Rachel Godek

Lipid Raft Disruption Alters Human Follicle Stimulating Hormone Receptor Signaling

Over 6.7 million people struggle with infertility each year. Studying signaling by reproductive hormones in fertility can allow us to gain a better understanding of the signaling pathways that must function correctly for proper fertility. Some infertility is due to incorrect human follicle stimulating hormone receptor (hFSHR) function. When follicle stimulating hormone (FSH) binds to hFSHR this begins a signaling cascade where the end product is the maturation of sperm by Sertoli cells in men, and egg development and production of estrogen through stimulation of granulosa cells in women. It has been determined that hFSHR is localized to microdomains of the cell membrane called lipid rafts which are characterized by higher concentrations of sphingolipids and cholesterol. This composition makes them less fluid than non-raft membranes, and it is believed that lipid rafts regulate signaling of proteins that reside in the rafts, including hFSHR. It was hypothesized that if the lipid rafts in cell membranes were removed then hFSHR signaling would be altered. To remove lipid rafts HEK293 cells stably expressing hFSHR were treated with sphingolipid synthesis inhibitors (Myriocin or Fumonisin B1) or a cholesterol withdrawing drug (Methyl-β-cyclodextrin). All treatments result in disruption of lipid rafts. hFSHR signaling from the cells with disrupted lipid rafts were compared to wild type hFSHR signaling by western blot. Two pathways were investigated: cAMP production and the activation of p44/42 MAP kinase (ERK1/2). cAMP production was measured indirectly by detecting phosphorylated cAMP Response Element Binding protein (p-CREB). p44/42 MAPK signaling was measured by directly detecting the phosphorylation of the kinase (p-p44/42). It was found that treatment with the lipid raft disrupting agents resulted in increased basal cAMP production (as measured by pCREB activation). However, time-dependent hFSH stimulation was decreased compared to cells with intact lipid rafts. In contrast, p-p44 signaling in the drug treated cells was not altered compared to untreated cells. These results suggest that G protein mediated activation of adenylyl cyclase to produce cAMP is lipid raft dependent while p44/42 MAPK activation is not. Further understanding of how lipid raft residency allows for functional hFSHR signaling would allow for the development of new treatments and pharmaceuticals for men and women struggling with infertility.

Tatyana Lynn

Investigating the Role of CRAC in FSH Receptor Function and Structure

Human infertility is a complex disorder that can often be attributed to a dysfunction of the endocrine system. Follicle-stimulating hormone (FSH) is one of many hormones that participate in a complex process in both women and men to regulate normal reproduction. The dysfunction of this hormone and its receptor are some of the many causes of infertility. FSH is secreted by the anterior pituitary and, in women, initiates a cascade of biological events that enable ovulation. FSH carries out its function by binding and activating specific receptors. The FSH receptor (FSHR) is a G protein-coupled receptor (GPCR) that is located in the cell membrane of target cells in the ovaries and testes. GPCRs often interact with a variety of molecules beyond their ligands including, in some cases, cholesterol in the plasma membrane. The Cholesterol Recognition/Interaction Amino Acid Consensus Sequence (CRAC) is one such binding domain and is represented by the sequence LV-XXXX-Y-XXXXX-K/R. The human FSHR contains a sequence consistent with the CRAC motif in the first intracellular loop L-TTLQ-Y-KLTVP-R. We hypothesized that mutating this domain will negatively affect the signaling of the receptor upon ligand activation. To test this hypothesis tyrosine 375 was mutated to phenylalanine (Y375F) which should render the CRAC inactive. After establishing a cell line stably expressing the mutant receptor, activation of the p44/42 (ERK1/2) pathway was measured. Preliminary data suggest that signaling of the hFSHR-Y375F mutant is decreased relative to the wild-type receptor. As we learn more about this receptor, we continue to gain knowledge about conditions such as infertility. This information can facilitate the development of innovative treatments which can help many families who struggle to conceive.

Cassidy Michalicka

The Effect of Cortisol Hypersensitivity Polymorphisms on Cortisol Activity in Obesity
Cortisol is a crucial part of the endocrine system; it has the capacity to affect nearly every organ and tissue in the human body. When functioning correctly, cortisol is known to regulate the body’s stress response, control metabolism, suppress inflammation, regulate blood pressure, regulate blood sugar, regulate our body’s circadian rhythm, and much more. When the concentration of cortisol in the blood is elevated for an excessive period, the body responds with symptoms such as hyperglycemia, hypertension, weight gain, and moon face. Commonly this is known as Cushing’s Syndrome (CS), and interestingly, we have seen a phenotypic resemblance when contrasted alongside Metabolic Syndrome, a subtype of obesity. Several specific nucleotide polymorphisms (SNPs) have been found along the glucocorticoid (GR) and mineralocorticoid (MR) receptors that are expected to present in a higher concentration amongst populations with obesity. Our current research is investigating the relationship between the allele frequency of four SNPs (rs7901695, rs12772424, rs3753519, and rs1051052), and obesity. Through a collaboration with Ellis Hospital Bariatric Care Center, we obtained the patients’ body mass index (BMI), blood glucose levels, serum triglycerides levels, HDL and LDL cholesterol levels, and systolic and diastolic blood pressures to evaluate. Amongst the SNPs tested, there was a significant difference between different genotypes of the rs3753519 SNP in original weight, diastolic pressure, extra body weight, and extra body weight percentage. This data supports the hypothesis that SNPs involved in the activity of cortisol can alter metabolic profiles and could be contributing to the development of diseases such as Metabolic Syndrome and CS. Furthermore, this could aid the deeper understanding of how these SNPs influence weight gain and could provide better predictions and outlooks on the success of weight loss significantly improving the overall quality of life for individuals with obesity.

