Title

A morphological study of the canine zona pellucida a heterogeneous ultrastructure and barrier

Date of Award

2011

Degree Name

Ph.D. in Biology

Department

Department of Biology

Advisor/Chair

Advisor: Shirley J. Wright

Abstract

The zona pellucida (ZP) which is an extracellular matrix surrounding the oocyte is the initial site of sperm-oocyte binding and is necessary for fertilization and preimplantation development. In all mammals studied, the ZP is punctuated by small fenestrations or pores. In the majority of mammals the ZP has a heterogeneous ultrastructure, with at least one smooth and/or compact morphology and at least one spongy less compact morphology. Few studies have analyzed the ZP in the canine. We investigated the canine ZP to determine if it had pores and a heterogeneous structure. The focus of our study had four objectives. The first objective was to develop and optimize a process to view the surface of the canine ZP using a scanning electron microscope. This was established by concurrent fixation and affixation of oocytes to a glass surface, and viewing gold-coated specimens under high vacuum with a low spot size. The second objective was to analyze the microarchitecture of the canine ZP. Over 400 zona-intact oocytes were isolated from ovaries, denuded of cumulus cells, fixed, critical point dried and viewed at 800 -- 35,000x with scanning electron microscopy. Four different ZP types were observed. The type I ZP had a smooth compact surface with only a few small pores (<0.5 μm). The type II ZP had many elliptical or spherical pores that were often conical and/or bifurcated going deeper into the ZP. The type III ZP had a rough surface with pores that were less spherical and more irregular in shape, but which still could be conical and/or bifurcated. The type IV ZP had a rough surface, but was covered in small fibers that clogged the pores making them hard to discern. The third objective was to determine what factors influence ZP ultrastructure. Dog donor characteristics (breed and size) did not affect morphology since most dogs had oocytes with two or three of the ZP types and one dog had all four ZP morphologies. Observation of living and dead oocytes (determined by oolema integrity) and oocytes from high and low quality cumulus-oocyte complexes revealed that the type I ZP and the type II ZP surrounded low quality oocytes and that the type III and IV ZP surrounded high quality oocytes. The final objective was to test the barrier function of the ZP to determine if the pores in the canine ZP would allow fluorescent microspheres (fluospheres) in the size range of nanoparticles and canine viruses to pass to the oolema. All three sizes of fluospheres (24, 100 and 210 nm) were able to traverse the canine ZP in a size-dependent manner in some oocytes and were located in the ooplasm. This research can aid in future studies working on optimizing in vitro maturation techniques and it can be applied to endangered canids. Also it brings to attention that scientists need to be aware of nano-sized particles and viruses because they are able to traverse the ZP and possibly enter the ooplasm and cause detrimental effects to canine oocytes.

Keywords

Zona pellucida Structure Research, Ovum Structure Research, Dogs Reproduction Research

Rights Statement

Copyright © 2011, author

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