No abstract

Multioocyte follicles (MOF) are follicles that enclose two or more oocytes. They have been described in many mammalian species, but there is no evidence about their activity in the ovaries. The aim was to estimate the prevalence of MOF and to compare the cell proliferation activity between follicles containing one or more oocytes in the ovaries of prepubertal and adult bitches. Eighty ovaries from prepubertal (n = 20) and adult bitches (n = 20) were obtained by elective ovariohysterectomy (OHE). Immediately after OHE, ovaries were immersed in Bouin's fixative for histological processing. 5 µm thick sections were mounted on histological slides and stained with periodic acid-Schiff (PAS) and hematoxylin. Cell proliferation was evaluated by immunohistochemistry using the proliferating cell nuclear antigen (PCNA). Monoclonal antibody PCNA (clone PC1O, 1 : 200 dilution, Biocare, Concord, CA, USA) was used according to manufacturer's instructions and an antibody diluent was used as a negative control. Slides were counterstained with hematoxylin and examined at 200× to 400× magnification under light microscope. Only cells showing PCNA signal exclusively in the nucleus were considered positive. The prevalence of MOF in the ovaries was compared using a Fisher's exact test (P < 0.05). In all females, the prevalence of MOF was 55% (22/40). MOF containing two or three oocytes were more abundant; however, multioocyte follicles with up to 12 oocytes were observed. The prevalence of MOF at the primordial stage was higher for prepubertal bitches (47 v. 28%) but adult bitches exhibited a higher frequency of secondary MOF (49 v. 25%; P < 0.05). There was no difference in the prevalence of MOF at primary stage between prepubertal and adult bitches (28 v. 23%; P > 0.05). Regarding the cell proliferation activity, PCNA immunoreactivity was detected in oocyte nucleus and granulosa cells of multioocyte follicles at different stages of development. Similarly to what was observed for follicles containing only one oocyte, all nuclei of oocytes within multioocyte follicles exhibited PCNA immunoreactivity and there was a gradual increasing of immunoreactivity in granulosa cells according to the stage of follicular growth. Expression of PCNA by granulosa cells of multioocyte follicles was higher in the secondary and antral stage of development; however, some primordial and primary follicles also exhibited some PCNA-positive cells. In conclusion, the prevalence of MOF at the primordial stage of development was higher in prepubertal bitches, whereas MOF at the secondary stage were more frequent in adult bitches. The PCNA expression pattern by the oocyte nucleus of multioocyte follicles was similar to that observed in follicles containing only one oocyte, which is suggestive of similar activity between these follicles. Furthermore, the presence of proliferative activity in granulosa cells of multioocyte follicles suggests an association of the PCNA expression with more advanced stages of follicular growth.