Michela Michielli

Cortisol Receptor Sensitivity as a Risk Factor for Depression

In 2020, the World Health Organization reported over 264 million people across the world were suffering from depression. Studies have demonstrated that one source of depression is a hormonal imbalance involved in the stress response. Cortisol is a stress hormone regulated by the Hypothalamic-Anterior-Pituitary (HPA) Axis. Its effects on the stress response and other metabolic activities in the body are exerted through the glucocorticoid and mineralocorticoid receptors (GR and MR respectively).
Our research has examined mutations known as single-nucleotide-polymorphisms (SNPs) relating to cortisol-receptor sensitivity and the behavior of cortisol in the body to investigate the link between cortisol activity and depression. The relationship between SNPs relating to receptor sensitivity and depression in patients will be established by analysis of SNPs in coordination with data from psychological inventory assessments collected for each patient. Patient samples and clinical information was collected from adult patients diagnosed with depression by collaborating physicians. Quantitative polymerase chain reaction (qPCR) was used for genotyping. Analysis of the data yielded no significant correlation between SNPs relating to GR or MR and depression as scored by the Beck Depression Inventory (BDI). However one SNP in GR (rs33389) and one SNP on the 11ß-hydroxysteroid dehydrogenase type 1 gene (rs12086634) correlated with scores on the Mindful Attention Awareness Scale and State Trait Anxiety scale, respectively.
Current studies focus on SNPs involved in the regulation of GR and MR activity that are not directly found in those receptors such as the FKBP5 GR co-regulator protein. Investigating other non-GR and MR SNPs could provide alternate explanations for cortisol sensitivity beyond the receptors themselves. In analyzing genetic differences relating to cortisol sensitivity in patients clinically diagnosed with depression, we aim to determine if an imbalance in an individual’s response to cortisol is linked to depression. If such a relationship can be established, this can provide physicians with a screening tool to better inform them of a patient’s risk factors for depression.

Ryan Sidorski

The Effect of Single Nucleotide Polymorphisms Related to Cortisol Activity in Obesity

Cortisol is a glucocorticoid hormone that is released from the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. Cortisol, a steroid hormone, regulates the expression of genes through nuclear glucocorticoid receptors (GR) that have a role in obesity and metabolic-related conditions such as Cushing’s disease, type 2 diabetes, and insulin resistance. There are currently no studies that have looked at how single nucleotide polymorphisms (SNPs) in genes related to cortisol activity correlate with the success rate of bariatric surgery as measured by weight loss and the weight regain percentage. Our study looked at the effect of two different SNPs on the TCF7L2 gene which has been shown to alter the behavior of cortisol receptors. Another SNP of interest is the rs3753519 located on the HSD11B1 gene which has been shown to alter the behavior of cortisol. DNA from buccal swabs collected from patients at the Ellis Hospital Bariatric Care Center were analyzed using quantitative polymerase chain reactions to determine the genotypic frequency for these SNPs. From the SNPs tested, there was a significant difference between genotypes for the rs3753519 SNP when it came to the original weight, diastolic blood pressure, excess body weight, and percent excess body weight lost. Understanding the relationship between variations in genes associated with cortisol activity and obesity could one day lead to more efficient treatment options for individuals who suffer from obesity and cortisol-related issues.

Katarina Zahedi

Mutations in Caveolin Binding Motif Alter Human Follicle Stimulating Hormone Receptor Signaling

Globally, there are about 48 million couples and 186 million individuals of reproductive age that are affected by infertility. Some cases of infertility in both men and women have been attributed to impaired follicle stimulating hormone (FSH) signaling. Lack of proper function of the cognate receptor for FSH (FSHR) could contribute to infertility since the biochemical signal generated by FSH binding to FSHR stimulates the production of a sperm-stabilizing protein in males and follicle maturation in females. It has been demonstrated that human FSHR (hFSHR) localizes to lipid rafts, which are rigid and detergent-resistant microdomains in the cell membrane. Their structure is in part due to having higher concentrations of cholesterol and sphingolipids compared to the surrounding plasma membrane. Additionally, some lipid rafts are characterized by the presence of the protein caveolin. Signaling pathways for some cell surface receptors have been demonstrated to be regulated by residency in lipid raft domains either by separating the related molecules inhibiting premature signaling or by colocalizing the related molecules to facilitate signaling. Based on evidence that FSHR forms direct protein-protein interactions with caveolin, it was hypothesized that disruption of the caveolin binding motif (CBM), a conserved peptide sequence present in proteins associated with caveolin, would affect hFSHR signaling. In hFSHR this sequence is found in transmembrane helix 4 between amino acids 479-489. The current study investigated the effect of mutations in the CBM on hFSHR signaling. The mutants were created via site-directed mutagenesis and expression vectors were transfected in HEK293 cells. Cells stably expressing the mutant FSHRs underwent western blot analysis for qualitative comparison of signaling. Mutations in all four conserved phenylalanine residues of the CBM resulted in much higher signaling compared to the wild-type and at a faster rate of activation. Elevated signaling at shorter FSH treatment times suggest that the mutated receptor might be more sensitive to FSH and may hint at the role of the caveolin interaction in regulating hFSHR signaling. Because current infertility treatments are costly, tend to have low success rates, and are not accessible to everyone, a greater understanding of FSH signaling pathways could lead to the design of more successful infertility treatment methods.