Leydig cells within the testis are the source of androgens that promote virility at both fetal and adult stages, but Leydig cell populations are distinct at each age. As such, adult and fetal Leydig cells function to synthesize testosterone in distinct cellular and endocrine environments.
The AIMs of this proposal are focused on regulation of Star, the known gatekeeper in controlling access of cholesterol to enzymatic activity of the series of steroidogenic enzymes required for conversion to testosterone. Here we focus on novel regulatory events that exert dynamic interactions with Star chromosomal loci that will explain how fluctuating Star transcript accumulation can relate to changes in androgen synthesis. Previously, we used high-resolution fluorescent in situ hybridization (HR-FISH) to localize and quantify a unique pattern for primary, spliced, and mRNA species accumulation for Star compared to other steroidogenic genes within single Leydig cells. We will use this and other innovative techniques to compare results from studies that investigate individual adult and fetal Leydig cells in vitro, within MA10 cells and primary cultures, and in vivo, within whole testes. Pulsatile LH stimulates primarily cAMP/PKA signals to promote testosterone synthesis in adult Leydig cells. While we have substantial means to explain how Star transcription is turned ON by LH/PKA, we understand little about what happens when the pulse is removed, and even less about the interpulse interval.
In AIM 1, we will test the hypothesis that the events occurring at Star loci during this interval are just as critical to controlling testosterone output as the initial stimulus. Meanwhile, the external stimuli that maintain androgen synthesis in fetal Leydig cells are less clear, but evidence points to paracrine signals, with PKA activity playing a role. Another paracrine factor, Sertoli cell-derived Desert Hedgehog (Hh) is known to initiate fetal Leydig cell differentiation, but its role in their maintenance has not been tested. Once differentiated, fetal Leydig cells produce androgens at a steadily increasing rate until late gestation. Therefore, in AIM 2, we will test the hypothesis that regulatory events on Star loci facilitate a controlled increase in androgens within the fetal Leydig cell that compare to those that occur during the interpulse interval in adult Leydig cells. Our findings have the potential to explain fundamental biology underlying steroidogenic control and will have a profound impact on our ability to explore mechanisms by which disturbances in testosterone synthesis, as in endocrine disruption, cause significant clinical ramifications in males from all stages of life.
In the adult male, low testosterone causes infertility, fatigue, and loss of bone and muscle mass. Excess testosterone, as highlighted by the recent surge in testosterone replacement therapy, can elicit testicular shrinkage, prostate swelling, and sleep apnea. In fetal stages, low or high testosterone can cause urogenital birth defects, including disorders of sex development, and subfertility later in life. Clearly, tight control of androgen levels is critical at both stages. Leydig cells within the testis are the source of androgens that promote virility at both fetal and adult stages, but their populations are distinct. As such, adult and fetal Leydig cells function to synthesize testosterone in distinct cellular and endocrine environments, but we have preliminary data that suggests that they share common regulatory steps. This proposal puts forth the conceptual innovation that one factor, STAR, is a critical node by which input from multiple levels is coordinated to control testosterone synthesis in Leydig cells of both adult and fetal testes. Successful completion of these aims will provide an overlay of transcript, loci, and transactivator/chromatin mark information for Star and other enzyme genes over space and time within single Leydig cells in response to no, continuous, or pulsatile stimuli. Further, significant new knowledge will be gained in our understanding of how testosterone synthesis is controlled in fetal Leydig cells and we will, for the first time, undertake direct comparisons between adult and fetal Leydig cell activities. In essence, we will deliver almost minute-to-minute images of molecular events in Leydig cells in culture and, more critically, within adult and fetal testis from whole animal and explant/primary culture approaches. This will provide the added benefit of facilitating comparisons of single Leydig cell activity depending on their immediate environment to learn how single cell function contributes to the whole of testosterone synthesis. These discoveries will help us to understand the biology of external control of testosterone synthesis in fetal and adult testes. In the fetal testes, new knowledge from these studies will help to explain birth defects and testis dysgenesis syndrome and their association with adult diseases. Finally, additional knowledge regarding adult Leydig cell testosterone synthesis regulation events will bring us closer to understanding causes of infertility in men.
| Jefcoate, Colin R; Lee, Jinwoo (2018) Cholesterol signaling in single cells: lessons from STAR and sm-FISH. J Mol Endocrinol 60:R213-R235 |
