Effect of preovulatory follicle size on cumulus cell and follicular wall transcript abundance in beef cows

Abstract

Gonadotropin releasing hormone (GnRH)-induced ovulation of small dominant follicles decreased pregnancy rates and increased late embryonic/fetal mortality in postpartum beef cows, which could be caused by inadequate oocyte competence and(or) maternal environment. Previous studies revealed that dominant follicle size at GnRHinduced ovulation in beef cows may affect oocyte competence, as higher fertilization rates and higher embryo quality were achieved as dominant follicle diameter at insemination increased. In addition, higher pregnancy rates and increased concentrations of circulating preovulatory estradiol and postovulatory progesterone were observed in recipient cows induced to ovulate large compared to small dominant follicles, which may affect the establishment of pregnancy. The objectives of the current study were to determine the effects of preovulatory follicle size and physiological status on the cumulus cell transcriptome (Experiment 1) and determine the effects of preovulatory follicle size and steroidogenic capacity (i.e. concentrations of circulating estradiol) on the dominant follicle wall transcriptome collected before the preovulatory gonadotropin surge (Experiment 2). In Experiment 1, ovulation was synchronized in suckled beef cows by administering an injection of GnRH on d-9, prostaglandin F2a (PG) on d-2, and a second injection of GnRH on d0. The dominant follicle was trans-vaginally aspirated on d1 and cumulus-oocyte complexes (COC) were collected. The cumulus cells were removed from the oocytes and assigned to one of the following follicle classifications based on follicle diameter and estrus expression: small (n=6; less than 11.7mm; no estrus expression), large (n=6; greater than 12.5 mm; no estrus expression), or spontaneous (n=5; 11.6-13.9 mm; estrus expression and endogenous gonadotropin surge). RNA was extracted from cumulus cells collected from pools of four oocytes and sequenced before being aligned to the Bos taurus genome (UMD3.1). When comparing the cumulus cell transcriptome of small versus large preovulatory follicles, 430 transcripts were more abundant at a false discovery rate (FDR) less than 0.10 in small follicles and 454 were more abundant in large follicles. The glycolytic pathway was enriched in the cumulus cells of large follicles compared to small follicles. In the small versus spontaneous preovulatory follicle comparison, 597 transcripts were more abundant in small follicles and 1012 transcripts were more abundant in spontaneous follicles. The steroid biosynthesis pathway (i.e. cholesterol synthesis) was enriched in the cumulus cells of spontaneous compared to small follicles. In the large versus spontaneous preovulatory follicle comparison, 541 transcripts were more abundant in cumulus cells from large follicles and 951 were more abundant in cumulus cells from spontaneous follicles. In summary, a greater abundance of transcripts encoding for members of the glycolytic pathway in large follicles and transcripts encoding for members of the steroid biosynthesis pathway in the cumulus cells of spontaneous compared to small follicles indicate that oocytes from small follicles may be less competent. Preovulatory follicle development was synchronized in Experiment 2 by administering GnRH on d-9 and PG on d-2 to non-lactating beef cows. The ovaries were harvested at slaughter 48 hr after PG (d0) and the dominant follicle collected. RNA was extracted from approximately half of the follicle wall and sequenced. Cows were divided into two classifications based on dominant follicle diameter at collection: small (n=4; less than 11.5 mm) and large (n=7; greater than 12.5 mm). Nine transcripts were more abundant in small follicles, and two transcripts were more abundant in large follicles. No significant pathways were found. The same 11 follicle walls were also divided into two classifications based on concentration of serum estradiol at 48 hr after PG: low (n=6; less than 4.0 pg/ml) and high (n=5; greater than or equal to 4.0 pg/ml). In the low estradiol classification, 281 transcripts were more abundant in the follicle wall and 40 were more abundant in the follicle wall of the high estradiol classification. No significant pathways were found. Transcripts were analyzed with PANTHER to find significant gene ontology (GO) terms. Differentially abundant transcripts in low estradiol follicles were more highly associated with mitosis, chromosome segregation, and regulation of biological processes. In summary, a small number of transcripts were differentially abundant in the follicle wall of small versus large dominant follicles prior to the preovulatory gonadotropin surge and no specific pathways were identified that might provide insight into how the physiological maturity of a dominant follicle can affect pregnancy rate. Comparisons made between follicle walls from follicles with low or high serum estradiol found a larger number of transcripts were more highly abundant in the low estradiol follicles, possibly indicating that a higher level of transcription is taking place in the low estradiol follicles, and therefore are less mature.