The effect of aging on gene expression and mitochondrial DNA in the equine oocyte and follicle
Date
2009
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Abstract
The decline in fertility of aged mares is linked to declining oocyte quality. Oocyte viability is dependant on the ability of oocytes to remain in meiotic arrest until the initiation of maturation and adequate cumulus communication. We hypothesize that aging is associated with quantitative and temporal differences in meiotic arrest and resumption in oocytes, decreased oocyte secretion of paracrine factors and lower mitochondrial numbers, ultimately resulting in a dissociation of oocyte and follicular maturation. The objectives of this study were to clone and determine quantitative and temporal differences in mRNA content of the LH receptor (LHR), amphiregulin (AREG) and epiregulin (EREG) in granulosa cells; PDE4 in cumulus cells; and PDE3A, GPR3, GDF9, BMP15, and mitochondrial DNA (mtDNA) in oocytes during in vivo maturation in young (3-12 yr) and old (>20 yr) mares. Oocytes and follicular cells were collected by transvaginal follicular aspiration. Follicle maturation was induced in estrous mares with a follicle >30 mm by injection of 750 µg of recombinant equine LH. Aspirations were conducted at 0, 6, 9, and 12 h after LH administration. Total RNA was isolated from single denuded oocytes and associated lysed cumulus and granulosa cells. For each gene, mean mRNA copy number for each time point and age group were compared by ANOVA and Fisher's LSD. Regression coefficients were generated to compare oocyte mitochondrial numbers and correlations between gene expression within age groups. Expression of LHR mRNA in granulosa cells was different (p<0.05) between age groups. Young mares displayed a significant drop in LHR mRNA between 0 h and 6, 9, and 12 h; while the pattern of expression in old mares was similar (p>0.05) among times and higher (p<0.05) at 6 h than in young mares. Expression of AREG mRNA in granulosa cells peaked (p<0.05) at 9 h; however, the magnitude of expression at 6 and 9 h was higher (p<0.05) in old than young mares. Similarly, EREG expression peaked (p<0.05) at 9 h in young and old mares but was higher (p<0.05) for old mares. Expression of PDE4D peaked (p<0.05) at 6 and 12 h in old and young mares, respectively. The patterns of expression of GPR3 for oocytes of young and old mares were different and peaked (p<0.05) at 9 and 12 h, respectively. Magnitude of expression of PDE3A for oocytes of old mares at 6 and 9 h was higher (p<0.05) than in young mares. Expression of GDF9 and BMP15 was different (p<0.05) between ages. Mean expression of both genes in the old group was similar over time; however, in young mare oocytes maximum expression was at 6 h (p<0.05). Correlation coefficients between GDF9 and BMP15 for old and young mares were 0.94 and 0.99, respectively. Numbers of copies of oocyte mtDNA did not vary in young mares; however, there was a temporal decrease (p<0.05) of oocyte mitochondrial copy numbers in old mares. The main effect for age for mtDNA was similar for old and young mares.
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Subject
aging
equine fertility
meiotic arrest
oocyte quality
mtDNA
animal sciences