Reproductive Competency and Mitochondrial Variation in Aged Syrian Hamster Oocytes

The hamster is a useful model of human reproductive biology because its oocytes are similar to those in humans in terms of size and structural stability. In the present study we evaluated fecundity rate, ovarian follicular numbers, ova production, mitochondrial number, structure and function, and cytoplasmic lamellae (CL) in young (2–4 months) and old (12–18 months) Syrian hamsters (Mesocricetus auratus). Young hamsters had higher fertilisation rates and larger litters than old hamsters (100 vs 50% and 9.3 +/- 0.6 vs 5.5 +/- 0.6, respectively). Ovarian tissue from superovulated animals showed a 46% decrease in preantral follicles in old versus young hamsters. There was a 39% reduction in MII oocyte number in old versus young hamsters. Young ova had no collapsed CL, whereas old ova were replete with areas of collapsed, non-luminal CL. Eighty-nine per cent of young ova were expanded against the zona pellucida with a clear indentation at the polar body, compared with 58.64% for old ova; the remaining old ova had increased perivitelline space with no polar body indentation. Higher reactive oxygen species levels and lower mitochondrial membrane potentials were seen in ova from old versus young hamsters. A significant decrease in mitochondrial number (36%) and lower frequency of clear mitochondria (31%) were observed in MII oocytes from old versus young hamster. In conclusion, the results of the present study support the theory of oocyte depletion during mammalian aging, and suggest that morphological changes of mitochondria and CL in oocytes may be contributing factors in the age-related decline in fertility rates

Age-Associated Metabolic and Morphologic Changes in Mitochondria of Individual Mouse and Hamster Oocytes

Background: In human oocytes, as in other mammalian ova, there is a significant variation in the pregnancy potential, with approximately 20% of oocyte-sperm meetings resulting in pregnancies. This frequency of successful fertilization decreases as the oocytes age. This low proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. In this study, we have examined mitochondrial biogenesis in both hamster (Mesocricetus auratus) and mouse (Mus musculus) ova as models for understanding the effects of aging on mitochondrial structure and energy production within the mammalian oocyte. Methodology/Principal Findings: Individual metaphase II oocytes from a total of 25 young and old mice and hamsters were collected from ovarian follicles after hormone stimulation and prepared for biochemical or structural analysis. Adenosine triphosphate levels and mitochondrial DNA number were determined within individual oocytes from young and old animals. In aged hamsters, oocyte adenosine triphosphate levels and mitochondrial DNA molecules were reduced 35.4% and 51.8%, respectively. Reductions of 38.4% and 44% in adenosine triphosphate and mitochondrial genomes, respectively, were also seen in aged mouse oocytes. Transmission electron microscopic (TEM) analysis showed that aged rodent oocytes had significant alterations in mitochondrial and cytoplasmic lamellae structure. Conclusions/Significance: In both mice and hamsters, decreased adenosine triphosphate in aged oocytes is correlated with a similar decrease in mtDNA molecules and number of mitochondria. Mitochondria in mice and hamsters undergo significant morphological change with aging including mitochondrial vacuolization, cristae alterations, and changes in cytoplasmic lamellae

Age-associated metabolic and morphologic changes in mitochondria of individual mouse and hamster oocytes.

BACKGROUND: In human oocytes, as in other mammalian ova, there is a significant variation in the pregnancy potential, with approximately 20% of oocyte-sperm meetings resulting in pregnancies. This frequency of successful fertilization decreases as the oocytes age. This low proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. In this study, we have examined mitochondrial biogenesis in both hamster (Mesocricetus auratus ) and mouse (Mus musculus) ova as models for understanding the effects of aging on mitochondrial structure and energy production within the mammalian oocyte. METHODOLOGY/PRINCIPAL FINDINGS: Individual metaphase II oocytes from a total of 25 young and old mice and hamsters were collected from ovarian follicles after hormone stimulation and prepared for biochemical or structural analysis. Adenosine triphosphate levels and mitochondrial DNA number were determined within individual oocytes from young and old animals. In aged hamsters, oocyte adenosine triphosphate levels and mitochondrial DNA molecules were reduced 35.4% and 51.8%, respectively. Reductions of 38.4% and 44% in adenosine triphosphate and mitochondrial genomes, respectively, were also seen in aged mouse oocytes. Transmission electron microscopic (TEM) analysis showed that aged rodent oocytes had significant alterations in mitochondrial and cytoplasmic lamellae structure. CONCLUSIONS/SIGNIFICANCE: In both mice and hamsters, decreased adenosine triphosphate in aged oocytes is correlated with a similar decrease in mtDNA molecules and number of mitochondria. Mitochondria in mice and hamsters undergo significant morphological change with aging including mitochondrial vacuolization, cristae alterations, and changes in cytoplasmic lamellae

Measurement of ATP levels (fmoles) in individual mouse (A) and hamster (B) oocytes.

<p>Note that there is a 38.4% decrease in ATP levels in old mice vs. young mice (<i>P</i><0.0001) and a 35.4% drop in ATP in old vs. young hamster oocytes. (<i>P</i> = 0.002). Data are also presented as median with 10^th, 25^th, 75^th, and 90^th percentiles and outliers in whisker plot inserts. Statistical analysis was performed by Student T-test and a <i>P</i> value less than 0.05 was considered significantly different between the groups.</p

Ultrastructure of mouse MII oocytes.

<p>A and B are representative TEM micrographs at 3,000X magnification. Note the mitochondria (M) and lipid droplets (LD). C and D are representative TEM micrographs from young (C) and old (D) mice at 12,000X magnification. Note the normal (Mn) and vacuolated (Mv).mitochondria (Bar: A&B, 2 µm; C&D, 500 nm).</p

Ultrastructure of hamster MII oocytes.

<p>A and B are representative TEM micrographs at 2,000X magnification, showing various cytoplasmic lamellae (CL) and numerous mitochondria (M) in this oocyte from a young animal (A) compared to the number in an oocyte from an old hamster (B). C and D are representative TEM micrographs of oocytes from young (C) and old (D) hamsters at 10,000X magnification. Note the dark mitochondria (Md) and mitochondria with clear cristae (Mc). The old ova also showed the presence of collapsed cytoplasmic lamellae (cCL). (Bar: A&B, 2 µm; C&D, 500 nm).</p

Correlation of ATP level and mtDNA copy numbers within single oocytes.

<p>Left panel (A), mouse oocytes and right panel (B), hamster oocytes. Statistical analysis was done by the Pearson correlation test.</p

Real-time PCR analysis of mtDNA copy numbers in individual mouse (A) and hamster (B) oocytes.

<p>mtDNA copy numbers were significantly lower in old oocytes compared to young oocytes in both mice and hamsters. There is a 44% decrease in mtDNA number in aged mouse oocytes and a 51.8% decrease in aged hamster oocytes. Data were also presented as median with 10^th, 25^th, 75^th, and 90^th percentiles and outliers shown in whisker plot inserts.</p