Date of Award

January 2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Turk Rhen

Abstract

Temperature-dependent sex determination (TSD) was first discovered in an African lizard over 40 years ago. TSD has since been shown to be exhibited by many vertebrates including some fish and amphibians, numerous lizards, turtles, and all crocodilians studied to date. Although numerous questions surround TSD, a major question focuses on understanding the genetic, physiological and molecular mechanisms underlying this process. However, the molecular mechanisms underlying TSD are not well known and the gene(s) that transduces a signal for ovary or testis development is not known in any species. Furthermore, it is well established that sex steroid hormones, androgens and estrogens, are important for sex determination and differentiation in TSD species. Yet, the role of androgens in these processes is not well understood. This dissertation addresses these questions in two parts. First, to identify unique, thermo-sensitive, genes involved in TSD and secondly, determine the role of androgens in sex determination and differentiation in the common snapping turtle, a reptile with TSD.

I used differential-display PCR to clone a candidate gene involved in TSD, the cold-inducible RNA binding protein (Cirbp). The temporal and spatial patterns of Cirbp mRNA and protein expression during and after sex determination were determined using quantitative real-time PCR, in situ hybridization, and immunohistochemistry. I used next-generation Illumina sequencing to identify small nucleotide polymorphisms (SNPs) to test for associations between Cirbp genotype, mRNA expression, and sex ratios.

To determine the role of androgens in sex determination and differentiation, snapping turtle embryos were treated with the androgen, dihydrotestosterone (DHT), the anti-androgen, flutamide, or a vehicle (ethanol) control. Whole mount in situ hybridization and immunohistochemistry were used to determine the effect of the treatments on sex differentiation of the male reproductive tracts. Quantitative real-time PCR was used to measure expression patterns of ovary-specific genes, testis-specific genes and steroidogenic genes in gonads from embryos treated with vehicle, DHT, or flutamide.

I found Cirbp was induced at a high female-producing temperature, but not at a low female-producing temperature. Cirbp is associated with TSD and expression of alternative Cirbp alleles is capable of transducing temperature differently for establishing a signal that directs ovaries versus testes development. I observed allelic specific expression and differences in allele frequencies between turtle embryos from northern and southern Minnesota, suggesting genetic adaptation to local thermal regime. I also found significant genetic associations between Cirbp genotype, Cirbp expression and sexual phenotype in a study that produced mixed sex ratios.

Androgens were capable of inducing ovarian development even at an all-male producing temperature, presumably by inducing aromatase expression and increasing endogenous estrogen production. I also observed among clutch variation in androgen responsiveness to produce females, suggesting there is a genetic basis for the response.

My findings provide the strongest evidence to date for a unique gene involved in TSD. Additional studies are required to define the functional role of Cirbp in sex determination. Androgens appear critical for ovarian development, but ongoing research is needed to understand androgen signaling and the genetic variation underlying this process.

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