Abstract
Previous studies of alligators from pesticide-contaminated lakes have shown decreased fertility in comparison to less polluted reference populations. Our research is focused on elucidating potential mechanisms of decreased reproductive success in alligators exposed to environmental contaminants during critical developmental periods. The localization and identification of germ cells during development can provide critical information in assessing future reproductive capability. Germ cells are the unique precursors of gametes, more commonly referred to as eggs and sperm. Specific cell types can be localized through immunohistochemistry (IHC), which involves the use of labeled antibodies to identify specific molecules present in the cells of interest. In this study we used standard IHC protocols to test two antibodies for use as potential biochemical markers of germ cells in hatchling alligator gonads. We ran parallel procedures on mouse gonads as a positive control for the validation of our techniques. Because the process of germ cell maturation is generally conserved among vertebrates, antibodies intended to identify germ cells in mice may bind to homologous antigens in alligator germ cells. Our results indicate that a polyclonal antibody to DDX4 binds to putative germ cells in mice and alligators. DDX4 is an RNA helicase expressed in germ cells and is thought to be expressed in all stages of germ cell development up to late meiosis. We also tested an antibody to SCP3, a gene involved in the stabilization of homologous chromosomes during prophase I. Our monoclonal antibody recognized germ cells in prophase I in mice, but it did not bind homologous antigens in the alligator. By identifying germ cells at various developmental stages, we can determine if contaminants affect development of alligator gonads in a way that would compromise future fertility. Further research will involve the validation of antibodies that identify germ cells in specific stages of meiosis.
Included in
Validation of Antibodies for the Immunolocalization of Germ Cells in Alligator Gonads.
Previous studies of alligators from pesticide-contaminated lakes have shown decreased fertility in comparison to less polluted reference populations. Our research is focused on elucidating potential mechanisms of decreased reproductive success in alligators exposed to environmental contaminants during critical developmental periods. The localization and identification of germ cells during development can provide critical information in assessing future reproductive capability. Germ cells are the unique precursors of gametes, more commonly referred to as eggs and sperm. Specific cell types can be localized through immunohistochemistry (IHC), which involves the use of labeled antibodies to identify specific molecules present in the cells of interest. In this study we used standard IHC protocols to test two antibodies for use as potential biochemical markers of germ cells in hatchling alligator gonads. We ran parallel procedures on mouse gonads as a positive control for the validation of our techniques. Because the process of germ cell maturation is generally conserved among vertebrates, antibodies intended to identify germ cells in mice may bind to homologous antigens in alligator germ cells. Our results indicate that a polyclonal antibody to DDX4 binds to putative germ cells in mice and alligators. DDX4 is an RNA helicase expressed in germ cells and is thought to be expressed in all stages of germ cell development up to late meiosis. We also tested an antibody to SCP3, a gene involved in the stabilization of homologous chromosomes during prophase I. Our monoclonal antibody recognized germ cells in prophase I in mice, but it did not bind homologous antigens in the alligator. By identifying germ cells at various developmental stages, we can determine if contaminants affect development of alligator gonads in a way that would compromise future fertility. Further research will involve the validation of antibodies that identify germ cells in specific stages of meiosis.