Determining If Serum Shock and Dexamethasone Initiate Circadian Rhythms in Bovine MAC-T Mammary Epithelial Cells
Open Access
- Author:
- Reineke, Rachel A
- Area of Honors:
- Veterinary and Biomedical Sciences
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Dr. Kevin John Harvatine, Thesis Supervisor
Dr. Lester C Griel Jr., Thesis Honors Advisor - Keywords:
- circadian rhythms
dexamethasone
serum shock
biological rhythms
dairy
nutrition
cell culture
in vitro
MAC-T
entrained rhythm
peripheral clock
bovine
mammary
epithelial
cells
circadian
rhythm
milk
production
gene expression
RPS9
CLOCK
BMAL1
PER1
PER2
CRY1 - Abstract:
- Biological rhythms allow organisms to coordinate changes in the external environment with behaviors and physiological processes. Daily changes are orchestrated by endogenous circadian timekeepers, which create repeating cycles of about 24 hours. The regulation of circadian rhythms has not been well-studied in the mammary gland, and may be important to milk production. Serum shock and dexamethasone are known to initiate the circadian clock of fibroblasts, mesenchymal stem cells, peripheral blood mononuclear cells, and peripheral tissues of Drosophilia and zebrafish, but their effects have not been studied in mammary cells. The objective of this study was to determine if serum shock and dexamethasone treatment could re-initiate the biological clocks in bovine mammary epithelial (MAC-T) cells. MAC-T cells were cultured in DMEM medium, exposed to serum starvation for 30 hours, and treated with either dexamethasone (DEX), 50% serum media (serum shock, SS), or serum-free control media (CON). The cells were harvested and lysed 0, 4, 8, 12, 16, 20, and 24 hours after treatment. The relative expression of the core circadian clock genes CLOCK, BMAL1, PER1&2, and CRY1 were measured using reverse transcriptase quantitative PCR (RT-PCR). Data were analyzed using the mixed model ANOVA procedure of SAS. Gene expression was normalized using the housekeeping gene Rps9 as a covariate in the model. A zero-amplitude test was conducted to determine the fit of the cosine function. A cosine function fit the relative expression of Per2, Cry1, and Clock in DEX (P<0.05), but not CON (P>0.10), indicating that dexamethasone initiated a 24-hour circadian rhythm of the expression of these genes. Alternatively, dexamethasone did not initiate a rhythm of Per1 or Bmal1 expression. A cosine function fit the relative expression of Cry1 in SS (P<0.05) but not in CON (P>0.10), indicating that serum shock initiated a 24-hour circadian rhythm of the expression of this gene. Serum shock did not initiate a rhythm in Clock, Per1, or Per2 gene expression. The results of this study suggest that dexamethasone re-initiates the damped biological clocks in cultured bovine mammary epithelial MAC-T cells, while serum shock appears unreliable for re-initiating rhythms of core clock gene expression.