of functional female protandrous anemonefish retain bipotency, which enables them
to re-differentiate into testicular tissue.

We found that mean plasma 11-KT levels significantly increased following AI treatment,
while mean plasma E2 levels were significantly lowered in the same treatment group.
This same decrease in the E2 plasma level was observed during female to male sex changes
in protogynous fishes; however, the increase in plasma 11-KT levels was less universal
16]–19], indicating that the decrease in estrogen levels may be more important to the sex
change than the increase of androgen. In addition, E2 compensation during AI treatment
suppressed the sex change in three-spot wrasse fish 18]. Additionally, treatment of ambisexual gonads with E2 after testicular differentiation
in anemonefish causes the disappearance of testicular tissue 20]. These results indicate that estrogen has an important role in ovarian differentiation
and in sex change in hermaphrodite fish.

Moreover, E2 treatments around the time of testicular differentiation suppressed the
differentiation of testicular tissue 26], 27]. In the protandrous black porgy, Acanthopagrus schlegeli, oral administration of AI suppressed aromatase activity and inhibited the natural
sex change from male to female 14], 28]–30]. Furthermore, we found that inhibition of E2 synthesis with AI was shown to induce
testicular differentiation in the developed ovaries of anemonefish, which agrees with
previous reports showing that high E2 levels restrict the ovarian transition to testis
in the black porgy. Thus, it is possible that high levels of estrogen act to maintain
ovarian tissue, whereas extremely low levels of estrogen enable testicular differentiation.
Therefore, there is a high possibility that some gonial germ cells and somatic cells
in the ovaries differentiate into testicular tissue under conditions of E2 depletion.