Ovarian Function/Dysfunction
Session: Poster Session A
Brooke Rudloff, BA (she/her/hers)
Graduate Student
University of Nebraska Lincoln
Lincoln, Nebraska, United States
Our laboratory identified 5 pubertal classifications in heifers from the UNL Research Herd: Typical (T), Early (E), Start-Stop-Start (SSS), Start-Stop-Discontinuous (SSD) and Non-Cycling (NC), which we have reported to be moderately heritable (0.38). If heifers were grouped as those that cycle and maintain cyclicity (E+T) compared to those that do not cycle or do not maintain cyclicity (delayed puberty; NC+SSD) then they are highly heritable (0.59). Non-Cycling heifers have delayed puberty due to reduced sexual maturity and inability to cycle during the collection period, resulting in reduced numbers of calves born in the first 21 days of the calving season. We hypothesized that (1) heifers have delayed puberty due to chronic inflammation and (2) Follicle Stimulating Hormone (FSH) exogenous stimulation may enhance granulosa cell gene function to advance follicle progression and granulosa cell function. To determine if there was chronic inflammation, monthly blood samples were collected from heifers born in 2019 and 2020 from weaning (October) to breeding (May) and complete blood counts were measured via a clinical veterinary Hematrue machine. When heifers were grouped into E+T vs NC+SSD, the NC+SSD had greater monocytes and blood platelets than T+E, indicating greater inflammation, as well as tendencies for reduced mean corpuscular volume and increased leukocytes and lymphocytes. To determine if granulosa cell gene expression was rescued in NC heifers compared to Typical with FSH stimulation; 1) estrous cycles were synchronized (2 X PGF2a 14d-apart) and ovariectomized 36hr after the second PGF2a (non-stimulated); or 2) Non-Cycling and Typical heifer estrous cycles were synchronized (2 X PGF2a, 14d-apart) with dominant follicle aspiration on day 10 of the estrous cycle, and then given FSH (250IU) ( 7 injections every 12h) FSH every 12hr for 7 injections (250IU) with PGF2a at last two injections. Ovariectomy occurred 24hr after the last injection of FSH/ PGF2a (stimulated). FSH increased the number of follicles > 7mm across pubertal classifications, compared to their non-stimulated counterparts (p < 0.05); however, FSH-stimulated Non-Cycling heifers had more follicles > 7mm (p < 0.05) and tended to have more > 10mm (P < 0.1) when compared to Typical FSH-stimulated heifers. Granulosa cells were isolated from estrogen-active dominant follicles at ovariectomy, and RNA was extracted for RNA sequencing. Poly-A+, mRNA libraries were sequenced using 150 bp mapped to ARS-UCD1.3 and quantified. Differential expression analyses were performed in R using DESeq2, with significance set to a P-value < 0.05. Ingenuity Pathway Analysis was used to determine canonical pathways, predicted upstream regulators, and gene networks. When comparing Typical FSH-stimulated females (n=5) to Typical non-stimulated (n=15), 205 genes were up-regulated and 122 were down-regulated, segregating into primarily upregulation of pathways related to second messenger signaling and immune function. In the granulosa cells from FSH-stimulated Non-Cycling heifers (n=5) compared to non-stimulated Non-Cycling (n=14) there were 96 up-regulated and 227 down-regulated genes. Interestingly, there was down-regulation of pathways related to immune response including phagosome formation and neutrophil degranulation. Taken together, these data indicate that Non-Cycling heifers have increased inflammatory markers; however, when treated with FSH, gene expression pathways regulating immune function are suppressed which may allow for partial rescue and formation of large antral follicles on Non-Cycling heifer ovaries.