Doctoral Dissertations

Date of Award

5-1999

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Animal Science

Major Professor

Hugo Eiler

Committee Members

Thomas Chen, Fred Hopkins, Charmi Mendis-Handagama, Jack Oliver

Abstract

The enzyme collagenase is involved in degrading uterine collagen during postpartum involution. It has been reported that cultures of rat and human myometrial smooth muscle cells produce collagenase when grown in medium containing fetal bovine serum (FBS) but not in medium containing newborn bovine serum (NBS). The substance in FBS that induces the cells to produce collagenase is 5-hydroxytryptamine (5-HT, serotonin). The concentration of 5-HT in FBS is between 30 µM-SG µM. In contrast, NBS has a concentration of approximately 1 µM or less. It has been proposed that 5- HT serves as a signal to initiate uterine collagen hydrolysis. Furthermore, a 5-HT transporter exists in the mouse and human placenta. The function of the transporter is not known, however it is speculated that it may act in growth and development of the fetus. Five-HT is known to act as a growth factor for many cell types yet its effect on placental cells is not known. In addition, the source of 5-HT found in placental tissue is unclear. It may be that the source for placental 5-HT is the fetal intestine since 95% of the body's 5-HT is located in the gastrointestinal tract and intestinal resection causes a drop in blood concentration of 5-HT.

The working hypothesis of this research was that 5-HT from the fetal intestine is a "proliferation factor" that supports placental cell growth and inhibits activity of matrix metalloproteinases (MMPs) during pregnancy. It is suspected that withdrawal of fetal 5- HT during partum causes placenta separation by cessation of cell proliferation and stimulation of MMP secretion. The objectives of this research were to: determine 5-HT concentrations in blood and tissues of bovine fetuses and neonates; determine whether or not 5-HT acts as a proliferation factor for cultured placental cells; determine whether 5- HT stimulates or inhibits placental MMP activity in cultured cells, isolated placentomes, and pregnant cows; and immunolocalize 5-HT and collagenase in placental tissue.

Blood, intestine, cotyledon, caruncle, and muscle were collected from mid-term and full-term gestation fetuses at an abattoir and from 24 hour and 48-72 hour old calves obtained from The University of Tennessee Dairy Farm. Blood was collected from the umbilical cord of cesarean section fetuses and from pregnant and non-pregnant cows. Samples were analyzed using an enzyme immunoassay procedure. Fetal blood 5-HT concentrations remained elevated from mid (54,111 nM) through full (51,640 nM) pregnancy, declined (P %le; 0.05) during delivery (13,625 nM), and stayed low in 24 hour old calves (24,460 nM), 48-72 hour old calves (18,400 nM), and in cows (8,004 nM). Five-HT concentration in intestine of mid (5,321 ng/g) and full (7,059 ng/g) gestation fetuses and 48-72 hour old calves (6,618 ng/g) was significantly higher (P ≤ 0.05) than 24 hour old calves (1,410 ng/g) and cows (3,049 ng/g). Concentration of 5-HT in placenta of mid (4,570 ng/g) and full (5,788 ng/g) gestation fetuses was significantly higher (P ≤ 0.05) than postpartum placenta (1,176 ng/g). Concentration of 5-HT in muscle of mid gestation fetuses (1,412 ng/g) and 24 hour old calf (1,759 ng/g) were significantly lower (P ≤ 0.05) than in full gestation fetuses (4,941 ng/g).

Cotyledon and caruncle cells were cultured from primary explants of cow placenta. Ten thousand cells were plated in each well of a 96-well plate. Cells were either used as control or treated with 5-HT ranging in concentration from 2.5 µM to 10 µM. The proliferative effect of 5-HT was determined by incorporation of 3H-thymidine into DNA of cells, a tetrazolium-based colorimetric proliferation assay, and cell count. Increasing concentrations of 5-HT were shown to stimulate incorporation of ^H-thymidine into DNA of cells; however, 5-HT was inhibitory of the reaction in the colorimetric proliferation assay. Five-HT had no effect on cell number when counted on a hemacytometer or by a Coulter counter.

Cultured cotyledon and caruncle cells were grown in serum-free medium, and medium supplemented with either FES or NBS and stimulated with 5 µM 5-HT. Media samples were analyzed for MMP activity using a fluorometric technique. Five-HT did not stimulate MMP activity; however, when all 5-HT samples were pooled (2.05 nM), there was significant (P ≤ 0.05) inhibition of MMP activity compared to control (3.81 nM). Isolated placentomes were: (1) perfused for 4 hours with blood containing 50 ≤M 5-HT then incubated for 4 hours with 5-HT, and (2) infused with 5 µM 5-HT and incubated for 4 hours to 11 hours. Matrix metalloproteinase activity was determined using a manometric technique and hydroxyproline and total protein analysis. There were no differences (P ≥ 0.05) in manometric pressure (force needed to separate cotyledon from caruncle) between control (118 mm Hg) and 5-HT (116 mm Hg) treated placentomes nor in the amount of total protein released in control (1.70 mg/dL) and 5- HT (1.61 mg/dL) treated placentomes. However, amount of hydroxyproline released was discretely higher (P ≤ 0.05) from 5-HT (2.06 µg/mL) treated placentomes than from controls (1.57 µg/mL). Fourteen near-term cows were induced to deliver with injections of dexamethasone and PGF2α. These injections also cause approximately 70% of cows to retain their placenta. Twenty-four hours later, 7 cows were injected with 50 µg/kg of 5-HT and 7 cows were injected with saline every 12 hours until delivery or for a total of 3 days. In each group, all but 1 cow retained placenta. Results of these experiments indicated that 5-HT does not stimulate MMP activity in the induced model.

Cotyledons and caruncles from mid and full gestation and naturally delivered placentae were fixed for light microscopy and electron microscopy study. Using light microscopy, 5-HT was localized to the surface membrane of epithelial cells and connective tissue of prepartum cotyledons and caruncles and postpartum cotyledons. Using electron microscopy, 5-HT was localized in close proximity to collagen fibers of postpartum cotyledons. We were unable to localize collagenase in the bovine placenta.

The results of this research support the following; (1) a pattern of 5-HT concentration change during pregnancy and parturition; (2) 5-HT may be a proliferation factor for placenta; (3) 5-HT did not stimulate MMP activity in placenta; (4) 5-HT is present in the bovine placenta during pregnancy and postpartum.

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