L-type calcium channel-dependent signaling impacts GnRH receptor function and intercellular communication in cultured gonadotropes
Date
2020
Authors
Drennan, Meggan L., author
Amberg, Gregory, advisor
Clay, Colin, committee member
Garrity, Deborah, committee member
Kelp, Nicole, committee member
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Abstract
The hypothalamic-pituitary-gonadal (HPG) axis is a negative feedback biological system critical in fertility, reproduction and development. Gonadotropin-releasing hormone (GnRH) is first released by the hypothalamus and binds to GnRH receptors (GnRH-R) on gonadotrope cells of the anterior pituitary gland where the receptors must mediate a variety of pulsatile signals. The gonadotropin hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH), are subsequently released by the pituitary and act upon the ovaries and testes, further producing gonadal steroids to be circulated throughout the body. GnRH pulse frequency and amplitude determine successful gonadotropin release, which is ultimately regulated by the GnRH-R. The GnRH-R is a heterotrimeric G-protein coupled 7-transmembrane domain receptor with Gα, β, and γ subunits. Ligand binding initiates an intracellular cascade that leads to a global increase of cytosolic calcium concentration by way of calcium influx through voltage-gated calcium (Cav) channels, and intracellular calcium release from endoplasmic reticulum (ER) stores. Gonadotropes depend on intracellular calcium concentration to carry out their specific physiological function, such as transcription of gonadotropin subunits, hormone biosynthesis and release. Calcium flux is a normal and important aspect of cellular function, including cell-cell communication. Calcium oscillations have been well documented in multiple cell types, with different patterns being induced with distinct treatments. Observations in this line of research include the following: different oscillatory patterns lead to different physiological outcomes, the rate at which internal calcium is secreted from the ER can greatly impact these patterns, and IP3 receptor clustering on the ER results in localized changes in calcium concentration rather than a marked global difference, implicating a spatial stochasticity. These oscillations have shown evidence of paracellular coupling at gap junctions, as well as synchrony following extracellular diffusion. Chapter two of this thesis details experiments investigating calcium oscillations using a membrane-targeted calcium indicator. Immortalized αT3-1 cells were transfected with a membrane-targeted GCaMP and TIRF microscopy was used to capture fluorescent calcium activity. Cells were treated with GnRH as well as various pharmaceutical treatments that would exploit L-type Cav channel function and manipulate normal intracellular calcium release. An array of observations was recorded. Qualitatively, there was an overall increase in calcium activity in the majority of cells after GnRH treatment. Drug-induced inhibition of calcium influx and intracellular calcium release diminished calcium activity entirely. Further, synchronized activity was captured among several cell groups, showing both pre-established synchrony and GnRH-induced synchronized peaking. Further research should be conducted to better understand the full mechanism underlying these behavioral responses, but these experiments provide a foundation for this work. Chapter three highlights experiments using a GFP-tagged GnRH-R in αT3-1 gonadotropes in order to investigate GnRH binding-induced receptor mobility and clustering. Treatment groups were identical to the previous chapter. SRRF and binary analysis were used to characterize receptor activity. Descriptively, clustering of receptors was seen, especially when calcium activity was limited, but more appropriate methods of quantitative analysis should be explored in order to go beyond these observations in processed images. This thesis concludes overall that GnRH-induced calcium oscillation patterning and receptor clustering are far more complex and difficult to study than initially thought. Much more research is needed to determine any conclusive findings, however, these experiments may serve as a stepping stone toward obtaining the answers sought.
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Subject
GnRH
gonadotropin-releasing hormone
receptor
gonadotrope
calcium oscillation
pituitary