Date of Graduation

12-2015

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Douglas D. Rhoads

Committee Member

Yuchun Du

Second Committee Member

Suresh K. Thallapuranam

Keywords

Biological sciences; Pathrings; Pathway; Primordial germ cells; Rna-seq; Specific markers; Transcriptomics

Abstract

Chicken primordial germ cells (PGCs) are derived from extraembryonic tissue of the embryo and first appear at stage X of development. They enter the bloodstream and migrate to the genital ridge, unite with somatic tissue to form a developing gonad, and then differentiate to sperm or ova (Fujimoto et al., 1976). Understanding molecular features of both male and female PGCs not only clarify the differentiation mechanism of such cells toward different germ lines, but will also help in selecting for highly productive types of commercial chicken. Most previous studies focused on the location of PGCs (Eyal-Giladi et al., 1981; Swift et al., 1914), but there are only a few reports describing unique properties of these cells compared to other embryonic cells. Germ cell specification is unclear due to the lack of reliable PGC markers. Most well known PGC identities are based on periodic acid-Schiff (PAS) reagent, which stains high glycoprotein content in PGCs (Fujimoto et al., 1976), and a cell surface glycoprotein, known as stage specific embryonic antigen-1 (SSEA-1). However, SSEA-1 is also expressed in other embryonic stem cells (Mozdziak et al., 2005). Thus, further research is warranted focusing on molecules, which underlie identities of chicken germ cells. In this study, we isolated PGCs from embryonic blood at three days of incubation. PGCs were cultured without feeder cells. PGCs were identified by generally accepted methods, including PAS staining, immunocytochemistry with anti-SSEA-1 antibody and germ cell-related gene expression. RNA purified from six male, six female PGC cell lines and chicken embryo fibroblast (CEFs) were reverse transcribed into cDNAs. cDNAs were then used to amplify candidate genes for PGCs markers, such as germline-specific genes (Cvh, Dazl), meiosis-related genes (Sycp, Stra8) and migration-related genes (Sdf-1). We compared the expression of these genes in PGCs with CEFs to ensure the unique characteristics of PGCs. These genes were not only highly expressed in chicken PGCs as compared to positive control from chicken juvenile testis tissue, but also were elevated in CEFs. Transcriptome using RNA-Seq in this study provided total gene expression profile of chicken PGCs, which elucidated unknown markers of PGCs as well as differences between male and female primordial germ cells.

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