Prolonged in vivo administration of testosterone-enanthate, the widely used and abused anabolic androgenic steroid, disturbs prolactin and cAMP signaling in Leydig cells of adult rats

Highlights

• Administration of testosterone-enanthate for 10-weeks increased prolactin in pituitary.

• Prolactin and increased Jak2/Jak3 could account for increased Hsd3b1/5 in Leydig cells.

• Elements of cAMP signaling were disturbed with remarkable up-regulation of PRKA.

• In vitro testosterone addition increase Prlr1, Prlr2, Hsd3b1/5 in Leydig cells.

• Prolactin signaling and PRKA contribute in a new adaptive response of Leydig cells.

Abstract

This study was designed to systematically analyze and define the effects of 1-day, 2-weeks, 10-weeks intramuscular administration of testosterone-enanthate, widely used and abused anabolic androgenic steroid (AAS), on main regulators of steroidogenesis and steroidogenic genes expression in testosterone-producing Leydig cells of adult rats. The results showed that prolonged (10-weeks) intramuscular administration of testosterone-enanthate, in clinically relevant dose, significantly increased prolactin, but decreased Prlr2 and Gnrhr in pituitary of adult rat. The levels of testosterone, Insl3, cAMP and mitochondrial membrane potential of Leydig cells were significantly reduced. This was followed by decreased expression of some steroidogenic enzymes and regulatory proteins such as Lhcgr, Prlr1/2, Tspo, Star, Cyp11a1, Cyp17a1, Dax1. Oppositely, Hsd3b1/2, Hsd3b5, Hsd17b4, Ar, Arr19 increased. In the same cells, transcriptional milieu of cAMP signaling elements was disturbed with remarkable up-regulation of PRKA (the main regulator of steroidogenesis). Increased prolactin together with stimulated transcription of Jak2/Jak3 could account for increased Hsd3b1/2 and Hsd3b5 in Leydig cells following 10-weeks in vivo treatment with testosterone-enanthate. In vitro studies revealed that testosterone is capable to increase level of Prlr1, Prlr2, Hsd3b1/2, Hsd3b5 in Leydig cells. Accordingly, testosterone-induced changes in prolactin receptor signaling together with up-regulation of PRKA, Hsd3b1/2, Hsd3b5, Ar in Leydig cells, could be the possible mechanism that contribute to the establishment of a new adaptive response to maintain homeostasis and prevent loss of steroidogenic function. Presented data provide new molecular insights into the relationship between disturbed testosterone homeostasis and mammalian reproduction and are important in terms of wide use and abuse of AASs and human reproductive health.

Introduction

Testosterone (T) and dihydrotestosterone (DHT) are the principal androgenic steroids essential for development and maintenance of the male phenotype including reproductive function [1], [2]. They bind to the androgen receptor (AR) to modulate gene transcription in target cells and/or interact with membrane receptor/second messenger cascades [2], [3]. Anabolic androgenic steroids (AASs), testosterone derivatives are originally designed to enhance muscular mass and they are used for the treatment of many clinical conditions and in contraception [2], [4], [5], [6], [7], [8], [9]. Recent estimations indicate an increasing misuse of AASs (beside professional and amateur athletes) by adolescents and rank the relative harm of AASs within a selection of 19 illicit drugs, including heroin, cocaine, ecstasy and cannabis [9]. Potential health risks associated with non-therapeutic use of AASs (such as decreased sperm production, testicular atrophy, gynecomastia, increased chance of heart attacks, mood changes, liver cancer, etc.) by large number of healthy individuals are believed to be high [2], [5], [8], [10]. AASs disturb the regular endogenous production of androgens and gonadotrophins which can in turn affect the ultrastructure of the testes that may persist for months after drug withdrawal [8], [11], [12]. In vivo administration of AAS impaired Leydig cells steroidogenesis, induced modulation of NO-cGMP signaling pathway and apoptosis in Leydig cells of adult rats [13], [14], [15].

Like all other steroid-producing cells, Leydig cells synthesize testosterone from a common precursor cholesterol using the steroidogenic machinery comprises of cholesterol transporters, steroidogenic enzymes and many regulatory molecules [16], [17], [18], [19], [20], [21]. The steroidogenic function of Leydig cell is predominantly regulated by pituitary luteinizing hormone (LH) and its receptor (LHCGR) through stimulation of adenylyl cyclases (ADCYs), accumulation in cAMP and activation of the cAMP-dependent kinase (PRKA). The cGMP signaling also stimulate testosterone production [14], while phosphodiesterases (PDEs) terminate cAMP/cGMP signaling and have regulatory function in Leydig cells [20], [21], [22], [23], [24]. Many other signaling molecules, including ERK, PRKC, PLC, osteocalcin, Ca²⁺ channels, Gonadotropin-regulated testicular RNA helicase also regulate androgen production [25], [26], [27], [28], [29], [30], [31]. Although Leydig cell steroidogenesis is mainly activated through activation of LH receptors, regulation itself is multi-compartmental process comprises of neural and complex endocrine, paracrine and autocrine signaling pathways (reviewed in [19], [32], [33], [34]). In addition, many transcription factors are involved in regulation of the steroidogenic machinery expression (reviewed in [35], [36], [37]). All mentioned molecules might be involved in the regulation of testosterone production, providing a molecular adaptive mechanism by which testicular structures, including Leydig cells, recover from disturbed homeostasis Fig. 1.

It has been recently published that abuse of AASs is growing public health problem, but mechanisms by which they exert their adverse health effects also remain unclear and need further investigation [2]. While human studies of these abused drugs are made difficult by their illicit nature, it was suggested that many of the AASs-induced changes should be recapitulated in animals as useful models for studying the molecular events that contribute to the adverse effects of AASs [38].

This study was designed to examine the in vivo effect of testosterone-enanthate (TE), widely used/abused AAS, applied in vivo, on components related to steroidogenic functions of Leydig cells. TE is the most extensively studied AAS for suppression of spermatogenesis and used in hypogonadal animals as replacement therapy, as well as the most commonly used form of testosterone by both athletes and bodybuilders [4], [5], [6], [13], [14], [15]. The in vivo experimental model and doses used in the study are comparable with those used in clinical applications or abused and all were published before [13], [14], [15]. The focus in our study was on molecular markers of Leydig cells functionality, as well as genes/proteins involved and/or related to testosterone synthesis and regulation including elements of cAMP signaling (the main regulator of steroidogenesis) and prolactin signaling in Leydig cells.