171 EXPRESSION OF THE OVULATION-INDUCING FACTOR/NERVE GROWTH FACTOR HIGH AFFINITY RECEPTOR IN THE OVARIES OF COWS DURING THE PERIOVULATORY PERIOD

Abstract

Ovulation-inducing factor/nerve growth factor (OIF/NGF) influences ovulation and corpus luteum (CL) size and function in camelids and, remarkably, cows. The ovulation effect in induced ovulators is mediated at the hypothalamo-pituitary-axis, but the site and mechanism of action of the luteotrophic effect is unknown. This mechanism may be an important aspect of OIF/NGF in spontaneous-ovulators. The objective of this experiment was to detect changes in expression of the high affinity OIF/NGF receptor (TrkA) in the ovary during the periovulatory period in cattle. Cows (n = 14) were examined daily by transrectal ultrasonography to determine the day of ovulation (Day 0), and were assigned randomly to be unilaterally ovariectomized on Day 2, 4, 6, or in the preovulatory period before or after the LH surge. Cows assigned to preovulatory groups were given a luteolytic dose of prostaglandin when the dominant follicle of the second follicular wave was ≥10 mm and growing. Cows assigned to the pre-LH group were ovariectomized 24 h after prostaglandin treatment. Cows assigned to the post-LH group were given LH 24 h after prostaglandin treatment and were ovariectomized 18–20 h later. Cows were allowed to rest for one complete interovulatory interval and re-assigned to a different day-group on which the remaining ovary was removed (n = 4 to 5 ovaries/day-group). Ovaries were fixed in paraformaldehyde, and 5-μm sections of ovarian tissue representing the dominant follicle, largest subordinate follicle, and the CL were treated for enzymatic antigen retrieval and blocked in 1% BSA for 1 h. Slides were incubated overnight with the primary antibody (rabbit anti-human TrkA) and for 2 h with the secondary antibody (goat anti-rabbit IgG). Slides were evaluated by fluorescence microscopy. Images of 3 arbitrarily chosen fields (40×) were captured for each structure for each day-group, and the average proportion of immunoreactive cells in the theca layer and luteal tissue was estimated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The intensity of individual cell immunofluorescence was also scored as no reaction (0), faint (1), weak to moderate (2), or strong (3). Data were compared among groups by two-way ANOVA. The proportion of immunoreactive cells was higher in the dominant follicle than in the subordinate follicle or the CL (P < 0.001); no effect of day-group or interaction was detected. The intensity score increased from faint on Day 2 to strong on Day 6, and remained strong in the pre- and post-LH groups. A discernible pattern of change in intensity score was not evident for the subordinate follicle or the CL. The greater proportion of TrkA positive cells and greater immunoreactive intensity in the maturing dominant follicle support the hypothesis that the luteotrophic effect of OIF/NGF in cattle is a result of a local increase in the expression of TrkA in the theca layer of the dominant follicle.

Research was supported by the Natural Sciences and Engineering Research Council of Canada.