Elizabeth Altman

Mutations in the Human Follicle Stimulating Hormone Receptor Caveolin Binding Motif Cause Increased Basal Activation

Over twelve percent of women aged fifteen to forty-five in America suffer with infertility, while over seven million women have used infertility services, such as intrauterine insemination and in vitro fertilization, to conceive. Dysfunctional human follicle stimulating hormone (hFSH) signaling may cause some cases of infertility. hFSH is released from the pituitary in the hypothalamic-pituitary-gonadal (HPG) axis and plays a role in spermatogenesis in males and follicle maturation and estrogen production in females. Therefore, dysfunctional signaling leads to decreased fertility in males and complete infertility in females. It has been shown that hFSHR interacts with lipid rafts, stiffer regions of the plasma membrane with higher sphingolipid and cholesterol concentrations, and with caveolin, a protein found in certain lipid rafts. This interaction may occur through a specific sequence of amino acids in the 4th transmembrane domain of hFSHR consistent with a caveolin binding motif (CBM). Previous work from our lab suggests that hFSHR signaling is regulated through residency in lipid rafts, such that recruitment into lipid rafts inhibits signaling. The current study investigated the effect of mutations of the CBM on hFSHR signaling. It was hypothesized that complex mutation of the CBM would cause increased hFSHR signaling by disrupting interactions with caveolin, preventing inhibition via residency in lipid rafts. Site-directed mutagenesis of the CBM allowed for creation of complex mutants and expression vectors were transiently transfected into HEK293 cells to qualitatively compare hFSHR signaling between mutants via western blot. Mutation of two sites in the CBM resulted in increased basal and induced hFSHR signaling, supporting the hypothesis. Mutation of three sites decreased signaling below that of the wild type receptor, suggesting that complex mutation of three sites may cause too large of a conformational change to allow for any receptor function. Further understanding of hFSHR signaling regulation would allow for development of new fertility treatments for men and women.

Molly Brogie

Characterization of Membrane Microdomains on a Human Granulosa Cell Line by Atomic Force Microscopy

Signaling via the Hypothalamic-Pituitary-Gonadal (HPG) axis regulates fertility in both men and women. Follicle stimulating hormone (FSH), secreted from the anterior pituitary, binds to a G protein coupled receptor (GPCR) on ovarian granulosa cells. The activation of this receptor triggers a cellular signaling pathway that leads to the promotion of follicle development and estrogen secretion. Our lab has previously demonstrated that the human FSH receptor (hFSHR) shows an affinity for residing in lipid rafts of HEK293 cells which may play a pivotal role in its signaling. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids, with higher stiffnesses than bulk membrane domains. To characterize lipid rafts on the human granulosa cell line hGrC1 we used atomic force microscopy. Atomic force microscopy is a technique used to produce nanoscale resolution topographical images. It can also be used for determining mechanical characteristics, such as stiffness and strain. Here, using contact and force-volume mode atomic force microscopy, we successfully generated topographical maps of hGrC1s and recorded membrane stiffness measurements. Areas of the granulosa cell membrane showed variable stiffness on a scale of 58.998 nm / pixel, providing the first direct evidence of lipid rafts in human granulosa cell membranes. Demonstration of lipid rafts contributes to the implications of the role of lipid rafts hFSH signalling. By understanding membrane dynamics on granulosa cells we hope to gain insight into how to manipulate hFSHR function.

Alexa Caruso

Impact of Single Nucleotide Polymorphisms on HPA-Axis Functionality in Depression
The hypothalamic-pituitary-adrenal (HPA) axis is responsible for regulating the secretion of the glucocorticoid hormone cortisol in response to stress. Diseases of cortisol dysregulation, such as Cushing’s syndrome (hypercortisolism) and Addison’s disease (hypocortisolism), share a common symptom of depression. Based on this we, and others, have hypothesized that single nucleotide polymorphisms (SNPs) in the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and other genes associated with cortisol regulation may contribute to depression. Our study investigated the genotypic frequency of several SNPs that affect GR and MR sensitivity to cortisol in a clinical population. DNA samples were acquired via buccal swab from patients suspected of suffering from depression. Extracted DNA was analyzed using a novel multiplex allele-specific polymerase chain reaction assay to determine the allelic frequency of SNPs associated with hypersensitivity or resistance to cortisol. Saliva samples were also collected from these patients via buccal wash and analyzed using an ELISA assay to measure cortisol levels. In addition, patients completed multiple measures of depression and anxiety. Preliminary results showed that mutant haplotypes of three specific alleles in the GR, BclI (rs41423247), N363S (rs56149945), and TthlllI (rs10052957), as well as rs120 (rs12086634) in 11-HSD, were seen more frequently in the patient population compared to the general population. Although depression (as measured by the Beck Depression Inventory) and salivary cortisol did not change significantly between clinic visits, there was a significant decrease in state anxiety (as measured by the State-Trait Anxiety Inventory) between visits for patients with the wild type or heterozygous BclI haplotype while patients with the homozygous mutant haplotype did not show a change in state anxiety.  This is the first demonstration that genotypic variation in cortisol sensitivity is associated with changes in stress anxiety. This could lead to more specific and successful treatments for depression with the goal of improving patient outcomes.

