Molecular signatures and cellular differences in granulosa cells from fertile and subfertile beef heifers
Type of DegreeMaster's Thesis
Restriction TypeAuburn University Users
MetadataShow full item record
Subfertility is a leading contributor to economic losses in the cow-calf sector of the beef cattle industry. Cow-calf producers invest significant time and financial resources in the selection and development of replacement heifers. Even so, a population of subfertile heifers remains unexplained by traditional phenotypic selection parameters such as body condition score, heifer age, and reproductive tract score. There are many well-known contributors to subfertility, including poor health status, immature reproductive tracts, and lack of estrous cyclicity. Some factors affecting fertility are challenging to identify, such as hypothalamic-pituitary-gonadal axis dysregulation, insufficient uterine receptivity, and poor ovarian function. Ovarian function and follicular dynamics are factors of the reproductive system which contribute substantially to fertility status. Granulosa cells within the follicle participate in highly coordinated biological processes, including proliferation and apoptosis, during follicular development, and they play a vital role in oocyte health status. However, not much information on cellular function and molecular signatures is available in the granulosa cells of beef heifers with differing fertility status. Therefore, in this study, granulosa cells were used to explore cellular function through proliferation and apoptosis assays and to identify molecular signatures through transcriptomic analysis between heifers of differing fertility status. To attain this information, Angus-Simmental crossbred heifers (n = 13) underwent an estrus synchronization (ES) and artificial insemination (AI) protocol (7-Day CO-synch + CIDR). Pregnancy determination was performed via ultrasound and rectal palpation by a veterinarian. Heifers bred to AI were classified as fertile (n = 8), and pregnancies were terminated to allow the resumption of normal estrous cyclicity. Heifers that remained non-pregnant following AI were exposed to a bull of proven fertility for 61 days. Heifers that remained non-pregnant after both AI and bull exposure were classified as subfertile (n = 5). Ovaries were collected, and follicles were aspirated from fertile and subfertile heifers at the time of harvest. Granulosa cells were then isolated from the follicular fluid. Granulosa cells were cultured and underwent 5-ethynyl-2'-deoxyuridine (EdU) and Terminal Deoxynucleotidyl Transferase-dUTP Nick End Labeling (TUNEL) assays to evaluate proliferation and apoptosis rates, respectively. No significant differences in granulosa cell proliferation or apoptosis rates were identified between fertile and subfertile heifers through the respective assays. Granulosa cells were further subjected to transcriptomic profiling through RNA sequencing. For transcriptomic analysis, total RNA was isolated from granulosa cells, quality checked, and subjected to the library preparation and sequencing on the Nova-Seq platform. Seventy-nine significantly differentially expressed genes (DEGs) were identified through the application of DESeq2 with p-values < 0.05 and log2 fold change > 0.2, including DNER, CALCRL, CXCL12, GADD45G, and PLCE1. DEGs were overrepresented in networks associated with the mitogen-activated protein kinase (MAPK) pathway, serine/threonine protein kinase pathway, major histocompatibility complex (MHC) binding pathway, adaptive thermogenesis, and plasma membrane signaling receptor complex. The overrepresented DEGs for these pathways overlap and create opportunity for further functional analysis of the effects of the differential expression of genes of interest. Five DEGs were selected for validation through RT-qPCR. CXCL12 was significantly upregulated in granulosa cells from subfertile heifers, confirming the results seen in the transcriptomic analysis. By further exploring molecular mechanisms and cellular functions of the differential expression of genes and cellular behavior of granulosa cells contributing to oocyte health and differing fertility status, factors underlying unexplained heifer subfertility can be further elucidated and utilized to improve our understanding of heifer fertility.