The srd a isoforms showed unique
The srd5a isoforms showed unique expression profiles in early FHM development (Fig. 2). Similar to amphibian embryos, there was a high abundance of both srd5a1 and srd5a3 at 1dpf in FHMs, suggesting that mRNA for these enzymes may be maternally deposited and that these enzymes may play a key role in early organization and patterning of the embryo. The expression of the two isoforms were significantly positively correlated to each other during early development (<0.01). Both srd5a1 and srd5a3 decreased in abundance at 3dpf relative to 1dpf and remained low until after hatch, at which time there appeared to be a modest increase in transcript abundance in srd5a1 in the newly hatched FHMs. This suggests that individuals may begin to transcribe srd5a1 relatively soon after hatch. In contrast, srd5a2 showed more of a consistent expression pattern over early development and did not appear to have high levels of maternally-deposited mRNA compared to srd5a1 and srd5a3. The srd5a2 transcriptional profile did not correspond to the Western clawed frog embryos as srd5a2 mRNA levels are the most abundant of the three isoforms. This suggests that there may be species differences in the ontogeny of the srd5a isoforms, although we point out that it is difficult to compare developmental landmarks between fish and frogs as the time points examined in each study may not correspond to the exact same developmental stage. Based upon the mammalian literature, srd5a1 is primarily associated with non-sexual organs, srd5a3 is ubiquitously expressed, and srd5a2 is associated with reproductive tissues in adults (reviewed in Langlois et al., 2010a). The low expression of srd5a isoforms early in FHM development may correspond to the lack of a need for reproductive tissues at this time. Tong et al. (2010) divided sex differentiation into four distinct stages in zebrafish. The first stage occurs around 10dpf and is referred to by the authors as the preparation stage, indicative of primordial germ 1,2-Dilauroyl-sn-glycerol sale of the bipotential gonads beginning to differentiate into gonocytes. At 12dpf, the female germ cells begin to grow rapidly; this time point is suggested to indicate the process of sexual dimorphism. At 14dpf, female germ cells differentiate via meiosis while testicular germ cells stay undifferentiated and retain their gonocyte appearance. In the FHM, pre-meiotic cells in the gonad can be detected at 10dph, indicating the start of ovarian differentiation (Van Aerle et al., 2004). We measured the expression of the srd5a isoforms at ∼9dph (14dpf), close to these initial stages of ovarian development. The srd5a isoforms may not be actively transcribed at this time of development as precursor testosterone may be required for 17β-estradiol (E2) production in order to mediate ovarian cell differentiation. In the study by Van Aerle et al. (2004), primary oocytes were detected at 20–25dph, while vitellogenic oocytes were first detected at ∼120dph. In contrast with the ovaries, the spermatogonia of the presumptive testes were first detected at approximately 40dph and did not enter meiosis until 90–120dph. Therefore, FHM embryos may not require a surge of srd5a expression before 20–40dph as sex determination and gonad formation have not yet reached a peak during the critical window. However, low expression of the srd5a isoforms may be required for regulating androgen biosynthesis, as the expression of other male-related transcripts such as androgen receptor (ar) and dmrt1 have been shown to peak between 10 and 16dpf in other small-bodied fish such as zebrafish (Jorgensen et al., 2008). Cell differentiation and growth are processes known to be regulated by androgens in early vertebrate embryogenesis. As pointed out above, sex differentiation in zebrafish begins at approximately 10dpf, and ovarian development progresses until 20dpf (Von Hofsten and Olsson, 2005). Testis development is initiated in males at approximately 30dpf which corresponds to the initiation of apoptosis pathways in the ovary. Based upon data in zebrafish and FHMs, the transcription of srd5a mRNA may surge later after hatch (i.e. 20–30dph) and this may contribute in part to the masculinization of the embryos.