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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C & D

W30 - Sex-specific effects of estradiol and dihydrotestosterone on clock gene expression patterns in cultured human fibroblasts

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

IS

Isabel Sagheb

Graduate Research Assistant
Tulane University
New Orleans , Louisiana, United States

Co-Author(s)

IP

Isabella Posey

Graduate Research Assistant
Tulane University, United States
Ashley Martier, PhD

Postdoctoral research scientist
Xavier University of Louisiana
New Orleans, Louisiana, United States

Primary Investigator(s)

MM

Mark Mondrinos

Assistant Professor
Tulane University
New Orleans, Louisiana, United States

Introduction: : Aging is a multifaceted biological process characterized by the progressive loss of physiological functions. With age, the gap between pathophysiological sex differences increases due to the significant decline in sex hormone levels; clinically defined as menopause in women and andropause in men. Declining sex hormone levels contribute to physiological deterioration and heightened susceptibility to disease. Hormone replacement therapy (HRT) is a popular treatment for aging patients seeking increased cardiovascular health and improved lifestyle. Despite the prevalence of HRT, the underlying cellular mechanisms remain poorly understood. Sex hormones influence clock genes that govern the circadian rhythm of normal functions in cellular physiology via direct control of the molecular clock by estrogen and androgen receptor-mediated gene expression patterns. Disturbance of the oscillatory expression of clock genes has been implicated in aging, cancer, and disease and decreased clock gene expression with age is believed to play a role in the regulation of cellular senescence, a significant hallmark of aging. Our long-term goal is to develop microphysiological systems for modeling how the interplay of sex hormones and clock genes influences hallmarks of cellular aging in culture. Here, we investigated the effects of estradiol (E2) and dihydrotestosterone (DHT) on clock gene expression in cultured primary human female and male cells, respectively.

Materials and

Methods: : Primary human lung fibroblasts (HLF) from both male (XY) and female (XX) donors were expanded and cultured in fibroblast growth media (FGM) containing 2% FBS. For experimental groups in hormone culture, media was changed to phenol-free media supplemented with charcoal stripped-serum (CSS). After a 24-hour starvation period, XX cells were treated with a saturating dose of 10nM estradiol (E2) and XY cells were treated with 10 nM dihydrotestosterone (DHT). Cells were passaged after reaching 80% confluency. Passaging cells induces artificial biological aging. In this study, we defined passage 2 as the ‘youngest” timepoint and passage 7 as the “oldest”. Clock gene expression in hormone supplemented CSS was compared to culture in standard FGM for equivalent passage numbers. To evaluate the influence of sex hormones on daily circadian oscillation expression of clock genes, XY cells were plated in 6-well plates in both FGM and CSS-treated media, and samples were collected every 12 hours for 48 hours via trypsinization. 10nM DHT was added to experimental groups grown in CSS media starting at hour 0. For all experiments, hormones were replenished every 24 hours and media was changed every 48 hours. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to measure fold change of expression of core clock genes: BMAL1, CLOCK, CRY, AND PER1. GAPDH was used as a housekeeping gene.

Results, Conclusions, and Discussions:: RT-qPCR analysis of clock gene expression revealed a pattern of upregulation in hormone-defined media (XY cells with DHT and XX cells with E2) compared to standard FGM culture. BMAL1 expression was upregulated by sex hormones as passage numbers increase to P6 (Figure 1A). Passage 6 XY HLF exhibited an oscillatory pattern of BMAL1 expression at 12-hour increments when cultured in standard FGM and in CSS medium with DHT (Figure 1B). While longer culture periods are required to determine if the CSS + DHT group would establish a regular oscillation, demonstrating daily rhythms of clock gene expression is a key step toward developing MPS models of chronobiology. In addition, we measured the expression of other core clock genes including CLOCK, CRY, and PER1 over a 48-hour period to more comprehensively profile hormone effects on molecular clock components (Figure 1C). Following the 36-hour time point, core clock gene expression levels are elevated across all targets in XY HLF cultured in CSS +10 nM DHT compared to cells grown in FGM. Collectively, the findings of this study confirm that sex hormones influence clock gene expression in primary human fibroblasts.

Future studies applying E2/DHT to both sexes in various ratios along with other sex hormones such as progesterone will more accurately mimic human sex hormone biology. Dose response studies will guide interpretation of findings in the context of known sex hormone levels in aging individuals and administered HRT doses. Sex hormone receptor gene expression profiling on aging cells undergoing hormone treatment will be necessary to interpret dose responses and potential donor variability.

In summary, we have taken first steps toward engineering microphysiological systems (MPS) for modeling sex-specific chronobiology. Future work will correlate multiple markers of the aging process including clock gene expression, hormone receptor expression, senescence-associated secretory phenotype, and telomere attrition in cultured cells of various origins. The development of multicellular 3D MPS models that incorporate the understanding of clock gene expression dynamics in the context of functional assay readouts will aid the development of therapeutic paradigms for aging and age-related diseases including fibrosis and cancer.

Acknowledgements and/or References (Optional): :