Identification of clathrin and dynamin II in the porcine ovary supports the presence of clathrin-mediated endocytosis
Date
2016
Authors
Bacon, Margaret Leese, author
Graham, James, advisor
Eckery, Douglas, advisor
Callahan, Gerald, committee member
Bruemmer, Jason, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
The feral swine population in the United States has grown to 6 million animals located in 41 states, and causing an estimated $1.5 billion annually in damages and control. Feral swine are well-known for spoiling crops, preying on smaller wildlife, spreading disease, and damaging the land’s ecology. In 2014, the federal government initiated the Animal Plant and Health Inspection Services (APHIS) National Feral Swine Damage Management Program to combat this overabundant wildlife population. One of five research areas identified as a key component in the advancement and improvement of tools and methods to manage feral swine is the development of reproductive inhibitors that can cause permanent sterility. Successful reproduction in mammals depends on an adequate number of healthy oocytes present in primordial follicles within the ovaries. Maintenance of the primordial follicular pool requires the coordinated actions of both oocyte survival factors and factors that maintain the follicles in a non-growing state until they are activated to grow. There is a finite number of primordial follicles in the ovaries of mammals, which if destroyed would leave the animal permanently sterile. Relatively little is known about the cellular communication mechanisms utilized by primordial follicles. The purpose of this study was to investigate whether primordial follicles express components of clathrin-mediated endocytosis (CME), the most common form of receptor-mediated endocytosis used in eukaryotes. This process, if present, could be exploited as a method to deliver chemosterilants to the primordial follicle pool. This study focused on determining the expression and localization of two key components of this CME, clathrin and dynamin II. Ovaries from 6 piglets and 6 gilts were bisected longitudinally, fixed in formalin, embedded in paraffin, and cut into 5µm thick sections which were mounted on microscope slides. Fluorescent immunohistochemistry using specific antibodies labeled with fluorescein isothiocynate was performed to determine the expression and localization of clathrin and dynamin II on the mounted tissue sections. Expression of clathrin and dynamin II was revealed in the cytoplasm of oocytes of all follicular stages examined, suggesting that CME could be a mechanism of cell signaling in porcine oocytes. A second aim of this study was to establish methods to visualize and characterize the internalization process in pig oocytes. The isolation of primordial follicles and oocytes and the live cell imaging of FM1-43 membrane probe uptake was completed. Primordial follicle isolation was attempted in piglet ovaries using a combination of chemical and mechanical methods. This process used enzymatic digestion and filtration of chopped cortical tissue from porcine ovaries. Mature oocytes were imaged over time after the addition of FM1-43. This established a protocol for live cell handling and imaging that would be useful in future studies. In addition, several ligands and their receptors that may utilize CME were investigated in porcine oocytes. The development of tools and methods to characterize cellular communication mechanisms in oocytes can contribute to the formulation of a chemosterilant to be used to cause non-lethal permanent sterility in feral swine.
Description
Rights Access
Subject
fertility control
primordial follicle
swine
ovary
clathrin-mediated endocytosis
reproduction