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Role of HPA and the HPG Axis Interaction in Testosterone-Mediated Learned Helpless Behavior

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Abstract

Affective disorders show sex-specific differences in prevalence, symptoms, and complications. One hypothesis for this discrepancy is the interaction between the hypothalamic-pituitary-adrenal (HPA) axis and hypothalamic-pituitary-gonadal (HPG) axis. The present study investigates the influence of androgen on the behavioral phenotype and explores how it interacts with HPA axis genes. Gonadectomized (GDX) and GDX rats treated with testosterone propionate (T) were tested for learned helplessness (LH) behavior and compared with tested controls (TC). Prefrontal cortex was used for analyses of HPG- axis-related genes (androgen receptor, (Ar); estrogen receptor-β (Er-β)) and HPA axis-related genes (corticotropin-releasing hormone, (Crh); glucocorticoid receptor, (Nr3c1); corticotropin-releasing hormone receptor 1, (Crhr1); corticotropin-releasing hormone receptor 2, (Crhr2); FK506 binding protein 5, (Fkbp5)). Promoter-specific CpG methylation in the Crh gene was determined by bisulfite sequencing. Chromatin immunoprecipitation (ChIP) assay was used for determining ER-β binding on the proximal promoter region of Crh gene. Serum testosterone levels confirmed a testosterone-depleted GDX group, a group with supraphysiological levels of testosterone (T) and another group with physiological levels of testosterone (control (C)). Unlike GDX rats, T group exhibited significantly higher LH score when compared with any other group. Crh and Fkbp5 genes were significantly upregulated in GDX group compared with controls, whereas Er-β showed a significant downregulation in the same group. Methylation analysis showed no significant differences in-between groups. ChIP assay was unable to determine a significant change in ER-β binding but revealed a notable contrast in Crh promoter occupancy between T and GDX groups. Altogether, the present study reveals an increased susceptibility to depression-like behavior due to chronic supraphysiological level of androgen via HPA axis inhibition.

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Abbreviations

HPA:

Hypothalamic-pituitary-adrenal axis

HPG:

Hypothalamic-pituitary-gonadal axis

GDX:

Gonadectomized

T:

Testosterone propionate

LH:

Learned helplessness

TC:

Tested controls

Ar:

Androgen receptor

ER-β:

Estrogen receptor-β

Crh:

Corticotropin-releasing hormone

Nr3c1:

Glucocorticoid receptor

Crhr:

Corticotropin-releasing hormone receptor

Fkbp5:

FK506 binding protein 5

ChIP:

Chromatin immunoprecipitation

PFC:

Prefrontal cortex

PND:

Postnatal day

ELISA:

Enzyme-linked immunosorbent assay

ET:

Escape test

IS:

Inescapable shock

DTT:

Dithiothreitol

qPCR:

Quantitative polymerase chain reaction

Gapdh:

Glyceraldehyde 3-phosphate dehydrogenase

cDNA:

Complementary DNA

PBS:

Phosphate buffered saline

PI:

Proteasomal inhibitors

RPM:

Revolution per minute

TE:

Tris-ethylenediaminetetraacetic acid

LiCl:

Lithium chloride

FST:

Forced swim test

AAS:

Anabolic androgenic steroid

3-β-diol:

5-Alpha-androstane 3beta,17beta diol

ERE:

Estrogen receptor elements

cAMP:

cAMP-response elements

AP-1:

Activator protein-1

PVN:

Periventricular nucleus

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Funding

The research was supported by grants from the National Institute of Mental Health (R01MH082802, 1R01MH101890, R01MH100616, 1R01MH107183-01) to Dr. Dwivedi. The sponsoring agency had no role in study design, collection, analysis, interpretation of data, and in the writing of the manuscript.

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  1. Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, SC711 Sparks Center, 1720 7th Avenue South, Birmingham, AL, 35294, USA

    Birgit Ludwig, Bhaskar Roy & Yogesh Dwivedi

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  1. Birgit Ludwig
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Correspondence to Yogesh Dwivedi.

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Research Involving Animals

This research involves the use of animals. The study was approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.

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Electronic Supplementary Material

Fig. S1

A schematic overview representing the groupwise distribution of rats under the present study. A total of 30 Long Evans rats were randomly assigned to treatments (GDX = 10 rats having undergone gonadectomy, T = 10 rats having undergone gonadectomy and daily supraphysiological testosterone injections, C = 10 naive controls). For behavioral testing, all 30 rats were further randomly assigned to be restrained only (tested controls (TC)) or restrained and exposed to inescapable shocks (IS). (PNG 23 kb)

Fig. S2

Methylation of Crh gene promoter. a The sequence represents 420 bp upstream region of rat Crh promoter with CpG sites highlighted, indicating their position relative to transcription start site (right arrow). b The checker board represents methylation status of individual CpG site as identified from each animal considering individual groups, all methylated cytosine (C) not converted thymine (T) are presented with double-plus symbol. Whereas, identification of both C and T peaks is represented with plus symbol, this indicates potentially incomplete bisulfite conversion. (DOCX 24 kb)

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Ludwig, B., Roy, B. & Dwivedi, Y. Role of HPA and the HPG Axis Interaction in Testosterone-Mediated Learned Helpless Behavior. Mol Neurobiol 56, 394–405 (2019). https://doi.org/10.1007/s12035-018-1085-x

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