Imaging Live Uterine Tissue Modulation Using Confocal Microscopy

Abstract

Pregnancy and childbirth are both natural occurring events, but still little is known about the signaling mechanisms that induce contractions. Throughout the world, premature labor occurs in 12% of all pregnancies with 36% of infant deaths resulting from preterm related causes [1]. Even though the cause of preterm labor can vary, understanding alternative signaling pathways which affect muscle contraction could provide additional treatment options in stopping premature labor. The uterus is composed of smooth muscle which is innervated with a plexus of nerves that cover the muscle fibers. Smooth muscle can be stimulated or modulated by many sources such as neurotransmitters [dopamine], hormones [estrogen], peptides [oxytocin] and amines. In this study, we are focusing on the biogenic monoamine tyramine which is produced in the tyrosine catecholamine biosynthesis pathway. Tyramine is known to be associated with peripheral vasoconstriction, increased cardiac output, increased respiration, elevated blood glucose and the release of norepinephrine. Our research has found tyramine, and its specific receptor TAAR1 [2], to be localized within mouse uterus and that this monoamine can induce uterine contractions at levels similar to oxytocin.Research that focuses on reproduction commonly use techniques involving immunohistochemistry or histological staining to identify localization of key modulators and receptors or they observe general morphology in fixed whole or sectioned tissue [ Fig. 1]. In studies involving muscle tissue, force transduction measurements allow the researcher to quantify differences in contractile force. These methods can be used with the addition of agonists and antagonists which help decipher the mechanisms that may modulate the contraction. A drawback of these methods is that muscle contraction is a complex system that relies on feedback mechanisms and without observing the tissue in real time, the researcher would be making interpretations based on random snapshots in time. To overcome this limitation, we have developed an imaging technique that can observe muscle signaling dynamics in real time.This protocol was approved through the IACUC protocol 15-1388T at Arizona State University and the mouse uterine tissues were derived from GFP LifeAct transgenic mice [3]. These mice contain a transgene encoding a 17-amino acid peptide called LifeAct, which binds F-actin. This peptide is coexpressed with GFP which enables the actin cytoskeleton to be imaged both in vivo and in vitro. Actin is a key component of muscle tissue and this transgenic model allows researchers the ability to visualize muscle contractions. Combining this tissue with the TAAR1 receptor antibody conjugated to a fluorescent probe [antibody labeling kit, Thermo Fisher, #A20186] is a tool that can be used to visualize the actual response and timing of a receptor mediated event. Imaging was conducted using a dipping lens on a Leica SP5 confocal microscope which allowed the tissue to continue normal peristaltic contractile activity. Time-...

Ultrastructural Modifications of the Mouse Uterus upon Biogenic Amine Stimulation

“…Tyramine and dopamine are both modulators of smooth muscle activity and are found around the smooth muscle of the mouse uterus. Dopamine is a documented neurotransmitter that inhibits smooth muscle contractions while a recent study has found that tyramine, and its specific receptor TAAR1 (trace amine associated receptor 1), are localized within the uterine plexus and can induce uterine contractions [2]. Although dopamine has greater affinity to the vesicular monoamine transporter (VMAT), both modulators can be taken up into synaptic vesicles [3].…”

The Role of Tyramine Within the Mouse Ovary

“…Understanding the signaling pathways that control the maturation and release of oocytes from the ovary may provide insight into how the ovarian cycle can be further regulated, especially for women experiencing infertility due to ovarian dysfunction. Previous studies have shown that the uterus, which is composed of innervated smooth muscle, can be induced to contract by the biogenic monoamine tyramine at levels similar to estrogen [1]. Tyramine is known to be associated with peripheral vasoconstriction, increased cardiac output, and increased respiration, which are all affected by smooth muscle activity [2].…”