Effect of ovarian stimulation on oocyte gene expression in cattle

Abstract

The objective was to analyze the impact of follicle stimulating hormone (FSH, ovarian stimulation) on the transcriptome of in vivo bovine oocytes three times around the luteinizing hormone (LH) surge. In vivo bovine oocytes were collected 2 h pre-LH surge, 6 h post-LH surge, and 22 h post-LH surge in both naturally ovulating and superovulated animals. To assess potential changes in gene levels, samples were hybridized using a custom bovine microarray. Two series of hybridizations were performed: the first comparing natural vs. stimulated cycles, the second according to time of collection. Among the potential candidates, 13 genes were selected according to their degree of differential expression and their potential link to oocyte competence. Measurements of their relative mRNA levels was made using QPCR. Gene candidates BTG4 (P = 0.0006), PTTG1 (P = 0.0027), PAPOLA (P = 0.0245), and LEO1 (P = 0.0393) had higher mRNA levels in oocytes treated with FSH for all collection times when compared to oocytes produced through the natural cycle. Among our selected candidates, only one gene, GDF9 (P = 0.0261), was present at a higher level in oocytes collected at -2 h and 6 h than 22 h post-LH for all treatments, regardless of the presence of FSH. Although the number of genes influenced by ovarian stimulation seemed low, the observed differences occurred at a time of minimal transcriptional activity and supported the potential impact on the future embryo. These impacts could have been epigenetic in nature, as embryo quality was not reported to be different from stimulated animals.

Introduction

Superovulation increases the number of offspring from genetically valuable cows and decreases the cost of embryo transfer by increasing embryo collection rates. Superovulation is also critical for other types of assisted reproduction technology in cattle [1], sheep [2], rabbits [3], monkeys [4], and mice [5]. However, the yield and quality of the embryos obtained after superovulation are variable and unpredictable, because of variations in ovarian response, fertilization rate, and embryo development [6]. Other factors, such as genetic background, age [7], breed [8], and reproductive and lactation status, may contribute to this variability. Perhaps some follicles in which growth is stimulated by the gonadotrophin contained rescued atretic follicles that remained aberrant. In addition, the shorter interval between luteolysis and the LH surge in gonadotrophin-stimulated compared to unstimulated cows could also be involved [9]. Variability in the FSH: LH ratios of gonadotrophin preparations is also thought to affect the superovulatory response.

Cytologic characteristics of oocytes recovered from cattle after superovulation were reported and no adverse effect on the pattern of nuclear maturation was detected. However, in subsequent reports, one third of oocytes aspirated from superovulated cattle during the preovulatory period had abnormal nuclear maturation [10]. In other species, aberrant DNA methylation of imprinted loci was detected in oocytes collected from superovulated women and mice [11], [12]. Regardless, in many studies, superovulation had no significant effect, other than a small increase in the number of transferable embryos [13]. Moreover, treatment with FSH provided good quality embryos in sheep [2], a higher percentage of embryos in rabbits [3], as well as a higher developmental potential for monkey embryos (through a 300 UI rhFSH regimen [4]). In the present study, cDNA microarrays and QPCR were used to determine the impact of hormonal stimulation on the bovine oocyte transcriptome or mRNA reserve.