Regulation of Cumulus Cell Numbers in the Course of the Canine Estrous Cycle

Abstract

In the canine, success rates of assisted reproduction techniques are very low. Since cumulus cells are known to play an essential role during the in vitro maturation (IVM) of oocytes, the aim of this study was to investigate the regulation of cumulus cell numbers in the course of the canine estrous cycle. Eighty‐two cumulus‐oocyte‐complexes (COCs) were obtained from 16 bitches undergoing elective ovariectomy during proestrus, estrus, metestrus and anestrus. First, mean cumulus cell numbers during the different stages of the cycle were compared. Then the incidence of programmed cell death (apoptosis) was detected by terminal deoxynucleotidyl transferase mediated dUTP labelling (TUNEL). Scanning electron microscopy (SEM) was used to confirm the presence of apoptotic cumulus cells. Additionally, mitosis was detected by using antibodies directed against the nuclear cell proliferation‐associated antigen Ki‐67. The mean number of cumulus cells in the COCs increased from proestrus to estrus (168.2 ± 9.2 to 265.5 ± 32.7, P < 0.01) and decreased from estrus to metestrus (265.5 ± 32.7 to 139.1 ± 10.7, P < 0.01) till anestrus (150.0 ± 8.3, P < 0.01). The percentage of apoptotic cumulus cells was generally very low during proestrus and oestrus (3.3% and 12.6%, P < 0.01) and distinctly increased during metestrus (83.1 %, P < 0.001). The predominance of apoptosis in the cumulus cells during metestrus was confirmed by SEM, which showed a high number of cells revealing cell shrinkage, loss of membrane integrity and cell disruption. Mitosis was highest during proestrus (2.7%) and oestrus (2.6 %) and decreased in the course of metestrus (1.8%) and anestrus (0.2%, P < 0.05).Our results imply that the distinct increase of cumulus cell numbers during proestrus and oestrus is due to increased mitosis and reduced apoptosis. The distinct reduction of cumulus cells during metestrus is predominantly regulated by apoptosis.