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Wallner, Sandra Elisabeth (2007): Untersuchungen zur Oozytenreifung beim Hund. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

The objective of this work was to examine follicular and oocyte growth in canine ovaries with light and electron microscopic techniques and to characterize canine oocytes during in vitro maturation. Ovaries of healthy bitches of different ages (4 months to 12.5 years)and breeds were used, which had undergone elective ovariohysterectomy at local veterinary clinics. The ovaries of 15 bitches were fixed in Bouin`s solution or paraformaldehyde (4%) for immunohistochemical studies and of three bitches in Karnovsky`s solution for electron microscopic evaluation. COCs and oocytes were recovered from 61 other bitches by slicing the ovaries. They were then examined before and after in vitro maturation (24 to 72 hours) in modified TCM-199 either by native evaluation or after fixation in paraformaldehyde (4%) and nuclear staining (propidium iodide/Hoechst 33342), immunofluorescence or glycohistochemistry. The evaluation of the fluorescence microscopic staining was performed by confocal laser scanning microscopy. Oocytes and COCs after 0, 24, 72 and 90 hours of in vitro maturation were also subjected to electron microscopic examination. The morphology of the canine ovary in light and electron microscopic aspects is comparable to that of other domestic animals. Primordial, primary, secondary and tertiary follicles were regularly seen. The diameter of the oocytes and of the germinal vesicle, as well as the thickness of the zona pellucida, clearly increases during oocyte development. Growing canine oocytes are characterized ultrastructurally by rapid growth in the number of cellular organelles, particularly mitochondria, smooth endoplasmatic reticulum and lipid droplets. Mitotic division starting at the primary follicle stage can be regularly observed by immunostaining with the proliferation marker Ki-67. Further immunohistochemical studies on ovaries indicate that estrogen receptors alpha and beta, as well as MMP-1, -2, -14 and TIMP-2 show a specific distribution in bitches. Canine oocytes could easily be isolated by slicing the ovaries. The number of recovered oocytes was clearly influenced by the age of the donor bitch but not by the breed, the reproductive status or the transportation time between time of surgery in the veterinary clinic and the recovery of the oocytes in the laboratory. 48% of all isolated oocytes had a dark homogenous ooplasm and multiple dense layers of cumulus cells. After in vitro maturation, morphological changes like the formation of vesicles and the loss of cumulus cells could be observed in most of the COCs. Immediately after recovery, the nuclei of all oocytes were at the germinal vesicle stage, although the chromatin showed different degrees of condensation. While first signs of the resumption of meiosis were seen after 24 hours of culture, only one oocyte in metaphase II could be seen after 72 hours. Nuclear and cytoplasmatic maturation could be detected by electron microscopy for up to 24 hours of in vitro culture, as well as signs of degeneration, which were even more prominent after longer culture periods. The immunoreaction of ZP3beta, alpha-Tubulin and Connexin 43 and the binding sites of the lectins WGA and SBA showed characteristic changes in canine oocytes and COCs before and after in vitro maturation.