The growth and atresia in equine follicles has received little attention although the mare is a
unique model because of the size of the animal, the size of the ovarian follicles and the
unusual ovarian anatomy. The size of the animal allows ultrasound scanning to monitor
follicle dynamics in vivo and the size of each follicle allows for several parameters to be
studied without having to pool follicles together and thereby potentially introducing errors.
Several studies in the equine species have focused on follicle dynamics, others on atresia and
follicular fluid hormone levels and yet others on the oocyte, but none have combined all of
these areas. The aims of this thesis were to establish whether granulosa cells of regressing
equine follicles degenerate via an apoptotic mechanism and whether the presence of
apoptotic cell death in granulosa cells was correlated with other indices of follicle health
such as steroid hormone levels and oocyte quality.
At first each parameter was investigated in detail at a microscopic level: oocyte chromatin
configuration, cumulus morphology, follicle wall atresia, granulosa cell apoptosis and steroid
content in follicular fluid. Then followed a whole animal approach with monitoring of
follicular growth via ultrasonography. In the end this information was put together when
studying ovaries recovered at different stages of the cycle in order to reveal when selection
took place and how this was reflected in the parameters of the individual follicle.
The temperature at which the ovaries were held did not significantly affect (P > 0.05)
chromatin configuration in oocytes (n = 73), but the length of time at which the ovaries were
held, did significantly change chromatin configurations (n = 222) after 6 hours (P < 0.001).
Health status of the follicle appeared to affect chromatin configuration (n = 118), but this
failed to reach significance (P = 0.08). Oocyte chromatin configuration tended to change
from even distribution within the germinal vesicle, through different degrees of
condensation, to metaphase stages with increasing apoptosis or atresia of the follicle. The
puzzling configuration condensed chromatin may be part of the normal development but
may also be an end-stage chromatin configuration as it tended to be most frequent in atretic
follicles and increased in frequency after long storage in the ovary. The relationship between
cumulus morphology and chromatin configuration (n = 149) was evident: compact cumulus
morphology was associated with immature oocyte chromatin configurations and fully
expanded cumuli were correlated with metaphase chromatin (P < 0.001). Each sequential
step of cumulus expansion represented a further step towards meiotic maturation.
Evaluation of follicle health based on granulosa cell morphology immediately upon retrieval,
follicular wall atresia and apoptosis showed that each of the methods to detect atresia and
apoptosis had advantages and limitations and that it was necessary to use the methods in
conjunction because they supplemented each other. Histological atresia and ethidium
bromide detection of apoptosis (n = 20) was significantly correlated (P < 0.001), and the
stereomicroscopic evaluation of freshly isolated granulosa cells were significantly correlated
(P < 0.001) with the histological findings (n = 33). Apoptotic granulosa cells and theca cells
were detected histologically, a phenomenon which has not previously been described in
equine follicles.
In a study with 293 follicles from 23 mares, it was found that apoptosis was not a definite
measure of death, because growing follicles, as determined by ultrasonography, could be
apoptotic and have no other signs of impending death of the follicle, although apoptosis
generally was associated with atresia of the follicle (P < 0.001). There was a population of
small, atretic follicles with expanded cumuli and oocytes with chromatin progressed towards
meiosis, and a population of large, healthy follicles, with young oocyte configurations and
no cumulus expansion, and finally a population of follicles larger than 30 mm, which were
healthy, but had started to move towards maturation in vivo, i.e. getting ready for ovulation.
Size of the follicle appeared to be more important than stage of cycle for predicting
chromatin configuration, cumulus morphology and atresia and apoptosis, but size of follicle
was correlated with stage of cycle. This led to the theory that despite the larger follicles
having stopped growing according to the follicle maps made from ultrasound scans,
regression may not initially be related to detectable apoptosis, and that overt apoptosis may
be a later event. The fact that most atretic follicles were small suggested that follicles
regressed and slowly became overtly atretic and contained low levels of oestradiol and
degenerate oocytes surrounded by expanded cumuli.
When used in combination with plasma hormone levels, daily mapping of follicles in 7
ponies and 5 mares throughout the cycle made it possible to detect days of changes in
follicular growth both of the individual large follicle and smaller follicles in groups in
relation to hormone changes. The growth of the largest follicle to a point where it was
capable of producing sufficient oestradiol, resulted in decreasing levels of FSH. The
IX
decreasing levels of FSH caused the smaller follicles to regress. The increased oestradiol
concentrations promoted increased LH production as well as partaking in luteolysis, with
resulting further increased levels of LH due to lack of negative feed back from progesterone.
When the inhibiting effect of oestradiol disappeared just before ovulation, FSH started to
increase, resulting in growth of a new wave of follicles, emerging on day 6 (P < 0.01).
Divergence in growth rates between the dominant follicle and the largest subordinate follicle
happened on day 9 and this became significant on day 12 of the cycle (P < 0.05). At this
point FSH levels were high, LH was low and progesterone was still produced. The cause of
deviation in this study remains unknown. The subordinate follicles grew until day 17 - 18, at
which point they started to regress. The follicle maps were a reliable method of determining
growth and regression, when keeping in mind that there are influences from regressing,
atretic follicles during periods of general growth and that monitoring of the individual small
follicle is not possible when there are many follicles.
In conclusion this thesis demonstrated that oocyte chromatin configuration, cumulus
morphology, follicular fluid steroid concentration and granulosa cell health are parameters,
which change with follicle health, and that ultrasound scanning is an important and
necessary tool to use in conjunction with the above measurements when studying follicle
growth, selection and regression. Death of follicles happens gradually, and selection for
dominance is a gradual process, where one follicle gains advantage over the competitors,
resulting in dominance at an early stage during the cycle.
