Identifying Gene Network Differences in Granulosa Cell RNA Between Bovine Pubertal Classifications with Altered Follicle Development

Authors

• Brooke Elizabeth Rudloff, University of Nebraska-LincolnFollow

First Advisor

Andrea S. Cupp

Committee Members

Jennifer Wood, John Davis

Date of this Version

5-2026

Document Type

Thesis

Citation

A thesis presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Master of Science

Major: Animal Science

Under the supervision of Professor Andrea S. Cupp

Lincoln, Nebraska, May 2026

Comments

Copyright 2026, Brooke Elizabeth Rudloff. Used by permission

Abstract

Our laboratory previously identified five pubertal classifications in heifers from the University of Nebraska–Lincoln Research Herd: Early (E), Typical (T), Start–Stop–Start (SSS), Start–Stop–Discontinuous (SSD), and Non‑Cycling (NC). These classifications reflect variation in the timing of pubertal onset and the ability to initiate and maintain estrous cyclicity. While individual classifications are moderately heritable (0.38), heritability increases substantially (0.59) when heifers that initiate and maintain cyclicity (E + T) compared with those that do not (NC + SSD). Non‑Cycling heifers exhibit delayed puberty, reduced sexual maturity, and impaired reproductive performance. The objective of this study was to determine whether differences in pubertal attainment are associated with altered ovarian responsiveness to follicle‑stimulating hormone (FSH), as reflected by granulosa cell gene expression. Estrous cycles were synchronized under Non‑Stimulated and FSH‑Stimulated conditions. Granulosa cells were collected from dominant follicles, total RNA was extracted, and polyA⁺ mRNA libraries were sequenced and mapped to the Bos taurus ARS‑UCD2.0 reference genome. Differential gene expression analyses were performed within and between pubertal classifications, and weighted gene co‑expression network analysis (WGCNA) was used to identify gene modules associated with pubertal classification, FSH stimulation, and age. Ovarian weight and number of follicles greater than 10 mm were increased by FSH stimulation in Early and Non‑Cycling heifers relative to Typical heifers. Despite this ovarian response, Non‑Cycling heifers exhibited substantially fewer differentially expressed granulosa cell genes following FSH stimulation compared with Early heifers, indicating reduced transcriptional responsiveness. Gene modules associated with Early versus Non-Cycling heifer pubertal classifications were significantly different by WGCNA including pathways related to actin cytoskeleton organization, intracellular signaling, and metabolic processes. Additional modules associated with FSH stimulation and age are presented. Collectively, these findings demonstrate that pubertal classifications, particularly Early and Non‑Cycling heifers, are characterized by distinct granulosa cell gene expression pattern signals and coordinated gene networks, suggesting persistent differences in ovarian regulation arising from altered pubertal development.

Advisor: Andrea S. Cupp