Untersuchungen zur Oozytenreifung beim Hund

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.