Alicia Clarke

Effects on Exercise & Meditation on Reported Stress and Cortisol in an Undergraduate Population

Stress is a daily factor that is directly correlated with negative physiological and psychological effects including stroke, heart attacks, and problem drinking.  A stressor that is perceived by the body activates the hypothalamic-pituitary-adrenal (HPA) axis which causes the synthesis and release of the glucocorticoid hormone cortisol from the adrenal cortex.  Studies have shown meditation or exercise programs help decrease stress and cortisol levels but this has not been tested in college-aged adults. In this study, undergraduate students were assigned to either meditation, exercise or control groups for four weeks to measure the effects of introducing either meditation or increased exercise on reported stress and physiological response to stress in the form of salivary cortisol levels.  Participants’ stress was measured using the Revised Undergraduate Student Hassles Scale (RUSH-S) which measures a variety of stressors including academic, social, and personal hassles. Students completed the RUSH-S 3 times at baseline and at 2-week intervals. Simultaneously, saliva samples were collected to analyze salivary cortisol levels using an enzyme-linked immunosorbent assay (ELISA). A one-way between subjects ANOVA was conducted to compare the effect of personal, academic and social stressors as measured by the RUSH-S. Both personal and social stressors for the meditation and exercise groups were reduced compared to the control (p=0.037 and p=0.052, respectively).  No significant reduction of academic stress was observed compared to the control. Cortisol levels tended to correlate with RUSH-S results. This study suggests that interventions such as meditation or exercise may be beneficial to reducing stress in undergraduate populations.

Scott Koszer

Developing an APEX Assay to Investigate Signaling and Protein Interaction of the Human Follicle Stimulating Hormone Receptor

Infertility affects millions of people in the United States. In addition to being unable to conceive, patients can face a variety of challenges ranging from mental health issues to increased economic costs. One cause of infertility is a defect in follicle stimulating hormone (hFSH). hFSH is a peptide hormone that is crucial for fertility in both males and females. In males, hFSH is involved in spermatogenesis. In females, hFSH is involved in estrogen production and ovarian follicle development. To better understand the role hFSH plays in fertility, our lab is interested in investigating the function of the hFSH receptor (hFSHR). hFSHR is a G protein-coupled receptor (GPCR); signaling from the receptor is of particular interest to us, because if we know how hFSHR signals, we can identify potential molecules to investigate that may be targets to improve fertility. However, investigating the signaling of GPCRs such as hFSHR is challenging. Ideal assays to investigate hGPCR signaling should be sensitive, simple to use, high throughput, and non-radioactive. Although assays for investigating GPCR signaling have historically been used, they have significant drawbacks. Assays using radioactive ligands are sensitive and high throughput, but are not simple to use and generate radioactive waste. Other assays such as G protein dependent functional assays are sensitive, but are only able to screen for the activity of one molecule. The APEX assay is a new assay for investigating GPCR signaling that has significant advantages over other assays. When cells expressing a GPCR-APEX fusion protein are incubated in culture media containing biotin-phenol, APEX tags signaling molecules and other proteins in a 20 nm radius with biotin-phenoxy radicals. Furthermore, this reaction can be quenched. Thus, the APEX assay can be used to investigate GPCR signaling with high spatial and temporal resolution. To develop the APEX assay to investigate hFSHR signaling, it was necessary to generate a plasmid containing the hFSHR-APEX fusion protein. Creation of this plasmid will allow us to have a high-throughput, time dependent assay to develop new therapeutic targets for infertility.

