Sex-Dependent Effect of Steroid Hormones on Neurodevelopment, Physiology, and Behavior

Sex Differences in Biomedical Research

The National Institutes of Health (NIH) is the largest supporter of medical research (more than $37 billion per year).1 As such, the NIH is a major driver of research priorities, including increased emphasis on sex as a biological variable.2 In 1993, the NIH Revitalization Act mandated the inclusion of women in clinical trials, and now women comprise half of all participants.3 This important achievement has broadened our understanding of diversity in biological responses across sexes in human research. However, not until January 2016 did the NIH extend similar consideration of sex as a biological variable to preclinical research involving cell and animal models.4 Proposed studies are now strongly encouraged to include both sexes or, if not, to include a legitimate scientific rationale for not doing so. Despite these recommendations, preclinical studies continue to favor eval uation of males five times more frequently than females. Even when both sexes are included, comprehensive studies are often performed in males with only confirmatory studies in females. Likewise, even if studies are powered to evaluate a particular outcome in both sexes, they are not necessarily powered to detect differences between the sexes. Another concern is that many investigators have not received any formal training in the proper evaluation of sex differences. Finally, evaluating both sexes increases time and cost of performing studies. Overcoming these challenges is critical for enhancing our fundamental understanding of the impact of sex on normal physiology and disease, thereby ensuring that new knowledge can be translated to both men and women.

Sex-Specific Differences in Neurodevelopment in Pre-Clinical Research

My research program focuses on sex-dependent effects of a variety of factors on early neurological development and their subsequent impact on physiology and behavior in adulthood. During my dissertation in the laboratory of Stuart A. Tobet, PhD, at Colorado State University, my research objective was to determine the sex-specific impact of glucocorticoid excess during fetal development on the development, vascularization, cellular composition, and function of specific hypothalamic nuclei.5,6 We found that glucocorticoid exposure had significantly different effects on male and female fetuses, including on the cellular com-position and function of the blood-brain barrier in specific nuclei of the hypothal-amus, as well as behavioral outcomes extending through early adulthood.

To gain further insight into the underlying mechanisms for these effects, I subsequently joined the laboratory of Donald B. DeFranco, PhD, at the University of Pittsburgh as a NIH T32-funded postdoctoral scholar. My research objective was to characterize how glucocorticoid exposure impacts gene regulation in the developing hypothalamus in a sex-specific manner, using systems-wide molecular genetic approaches.7,8 In doing so, we identified and characterized the sexually dimorphic dexamethasone transcriptome in mouse cerebral cortical and hypothalamic embryonic neural stem cells. The results provided insight into the mechanisms of fetal glucocorticoid exposure on hypothalamic neurodevelop-ment, as well as their long-term behavioral and physiological consequences in both males and females. These results also highlight the importance of sex as a biological variable in biomedical research.

These studies also have important implica-tions for future research. Specifically, these sex-dependent effects of glucocorticoids on early neurodevelopment and subsequent physiology and behavior support the possibi lity of broader and more long-term effects on a variety of outcomes later in life. In particular, glucocorticoids are well known to have profound effects on energy and metabolic homeostasis, suggesting that these same processes in early develop ment may profoundly affect risk of obesity and metabolic disease in adulthood. Researchers at the University of Pittsburgh have recently identified a novel obesity-risk variant that is highly associated with BMI and obesity risk in a specific human population.9 The gene harboring this variant has been shown to influence glucocorticoid receptor stability and responses to stress in preclinical models.10 Through an NIH Mentored Research Scientist Career Development Award, I am now using my background 
in neuroanatomy/development, neuro-endo crine function, physiology and behavior, sex-differences, and systems genetics approaches to examine the impact of this gene and its risk variant on glucocorticoid receptor action within the hypothalamus in a sex-dependent manner. These studies are likely to reveal novel insights into the interaction between 
sex and glucocorticoid action in early development and their impact on metabolic outcomes in adulthood.

Collaborative and Supportive Research Environment at the University of Pittsburgh

During my postdoctoral training at the University of Pittsburgh, I was fortunate to be supported by a well-established institutional training program and by the NIH. I was initially recruited to the University with the support of the NIH T32 Research Training Program in Endo - crinology, Diabetes, and Metabolism. This training grant has been in place for more than 40 years and is the longest running training grant at the University of Pittsburgh. This opportunity provided me with an instant network of experienced scientists, protected time to focus on my new research project, structure and resources to acquire new technical skills, and a rich environment to expand my knowledge. I presented my research quarterly during the Research in Progress sessions to other T32 scholars and faculty from within the Division and across the University, met with my committee annually to review my progress and future goals, and participated in grant writing and career development workshops to further my scientific development. The T32 also provided a stipend, research funds, and financial support to attend scientific meetings. This support ensured that I would devote the majority of my time to my research and career development.

