The Regulation of the Angiogenic Inducer, Cellular Communication Network Factor 1, by Angiotensin II, in the Bovine Corpus Luteum

Date of Award

Fall 2024

Project Type

Thesis

Program or Major

Genetics

Degree Name

Master of Science

First Advisor

Paul C. Tsang

Second Advisor

Sarah Walker

Third Advisor

W. Kelley Thomas

Abstract

The female reproductive tract is one of the few places in the body that exhibit physiological angiogenesis. Angiogenesis, the formation of new blood vessels from existing ones, is essential for the proper functioning of ovarian follicles and the corpus luteum. Various growth factors produced by ovarian follicular and luteal cells play a role in this angiogenic process. One such factor is the angiogenic inducer, Cellular Communication Network Factor 1 (CCN1), which is expressed in the bovine corpus luteum during the estrous cycle. While we have learned more about the regulation of CCN1 in the corpus luteum, we do not fully understand whether the ovarian renin-angiotensin system has a role. Therefore, utilizing primary cultures of cells obtained from cow early cycle and midcycle corpora lutea, the objectives of the present study were to determine if Angiotensin II (Ang II) is a potential regulator of CCN1 and the matrix metalloproteinases, MMP-2 and MMP-9. Corpora lutea were dissociated and luteal cells were seeded into 6-well plates, where they were treated with Ang II and calcium ionophore, A23187, either alone or in combination, for 2 hours. Total ribonucleic acid (RNA) was extracted from cultured cells, followed by complementary deoxyribonucleic acid (cDNA) generation. The samples were analyzed using quantitative polymerase chain reaction (qPCR) and normalized to the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). During the treatment period, the presence of fetal bovine serum (+FBS) served as the positive control, while its absence (-FBS) served as the negative control. Data was analyzed using repeated measures analysis of variance (ANOVA) followed by Tukey’s test of pairwise comparisons to determine differences between groups treated with Ang II (10nM and 1nM) increased (p<0.05) CCN1 mRNA expression in early cycle bovine luteal cells by 1.7- and 2.0-fold, respectively, while there was no change (p>0.05) in CCN1 expression in the treated midcycle luteal cells, compared to the negative control. The calcium ionophore, A23187, increased CCN1 mRNA expression in early cycle luteal cells with all tested concentrations (1µM, 0.5µM, and 0.1µM) by 2.9-, 2.7- and 2.4-fold, respectively. Further, in midcycle luteal cells, A23187 (1µM and 0.5µM) increased CCN1 expression by 2.12- and 2.1-fold, respectively. Notably, luteal cells from early cycle and midcycle corpora lutea treated with a combination of Ang II (1µM, 0.5µM, and 0.1µM) and calcium ionophore (1µM), had no additive effect on CCN1 mRNA expression, compared to calcium ionophore alone. The second objective of the present study was to determine if Ang II regulated the expression of matrix metalloproteinases (MMP)-2 and -9. Our findings showed that Ang II does not regulate MMP-2 or -9 in either early or midcycle bovine luteal cells. In conclusion, the expression of CCN1 in the early cycle bovine corpus luteum is increased by Ang II. Further, the calcium ionophore, A23187, also increased CCN1 expression, but in steroidogenic luteal cells obtained from the early and midcycle bovine corpus luteum. Collectively, Ang II and calcium may potentially regulate angiogenesis in the bovine corpus luteum, especially during its early development during the estrous cycle.

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