All ETDs from UAB

Advisory Committee Chair

Thane Wibbels

Advisory Committee Members

Asim Bej

Chris Murdock

Turk Rhen

Douglas Watson

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences

Abstract

The red-eared slider turtle (Trachemys scripta) possesses temperature-dependent sex determination (TSD) in which the incubation temperature of the developing embryo determines gonadal sex. Although a number of mammalian gene homologues have been identified in the sex determination/differentiation cascade of reptiles with TSD, the exact trigger(s) is not well understood. As such, this dissertation research addresses the mRNA expression profiles of putative sex-determining/differentiation genes (Dmrt1, Sox9, Amh, Lhx9, and Foxl2) with an emphasis on the male cascade in embryonic gonads of T. scripta. Additionally, this research addresses the influence of exogenous estrogen and aromatase inhibitors on gene expression. Lastly, this dissertation is the first study to address epigenetic regulation during TSD by assessing alterations in Dmrt1 expression upon application of a global DNA methylation inhibitor, zebularine. The chronology of gene expression during the sex determination/differentiation period demonstrates that Lhx9 was expressed prior to the temperature-sensitive period, increased throughout all stages, and showed no sex specific differences in expression throughout embryonic development. Following Lhx9 expression at male-producing temperatures, Dmrt1 was expressed earlier and greater than both Sox9 and Amh, suggesting a testis-specific role for all three genes. Conversely, Foxl2 mRNA displayed female-specific expression at embryonic stage 23 after the TSP. Treatments with 17ß-estradiol appeared to inhibit or delay gonadal differentiation and resulted in down-regulation of Dmrt1, Sox9, Amh, and Lhx9, but up-regulation of Foxl2. Whereas, treatment with an aromatase inhibitor, letrozole, resulted in inconsistent degrees of sex reversal; therefore, no significant differences in gene expression for Dmrt1, Amh, and Lhx9 were detected. Lastly, within 12 hours of zebularine treatment, Dmrt1 expression increased at both male- and female-producing temperatures. Due to the close evolutionary relationship between birds and turtles, Dmrt1 was of particular interest when addressing potential molecular triggers of TSD. In T. scripta, Dmrt1 was sexually dimorphic, was expressed earlier than any other gene analyzed, and may be regulated by DNA methylation. This is consistent with the hypothesis that Dmrt1 may be an essential factor for testicular development across amniotic vertebrates, and potentially a sex-determining trigger in T. scripta. Collectively, these studies contribute some understanding of the molecular mechanisms of TSD.

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