1 DIFFERENCES IN RESUMPTION OF OOCYTE MATURATION IN YOUNG AND OLD MARES

Abstract

The decline in fertility of aged mares is linked with declining oocyte quality. Oocyte viability is dependent on the ability of oocytes to remain in meiotic arrest until the initiation of maturation. We hypothesize that aging is associated with quantitative and temporal differences in meiotic arrest and resumption in oocytes, ultimately resulting in a dissociation of oocyte and follicular maturation. The objectives of this study were to determine temporal differences in the mRNA content of amphiregulin and epiregulin in granulosa cells; PDE4 in cumulus and granulosa cells; and PDE3A, GPR3, GDF9, and BMP15 in oocytes during in vivo maturation in young (3–12 years) v. old (>20 years) mares. Oocytes and follicular cells were collected by transvaginal follicular aspiration. Maturation was induced in estrous mares with a follicle >30 mm by injection of 750 ¼g of recombinant equine LH. Aspirations were attempted at 0, 6, 9, and 12 h after LH administration. Six oocytes and follicular cell samples from each age group and time point were collected and stored immediately after aspiration. Total RNA was isolated from single denuded oocytes and lysed cumulus and granulosa cells. A fraction of the total lysate was used to determine cell numbers from the DNA copy number of the equine CG² subunit gene. DsRED RNA was added to each RNA isolate to serve as an exogenous standard. Quantitative RT-PCR was performed from cDNA with equine primer pairs. Copy numbers were calculated with an intra assay standard curve of plasmid containing the specific gene and corrected with the exogenous DsRED RNA and cell number. For each gene, mean mRNA copy numbers for time points and age groups were compared by ANOVA and Tukey's HSD test. Expression of PDE4D in cumulus cells was similar between young and old mares and time points. However, PDE4D peaked (P < 0.05) at 6 h in granulosa cells from young, but not old mares. Amphiregulin expression in granulosa cells of young mares peaked (P < 0.05) at 9 h and did not increase in the old mares. Epiregulin expression in granulosa cells peaked (P < 0.05) at 9 h and 6 h in young and old mares, respectively. The pattern of expression of PDE3A for oocytes of young and old mares was similar with an increase (P < 0.05) at 9 h. There was an interaction (P < 0.05) in the expression of GPR3 for age × time. Expression peaked at 9 and 6 h in young and old mares, respectively. Pattern of expression of GDF9 was similar between young and old mares except for a decrease (P < 0.05) in expression in old mares at 9 h. There was an interaction (P < 0.05) in the expression of BMP15 for age × time. Expression in young mares peaked at 9 h while that in old mares peaked at 6 h and decreased at 9 h. These results suggest that key gene expression patterns involved in oocyte and follicular maturation cascades are asynchronous for young versus old mares and could explain some aspects of the age-associated decline in fertility.