Abstract
The ability to breed from juvenile sheep and cattle can increase their lifetime productivity and the rate of genetic gain, and thus increase the profitability of the agricultural industry. Unfortunately, oocytes from prepubertal animals have limited potential to undergo normal embryogenesis and produce viable offspring, and little is known about the underlying causes of this reduced competence. The correct quantity, activity and cytoplasmic distribution of organelles are essential for oocyte maturation, fertilisation and subsequent embryo development. However, very few studies have attempted to quantify differences in the cytoplasm of prepubertal and adult oocytes to understand the underlying causes of the reduced quality. Therefore, the aims of this project were firstly; to determine if there were differences in the volumes, morphology or distribution of organelles between oocytes from prepubertal (lamb) and adult ewes both before and after in vitro maturation; and secondly, to determine if treatment of lamb oocytes in vitro with factors that may alter the volume or distribution of these organelles can improve embryo development.
Embryos were produced from oocytes from slaughterhouse derived ovaries using standard sheep in vitro embryo production methods to determine blastocyst rate. Oocytes were fixed and processed for electron microscopy and stereology techniques were used to measure the distribution, density and volumes of vesicles, mitochondria and lipid droplets in 12 oocytes per age and time point. The volume of vesicles before maturation was greater in adult ewe compared to lamb oocytes (P = 0.018), and decreased during maturation in both age groups. The volume of mitochondria in adult ewe oocytes increased during maturation (P = 0.005). This was due to an increase in mitochondrial number and was greater than the mitochondrial volume (P = 0.026) and number (P = 0.003) of the mature lamb oocytes, which did not change. There were also differences in the morphology of the mitochondria and the distribution of mitochondria and lipid droplets between adult and lamb oocytes.
Studies have attributed improved oocyte competence to an increased number of mitochondria and altered cytoplasmic mitochondrial distribution. However, estimates of mitochondrial number have mainly been based on mitochondrial DNA (mtDNA) copy number performed by qPCR methods while the actual number of copies of mtDNA per mitochondria is not known. Therefore, the current study compared the mtDNA copy number, measured by qPCR, with mitochondrial number estimated by stereology in oocytes from the same cohorts of adult ewes and lambs. Unlike the mitochondrial number estimated by stereology, the mean number of copies of mtDNA did not differ between the two ages either before or after in vitro maturation, but decreased in the lamb oocytes during maturation. These results indicate that mtDNA copy number does not represent actual mitochondrial organelle number.
L-carnitine has been shown to increase blastocyst rate and alter mitochondrial distribution when cultured with oocytes during in vitro maturation. It has also been shown to alter expression of mitochondrial biogenesis genes in other cell types. In the current study lamb oocytes were treated with acetyl-L-carnitine during in vitro maturation and this doubled the blastocyst rate compared to untreated oocytes (P = 0.028). Stereology results showed vesicle volume and size, and lipid droplet distribution appeared to be altered. There was no effect of L-carnitine on mitochondrial volume, number or distribution, or mtDNA copy number.
In conclusion the results of this research suggest a lower volume of storage vesicles prior to maturation and altered mitochondrial replication, distribution and morphology may underlie the reduced developmental competence of prepubertal lamb oocytes. Treatment with L-carnitine during in vitro maturation can also be used to improve in vitro embryo production methods for oocytes from juvenile lambs.