I personally benefited from these established and routine interactions set up through the T32. During grant submissions, I had a network of well-established researchers who were leaders in their fields and had observed my progression from the beginning of my postdoctoral experience. I am confident that these consistent and valuable rela- tion ships provided through the T32 were instrumental in securing an NIH Loan Repayment Award, an American Diabetes Association Postdoctoral Fellowship, and an NIH Mentored Research Scientist Development (K01) Award. In addition to the supportive and collaborative research environment both within and outside the Division of Endocrinology and Metabolism, the University of Pittsburgh provides outstanding research infrastructure and core resources that helped drive my research and career development forward. This includes resources for genome-wide studies, DNA and protein synthesis and sequencing, animal care, proteomics, imaging, biostatistics, and metabolic studies. I continue to benefit from ongoing multidisciplinary collaborations with expert researchers in a variety of fields, including neuroscience, pharmacology, immunology, vascular medicine, bioinformatics, and genetics. Thus, the Division of Endocrin-ology and Metabolism at the University of Pittsburgh provides an exceptional environment for career development and research success.

I look forward to a long career tackling the most critical questions related to the impact of sex-dependent effect of steroid hormones on neurodevelopment, physiology, and behavior.

Krystle A. Frahm, PhD, is an Instructor in Medicine in the Division of Endocrinology and Metabolism at the University of Pittsburgh and is currently funded by an NIDDK Mentored Research Scientist Career Development Award. Dr. Frahm also received the Endocrine Society Early Investigator Award at the annual meeting in March 2018.

References

1 https://www.niddk.nih.gov/health-information/health-statistics/overweight-obesity

2 Mauvais-Jarvis F. Sex Differences in Metabolic Homeostasis, Diabetes, and Obesity. Biol Sex Differ. 2015 Sep 3; 6: 14.

3 https://grants.nih.gov/grants/guide/notice-files/NOT-OD-15-102.html

4 McGarvey ST. Obesity in Samoans and a Perspective on Its Etiology in Polynesians. Am J Clin Nutr. 1991 Jun; 53(6 Suppl): 1586S-1594S.

5 Frahm KA, Handa RJ, Tobet SA. Embryonic Exposure to Dexamethasone Affects Non-Neuronal Cells in the Adult Paraventricular Nucleus of the Hypothalamus. J Endocr Soc. 2017 Dec 28; 2(2): 140-153.

6 Frahm KA, Tobet SA. Development of the Blood-Brain Barrier Within the Paraventricular Nucleus of the Hypothalamus: Influence of Fetal Glucocorticoid Excess. Brain Struct Funct. 2015 Jul; 220(4): 2225-34.

7 Frahm KA, Peffer ME, Zhang JY, Luthra S, Chakka AB, Couger MB, et al. Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells. Mol Endocrinol. 2016 Jan; 30(1): 144-54.

8 Frahm KA, Waldman JK, Luthra S, Rudine AC, Monaghan-Nichols AP, Chandran UR, DeFranco DB. A Comparison of the Sexually Dimorphic Dexamethasone Transcriptome in Mouse Cerebral Cortical and Hypothalamic Embryonic Neural Stem Cells. Mol Cell Endocrinol. 2018 Aug 15; 471: 42-50.

9 Minster RL, Hawley NL, Su C, Sun G, Kershaw EE, Cheng H, et al. A Thrifty Variant in CREBRF Strongly Influences Body Mass Index in Samoans. Nat Genet. 2016 Sep; 48(9): 1049-1054.

10 Martyn AC, Choleris E, Gillis DJ, Armstrong JN, Amor TR, McCluggage AR, Turner PV, Liang G, Cai K, Lu R. Luman/CREB3 Recruitment Factor Regulates Glucocorticoid Activity and Is Essential for Prolactin-Mediated Maternal Instinct. Mol Cell Biol. Dec; 32(24): 5140-50.

Krystle A. Frahm, PhD 
Research Instructor in Medicine 
Division of Endocrinology and Metabolism