Jillian Love

Modulating Human Follicle Stimulating Hormone Signaling Activity Using Small Molecules

Human follicle stimulating hormone (hFSH) is a gonadotropin hormone involved in maturation of ovarian follicles and estrogen production in females and spermatogenesis in males. Its release is regulated via the hypothalamic-pituitary-gonadal axis. Proper regulation and function of hFSH in females is necessary for fertility, so its activity has been a focus in female fertility treatments and contraception. The hFSH receptor (hFSHR) is a GPCR found in granulosa cells in females and Sertoli cells in males and is responsible for initiating a complex downstream signaling cascade in both cell types. It has recently been discovered that hFSHR is capable of biased signaling, meaning it may be possible to selectively activate some downstream signaling pathways without affecting others. This biased signaling may be achieved through the addition of negative allosteric modulators (NAM’s), notably the small molecules ADX68692 and ADX68693, which bind to hFSHR and alter the receptors conformation. Both NAM’s have been shown to decrease FSH-dependent ovulation in vivo although have differing effects on steroid hormone production. The specific molecular mechanisms by which this occurs is unclear. Utilizing the human granulosa cell line hGrC1 we have observed differences in p44/42 MAPK signaling after treatment with NAM’s. In control conditions, activation of p42/44 MAP kinase peaked five minutes following FSH stimulation, but in the presence of NAM’s there was a continuous increase in signaling over thirty minutes. Additionally, signaling through protein kinase A (PKA) in the presence of ADX68692 showed increased activation of some substrates but in the presence of ADX68693 basal levels of the same substrates were initially higher and showed a decrease over time. Interestingly, some PKA substrates showed no difference in hFSH activation between untreated and NAM treated samples. The effects of treatment with NAM’s show their applicability in the development of novel non-steroidal contraceptive methods that could be associated with fewer side effects. Studying the complex biased signaling mechanisms of hFSHR could identify ways to manipulate hFSHR for not only improved methods of contraception but also fertility treatments.

Margot O’Brien

Impact of Single Nucleotide Polymorphisms in HPA Axis Functionality in Obese Populations

The human body’s primary stress response is regulated through the secretion of the glucocorticoid hormone cortisol through the hypothalamic-pituitary-adrenal (HPA) axis. Utilizing glucocorticoid receptors (GR), cortisol regulates the expression of certain genes that play a role in metabolism regulation and obesity related conditions such as type 2 diabetes, insulin resistance, and elevated serum triglycerides. Based on this, our lab and others have identified the presence of mutations in the genes for the glucocorticoid receptor (GR), the closely related mineralocorticoid receptor (MR), and regulatory proteins associated with cortisol or GR function (heat shock protein 90, 11ß-hydroxysteroid dehydrogenase type 1, and FK506 binding protein (FKBP)) to be of higher frequency in obese populations. Our study continued to investigate the genotypic frequency of these single nucleotide polymorphisms in the clinical population of bariatric patients from Ellis Hospital in hopes to better understand the role of genotypic variation in cortisol regulation and obesity. DNA samples were extracted from buccal swabs of Ellis Hospital participants and analyzed using allele specific polymerase chain reactions to determine the genotypic frequency of SNPs associated with hypersensitivity or resistance to cortisol. From the polymorphisms tested, there were no presence of mutations for either the TthIIII or FKBP SNPs. In addition, our study designed a new multiplex assay in which wild type and mutant alleles can be tested in a single PCR reaction to greatly increase the efficiency of data collection. With a greater sample population, more significant correlations can be made between  SNPs and obesity to aid in the steps towards personalized medicine based on one’s genetic information.

Brandon Zahler

Investigating the Lipid Raft Residency and Caveolin Binding of the Human Follicle Stimulating Hormone Receptor

Follicle Stimulating Hormone is involved in male and female fertility as a component of the HPG Axis. Its functions include spermatogenesis in males and ovarian follicle development in females. To better understand the physiology of this hormone and its effects, we focused on the receptor for hFSH (hFSHR) and its location on the cell membrane. Previous work in our lab has demonstrated that hFSHR localizes in lipid rafts. These are dense regions of the cellular membrane with high levels of cholesterol and sphingolipids. What is unclear is how the receptor gets into the lipid rafts. To study the interaction between hFSHR and caveolin of lipid rafts, I examined the properties of the lipid rafts and their sensitivity to various detergents. This was done to verify that regions of the membrane where hFSHR was located were detergent resistant. This study was performed using a discontinuous Optiprep Density Gradient and then verifying the location of the hFSHR using western blot analysis. The results showed that lipid rafts are resistant to some mild detergents such as Triton, but were disrupted by stronger ones such as SDS. I also used the methods of immunoprecipitation and western blot analysis to directly study the interaction between caveolin proteins and hFSHR. Experiments have suggested that the caveolin protein is closely linked with the receptor. Future experiments will build off these results and make use of caveolin mutants produced in the Cohen Lab to determine how changes to caveolin interfere with hFSHR function by measuring levels of downstream signaling molecules. This has potential implications in developing contraceptives and possible treatments for fertility treatments involving the action of FSH and its receptor.