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Thesis (Ph. D.)--Washington State University, May 2009. / Title from PDF title page (viewed on June 19, 2009). "School of Molecular Biosciences." Includes bibliographical references.

Effective reproduction is essential for species survival. Sexual reproduction depends upon functional gonads and reproductive ducts. Zebrafish (Danio rerio) is a popular model organism, but the genetic basis of zebrafish sex determination, gonad development, and reproductive tract development are not fully understood, and understanding this basis could inform about the evolutionary conservation of these genes and the use of zebrafish to investigate and treat reproductive diseases. In chapter I, I give a overview of sex determination systems, gonad development, and reproductive duct development in mammals and fish, and ask how sex is determined and how reproductive ducts develop in zebrafish. In chapter II, I used genome wide association studies (GWAS) to investigate if the genetic basis of sex determination in a variety of zebrafish strains -- two ‘wild-type’ strains cultured for about 30 years in the lab, and four ‘natural’ strains, wild-type strains isolated directly or recently from nature in India -- and identified a sex-associated region on zebrafish chromosome 4 in natural zebrafish strains that was lacking in the lab strains. In chapter III, I investigated whether or not wnt4a is important for zebrafish ovarian development, and found that wnt4a is expressed in the early bipotential gonad and that loss of wnt4a results in male-biased sex ratios, indicating that wnt4a is important for zebrafish ovarian development. In chapter IV, I investigated whether or not wnt4a is important for male reproductive duct development, and found that loss of wnt4a slows the formation of male reproductive ducts and prevents the male fused ducti deferens from connecting to the genital orifice in zebrafish males. Results further showed that wnt4a is expressed in tissue around the site where this connection should occur before and after the connection is formed, revealing a novel wnt4a phenotype in zebrafish that hasn’t been seen thus far, and indicating that wnt4a is necessary for proper male reproductive duct development in zebrafish. This dissertation contains both published and unpublished co-authored material.

Inaug.-Diss.--Heidelberg. / Lebenslauf. "Benutzte literatur": p. [21]-22.

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Multiplex polymerase chain reaction (PCR) assays were designed that incorporated primer pairs for the sex determining region on the Y chromosome, Sry, and one or two control sequences. A triplex and a duplex assay were created involving Sry and Dax, a single copy X chromosome gene which is involved in the female sex determination pathway. The third sequence in the triplex assay was a repetitive Y chromosome sequence, YR. A minimum of 2.5 x 10-3 to 2.5 x 10 -4 m g/ m L of male DNA and 2.5 x 10-5 m g/ m L of female DNA was required if a single multiplex PCR was performed. To demonstrate that sex determination of preimplantation porcine embryos is possible, morulae were collected 5 d post insemination from pregnant mare serum gonadotrophin (PMSG)/human chorionic gonadotrophin (hCG)-treated 60--70 kg prepubertal gilts. Embryos were biopsied using a micromanipulator and cells were placed into individual microcentrifuge tubes for further analysis. Embryos were then cultured to the blastocyst stage. A total of 315 embryos were sexed via the PCR assay with a resultant female/male (%) ratio of 73/27. When 94 embryos from heavier gilts (90--100 kg) were sexed in a similar assay, the resultant female/male ratio was 60/40. Attempts were made to correlate these results with karyotypes. Five transfers of sexed embryos into synchronized recipients were attempted. None of these resulted in pregnancies; although return to estrus was delayed by two to eight days, in four out of the five recipients. Our findings suggest that PCR amplification of the Sry gene can be a reliable method for sexing porcine embryos. It does appear that embryo quality is critical for both the PCR assay and subsequent successful culture. (Abstract shortened by UMI.)