Abstract
Orexin A (OxA) is a neuropeptide produced in the lateral hypothalamus that performs pleiotropic functions in different tissues, including involvement in energy homeostasis and reproductive neuroendocrine functions. The role of OxA is particularly important given the well-studied relationships between physiological mechanisms controlling energy balance and reproduction. The enzyme P450 aromatase (ARO) helps convert androgens to estrogens and has roles in steroidogenesis, spermatogenesis, and energy metabolism in several organs. The goal of this study was thus to investigate the role of OxA in ARO activity and the effects of this regulation on reproductive homeostasis in male gonads from healthy and cryptorchid dogs. The cryptorchidism is a specific condition characterized by altered reproductive and metabolic activities, the latter of which emerge from impaired glycolysis. OxA helps to stimulate testosterone (T) synthesis in the dog testis. We aimed to investigate OxA-mediated modulation of 17β-estradiol (17β-E) synthesis, ARO expression and metabolic indicators in testis of normal and cryptorchid dogs. Our results indicate putative effects of OxA on estrogen biosynthesis and ARO activity based on western blotting analysis and immunohistochemistry for ARO detection and in vitro tests. OxA triggered decrease in estrogen production and ARO activity inhibition; reduced ARO activity thus prevented the conversion of T to estrogens and increasing OxA-mediated synthesis of T. Furthermore, we characterized some metabolic and oxidative modulations in normal and cryptorchid dog's testis. The steroidogenic regulation by OxA and its modulation of ARO activity led us to hypothesize that OxA is a potential therapeutic target in pathological conditions associated with steroidogenic alterations and OxA possible involvement in metabolic processes in the male gonad.
Highlights
Spermatogenesis is a biological process in animals that requires additional energy stores for performance
We recently demonstrated a possible relationship between Orexin A (OxA) and ARO expression in alpaca testis [48], it is evident that OxA, is able to significantly decrease basal 17β-E secretion and OxA acts through decreasing ARO activity
Rabbit anti-glucose transporters (Glut) 3 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, United States); anti-rabbit phopsho AKT (Ser473) (#4060), anti-mouse AKT (#2920) and antirabbit superoxide dismutase (SOD)2 (D3X8F) (#13141) antibodies were from Cell Signaling Technology (Danvers, MA, United States), biotinylated goat anti-rabbit (BA-1000) secondary antibody, peroxidase-conjugated rabbit anti-goat (PI-9500) IgG, VECTASTAIN ABC kit (PK-6105), and 3,3′-diaminobenzidine tetra-hydrochloride (DAB) solution were obtained from Vector Laboratories (Burlingame, CA, USA); peroxidase-conjugated goat anti-rabbit IgG (111-035-003) and peroxidase-conjugated goat anti-mouse IgG (115-035-003) were purchased form Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, United States)
Summary
Spermatogenesis is a biological process in animals that requires additional energy stores for performance. Androgens, estrogens, and other testicular factors appear to have important roles in metabolic process control of testis. ARO is an enzymatic complex composed of a ubiquitous NADPH-cytochrome P450 reductase and a cytochrome P450 aromatase, which contains the steroid-binding site [9] This enzyme complex is localized to the endoplasmic reticulum of many different areas of the body, including the testis [10], in which its distribution changes during development, being primarily located within Sertoli cells in immature animals and in Leydig and germ cells of mature animals [7, 9, 11, 12]. In light of its importance, ARO activity must be finely regulated [3, 20]
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