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The protandrous black porgy, Acanthopagrus schlegeli, has a striking life cycle, with sex differentiation at the juvenile stage, mono-male development, a bisexual gonad during the first 2 yr of life, and a male-to-female sex change (with vitellogenic oocytes) at 3 yr of age. In the present study, we investigated the possible roles of amhand amhr2in gonadal development in a nonmammalian model organism (protandrous black porgy), especially in relation to sex differentiation, testicular and ovarian growth, and sex change. Fish of various ages were treated with estradiol or an aromatase inhibitor to induce the fish to become female. Furthermore, a natural sex change (2+-yr-old [>2 yr and <3 yr] fish) and a nonchemical method to surgically remove one of the pair of gonads to examine the possible roles of amhin the natural sex change were conducted. We present integrative in situ hybridization, immunohistochemical, cellular, and molecular data describing these phenomena. During gonadal sex differentiation, an increase in amhand amhr2expression was detected. Higher levels of amhand amhr2transcripts were observed in the testicular tissue when compared to the ovarian tissue in the bisexual gonad of 1+-yr-old (>1 yr and <2 yr) fish. Transcripts of amhreached peak levels in November (prespermatogenesis period) and then declined to the lowest levels in January (spawning period). Chemical-induced ovarian tissue had very low amhtranscript levels but high levels of amhr2. Active testes had significantly higher amhand amhr2expression levels as compared to inactive testes. In contrast, no difference in the expression of amhand amhr2between active and inactive ovarian tissues was found. Transcripts of amhwere expressed in the somatic cells of the spermatogonia and vitellogenic oocytes, and amhr2was expressed in the somatic cells of the spermatogonia. Transcripts of amhdecreased in the testicular tissue 5 mo before occurrence of the sex change into a female. In contrast, testicular amhexpression remained high if the fish remained male. Human chorionic gonadotropin regulated amhand amhr2expression in the testicular tissue but not in the ovarian tissue. The present results suggest that amhplays important roles in early testicular and ovarian development, late ovarian growth (e.g., vitellogenic oocytes), and natural sex change in the protandrous black porgy.

Inhermaphroditic fish, the ovotestis can respond to external stimuli so that only one type of gonadal tissue (either ovarian or testicular tissue) will remain reproductively active and the other will recede to a rudimentary stage. However, the molecular mechanism for sexual fate determination is still poorly understood in hermaphroditic fish. In the present study, we examined whether sexual fate determination with respect to testis development is due to differential expression of dmrt1. Expression of dmrt1was limited to the spermatogonia-surrounding cells (Sertoli cells) throughout testis development. Testicular dmrt1was differentially expressed in fish (black porgy [Acanthopagrus schlegeliBleeker]) depending on if fish were destined to be female or male. Expression of dmrt1in Sertoli cells did not require germ cell factors with busulfan treatment. To examine the role of dmrt1, we used virus-based RNA interference. Deficiency of dmrt1resulted in a reduced number of germ cells in the testis and stimulated a male-to-female sex change. Higher serum luteinizing hormone levels were detected in 2+- to 3-yr-old male fish as compared to sex-changing female fish.Furthermore, we showed that fish treated in vivo with gonadotropin-releasing hormone (Gnrh) and fish treated in vitro with gonadotropin (Gth) had higher dmrt1expression in the testis, suggesting that these endocrine factors may affect the male-to-female sex change. Therefore, our data suggest that dmrt1plays a key role in initial testis differentiation and in later maintenance of male development. We show, to our knowledge for the first time, the functions of dmrt1in hermaphroditic fish, which indicate that male-phase maintenance may be regulated by the brain-pituitary-gonadal axis via the Gnrh-Gth-Dmrt1 axis.