Reduced antral follicle count in dairy cows of reproductive age is associated with a high incidence of aneuploidy and alterations in the localization of PGRMC1 and AURKB

Premature ovarian senescence as judged by a reduced number of antral follicles is a common cause of infertility in both woman and diary cattle. In addition, oocytes isolated from cattle with reduced numbers of antral follicles have an impaired capacity for embryonic development. The mechanism responsible for the reduced developmental competence of these oocytes may be related to alterations in the mechanism that directs the proper segregation of chromosomes. Interestingly, Progesterone Receptor Membrane Component 1 (PGRMC1) and the phosphorylated (active) form of Aurora Kinase B (AURKB) are known to co‐localize at the centromere of chromosomes in metaphase II oocytes. Moreover, alterations in localization and function of PGRMC1 during oocyte maturation may account for the reduced development competence, since injection of a PGRMC1 antibody impairs the ability of oocytes to successfully mature and causes abnormalities in chromosomal segregation. Therefore, the present study was designed to determine if the decrease in oocyte developmental competence observed in dairy cows with a reduced numbers of antral follicles is due to 1) a higher incidence of aneuploidy and 2) whether alterations in PGRMC1 contributes to the incidence of aneuploidy. For these studies, ovaries were obtained from the abattoir and oocytes collected from ovaries with reduced antral follicles counts (AFC; <10 follicles of 2‐6 mm) and aged‐matched controls (>10 follicles of 2‐6 mm). Oocytes were matured in vitro and the occurrence of aneuploidy determined. The level of mRNA and localization of PGRMC1 were also monitored using real‐time PCR and immunocytochemistry, respectively. All data were analyzed by either Fisher's exact test or Student t test when appropriate. Although oocytes from ovaries with reduced AFC were capable of undergoing meiosis in vitro, these oocytes showed a 3‐fold increase in aneuploidy compared to oocytes isolated from ovaries with the controls (60% vs 21%, P<0.05). Although PGRMC1 mRNA levels were not reduced in oocytes of ovaries with low AFC, immunolocalization studies revealed PGRMC1 failed to localize to precise focal points on metaphase II chromosomes as observed in oocytes of control ovaries (82% vs 44%, P<0.05). Similarly, AURKB failed to properly localize on the metaphase chromosomes of oocytes from ovaries with reduced AFC (70% vs 30%, P<0.05). Further, when oocytes of control ovaries were cultured with an inhibitor of AURKB activity (ZM447439), the ability of oocytes to mature was not affected (85% vs 95% control) but the chromosomal organization of the metaphase II plate was disrupted and PGRMC1 was not properly localized to the chromosomes (91% vs 45% control, P<0.05). Taken together, these results strengthen the hypothesis that PGRMC1‐AURKB interaction plays an essential role in the process of chromosome segregation during oocyte meiosis and that alterations in the localization of PGRMC1 and AURKB account in part for the increased aneuploidy and low development competence of oocytes of ovaries with reduced AFC. This work was supported by grants from OECD Co‐operative Research Programme: Biological Resource Management for Sustainable Agricultural Systems (Contract: JA00064361, AML); by Marie Curie Actions FP7‐Reintegration‐Grants within the 7th European Community Framework Programme (Contract: 303640,'Pro‐Ovum', VL) and by “Dote Ricercatori e Dote Ricerca Applicata”, FSE, Regione Lombardia, Italy (VL, IT).