Retinoic Acid Metabolic Genes, Meiosis, and Gonadal Sex Differentiation in Zebrafish

Abstract

To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that cyp26a1 in zebrafish and its paralog Cyp26b1 in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish.

Germ Cell Commitment to Oogenic Versus Spermatogenic Pathway: The Role of Retinoic Acid

“…In mice, Cyp26b1 is initially expressed in the gonads of both sexes, but by 12.5 dpc, expression is lost from the ovary and becomes specific to the Sertoli cells of the fetal testis (Bowles et al 2006). A similar localization pattern is also present in anuran amphibians, with CYP26B1 protein present in the developing testis but not ovary (Piprek et al 2013), whereas in zebrafish, Cyp26a1 expression is upregulated in somatic cells of the testes during the developmental period critical for sex determination (Rodriguez-Mari et al 2013). Global deletion of Cyp26b1 in mice leads to late embryonic lethality or the animals die shortly after birth (Abu-Abed et al 2001;Sakai et al 2001;Yashiro et al 2004), therefore allowing for the examination of fetal testis development in the absence of CYP26B1 enzymatic activity.…”

“…As a result, germ cells in a fetal testis are never exposed to RA during the window of plasticity that would allow them to enter meiosis and are instead set upon the male program. While data now exist to support this model in multiple vertebrate species, although the location of RA synthesis does differ (Feng et al 2015;Piprek et al 2013;Rodriguez-Mari et al 2013;Smith Fig. 6.3 RA and CYP26B1 function in the mouse embryonic gonad.…”

“…(a) In the female urogenital ridge, RA generated by aldehyde dehydrogenase (ALDH) activity in the mesonephros drives the expression of Stra8 and the onset of meiosis in the oogonia (red circles). (b) In the male urogenital ridge, RA is still produced in the mesonephros; however, the action of CYP26B1 within the testis cords acts to degrade this RA (marked by red crosses), thereby preventing Stra8 expression and the onset of meiosis et al 2008) and the action of STRA8 does not appear to be conserved in all vertebrates (Feng et al 2015;Rodriguez-Mari et al 2013), this model has recently been challenged ) and the following sections will discuss the evidence for and against the role of RA in initiating meiosis in the developing ovary.…”

“…While there is debate regarding the action of RA in the fetal ovary, there is clear evidence that CYP26B1 is a testis-specific factor required to inhibit male germ cells from entering meiosis during fetal development, although in fish belonging to the teleost class, e.g., zebrafish and tilapia, RA degradation within the gonad relies on CYP26A1 instead of CYP26B1 (Feng et al 2015;Rodriguez-Mari et al 2013). In mice, Cyp26b1 is initially expressed in the gonads of both sexes, but by 12.5 dpc, expression is lost from the ovary and becomes specific to the Sertoli cells of the fetal testis (Bowles et al 2006).…”

The Reversible Sex of Gonochoristic Fish: Insights and Consequences

“…The cyp26a1 or its paralog cyp26b1 genes are considered as meiosis-inhibiting factors of the Cyp26 RA-degrading enzyme and have been shown to have an important role in the onset of germ cell meiosis in fish ovaries [Yamaguchi and Kitano, 2012;Rodríguez-Marí et al, 2013;Feng et al, 2015]. In temperature-induced males of the Japanese flounder, cyp26b1 was upregulated by cortisol, leading to a delay in germ cell meiosis [Yamaguchi and Kitano, 2012].…”