Effects of excessive FSH dose on granulosa cell, cumulus cell and oocyte transcriptomes in small ovarian reserve heifers

To evaluate the mechanisms by which excessive FSH doses during ovarian stimulation are detrimental to ovarian follicle function and assisted reproduction outcomes, small ovarian reserve (SOR) heifers were subjected to ovarian stimulation with either a standard, 70 IU, or excessive, 210 IU, dose of commercial porcine FSH (cpFSH) preparation, per injection. The ovaries were recovered at the end of the ovarian stimulation protocol, consisting of eight cpFSH injections at 12 h intervals. In excessive dose treated heifers, heterogeneity was observed with regards to follicle phenotype including cumulus cell-oocyte complex (COC) morphology and intrafollicular steroid hormone milieu. In contrast, standard-dose treated heifers exhibited one predominant follicle phenotype which was selected as the control follicle type for all comparisons herein. RNA-seq analyses of granulosa, cumulus cells and the oocyte from individual follicles of these defined phenotypes was used to identify changes in the transcriptomes of these cell types in excessive dose treated individuals. RNA-sequencing of the oocyte, cumulus and granulosa cells collected from individual ovulatory-size (≥ 10 mm) follicles from the ovaries of small ovarian reserve (SOR) heifers subjected to an ovarian stimulation protocol with either 70 IU (n = 7 heifers) or 210 IU (n = 6 heifers) cpFSH per injection. Follicles were classified into follicle types and one follicle per follicle type per heifer was randomly selected and RNA-libraries prepared. Samples from Type 1 follicles were treated as controls for subsequent comparisons because these follicles displayed the published characteristics (e.g., estrogen-active with a compact COC) of the ovulatory follicle in cattle and the fact that these samples were collected from standard, 70 IU, cpFSH dose-treated heifers.