Characterization of NADPH Oxidases in Uterine Smooth Muscle during Pregnancy

During gestation, the uterine smooth muscle, or myometrium, transforms from a quiescent tissue to a contractile muscle required for parturition. Immune system activation is a crucial precursor to labour during which the myometrium is infiltrated by immune cells. Immune and myocyte activation rely on signal transduction via oxidation-reduction reactions and generation of reactive oxygen species (ROS). Nicotinamide adenine dinucleotide phosphate oxidases (Nox) are proteins responsible for the transport of electrons across biological membranes. It was hypothesized that Nox proteins and their regulatory subunits would be readily expressed in the rat myometrium during pregnancy to help prepare the tissue for parturition. Using rat myometrial tissue collected from pregnant rats throughout gestation, an immortalized human myometrial cell line, as well as immunoblot and immunofluorescence analyses, the spatial and/or temporal expression pattern of Nox1, Nox4, as well as their regulatory subunits NoxO1, NoxA1 and p22phox were examined, including possible regulation by uterine distension and inflammation. Nox1 was prominently localized to myocyte cytoplasm, with some plasma membrane association, and significantly expressed during the contractile and labour phases. NoxO1 was similarly expressed during these periods and was observed to have a general cytoplasmic staining pattern in situ. Nox4 was prominently localized in peri-nuclear regions, again with some plasma membrane association, and significantly expressed during the proliferative and synthetic phases. p22phox was similarly expressed during both phases while NoxA1 expression was only elevated during the proliferative phase. Stimulation of inflammation in hTERT-HM cells using the pro-inflammatory cytokine IL-1B only induced Nox1 expression in hTERT-HM cells while uterine distension did not significantly affect expression of any Nox enzyme or subunit examined. It is speculated that Nox1 could form signaling platforms during late pregnancy and labour at caveolae to help prime the uterine contractile apparatus prior to labour while Nox4 could participate in cellular signaling events that lead to myometrial proliferation during early pregnancy. This study was the first to methodically characterize the expression of Nox1 and Nox4 enzymes as well as their regulatory subunits in the rat myometrium throughout gestation. The results highlight the potential importance of these proteins in the myometrium for pregnancy and parturition.

Name thesis:degree_name

Master of Science (M.Sc.)

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Veterinary Biomedical Sciences

Grantor

University of Saskatchewan

Year dc:date.issued

2020

Author dc:creator

• Waller, Mikayla Dawn Ward

Advisor dc:contributor.advisor

• MacPhee, Daniel

Committee members dc:contributor.committeemember

• Unniappan, Suraj
• Carvalho, Carlos
• Dadarwal, Dinesh

• Uterus
• myometrium
• NADPH oxidases
• reactive oxygen species
• pregnancy

Handle dc:identifier.uri

https://hdl.handle.net/10388/13091

OAI identifier oai:identifier

oai:harvest.usask.ca:10388/13091