Major Projects

Anatomical and functional identification of a novel hypothalamic-testicular pathway.

Mapping of a novel neural pathway. The ability of various stressors to lower plasma testosterone (T) levels is well known. It has usually been attributed to the inhibitory effect of these stressors on LH release, itself a consequence of impaired production of the hypothalamic peptide luteinizing hormone-releasing hormone (LHRH) (also called GnRH). However, there are many cases of decreased T secretion in the absence of measurable changes in LH production. In the past, stress-induced testicular synthesis of factors that inhibit Leydig cell function, such as opiates and CRF, has been invoked to explain the ability of homeostatic challenges to act directly at the level of the male gonad to impair steroid synthesis. We recently made a discovery that suggests the involvement of another mechanism. Specifically, we demonstrated that the intracerebroventricular (icv) injection of pro-inflammatory cytokines, CRF, catecholamines or a small amount of alcohol produced a profound inhibition of Leydig cell responsiveness to gonadotropin.  These responses were not altered by pretreatment of the animals with a potent GnRH antagonist, thereby demonstrating that the influence of icv administered signals did not involve pituitary LH. The extreme rapidity of this response suggested that icv treatments activated a neural pathway, possibly originating in the hypothalamus, that controls testicular activity independently of the pituitary. The observation that the icv injection of adrenergic antagonists interfered with the inhibitory effect of the treatments listed above, suggested that this pathway was dependent on catecholamines. We recently mapped this neural circuit with tracing techniques based on the intratesticular injection of Pseudorabies virus (PRV), and showed that it plays an important role in the ability of alcohol to lower T levels. In rats, spinal cord injury virtually abolished the appearance of PRV in the brain and prevented the inhibitory effect of icv-injected CRF and IL-1b on the T response to hCG. Very recently, we identified the paraventricular nucleus (PVN) of the hypothalamus as an important component of the proposed circuit. We showed that microinfusion of CRF or the a-adrenergic agonist isoproterenol into the PVN mimicked the effect of the icv injection of these secretagogues. Reciprocally, PVN lesions significantly blunted the ability of icv CRF or isoproterenol to decrease hCG-induced T release. We therefore investigated the role of catecholamines in the PVN and functional connected regions such as the locus coeruleus, in mediating the inhibitory influence of stress-related signals on testicular activity. The icv injection of 5 ml of 200 proof EtOH (86 mmole) did not result in detectable levels of the drug in the general circulation and did not induce neuronal damage, but rapidly blunted hCG-induced T release while not decreasing LH levels, nor altering testicular blood flow. This was a particularly important finding because many investigators had suggested that at least part of the inhibitory effect of icv-injected agents on testicular activity might be due to altered testicular blood flow. Our results conclusively showed that this was not the case. We also found that EtOH significantly upregulated transcripts of the immediate early gene c-fos in the PVN of the hypothalamus. Lesions of the PVN blocked the inhibitory effect of IL-1b on T, but only partially interfered with the influence of EtOH. PVN catecholamine turnover significantly increased following icv injection of IL-1b, but not EtOH. Brain catecholamine depletion due to the neurotoxin 6-hydroxydopamine did not alter the ability of hCG to induce T release, but significantly reversed the inhibitory effect of icv EtOH or IL-1b on this response. Collectively, these results indicate that icv-injected IL-1b or EtOH blunt hCG-induced T secretion through a catecholamine-mediated mechanism that does not depend on either peripherally-mediated effects or pituitary LH, and that the PVN plays a role in these effects.

Site of action of alcohol. While they answered several questions, these experiments also begged the issue of whether alcohol consumed via the normal stomachal way inhibits testicular function primarily through a central site of action, a testicular site of action, or both. It is well established that systemic alcohol exposure lowers plasma levels in adult males, but the relative role of impaired LHRH synthesis and decreased pituitary LH release versus that of a direct ability of circulating alcohol to inhibit testicular steroidogenesis remains poorly understood. We had reported preliminary evidence that alcohol might stimulate a pituitary-independent, neural pathway between the hypothalamus and the testes whose activation blunts T secretion in response to hCG. We then further investigated the influence of alcohol on this pathway by comparing the influence of the intragastric (ig) and icv injection of alcohol on the T response to hCG.  Both treatments significantly blunted the T response to hCG, although only if injected alcohol led to detectable drug levels in the circulation. This effect was comparable in 40- and 65-d-old rats, which indicated that the pathway appears functional in young rats. Subsequent experiments done in prepubertal  (35-d-old) rats injected with CRF icv also supported the concept that stimulation of the neural brain-testicular pathway can already inhibit Leydig cell activity at a very early age.  As observed before, neither prior blockade of LHRH receptors with a potent LH releasing hormone antagonist nor immunoneutralization of endogenous CRF altered the inhibitory effect of alcohol injected icv or ig on T secretion. Collectively, our results indicate that alcohol can act within the brain to influence testicular activity independently of LH, hormones of the HPA axis and/or of the presence of the drug in the circulation.

Role of altered testicular steroidogenic enzymes. We recently turned to the identification of the testicular molecules that may mediate the influence of this pathway. In other words, what are the neurotransmitters or peptides that brain stress signals release in the testes that interfere with steroidogenesis? To elucidate these signals, we investigated the influence of icv-injected ISO, CRF or EtOH on levels of the steroidogenic acute regulatory (StAR) protein, the peripheral-type benzodiazepine receptor (PBR) and the cytochrome P450 side-chain cleavage enzyme (P450scc) in semi-purified Leydig cells. ISO (10 mg), CRF (5 mg) or EtOH (5 ml of 200 proof, a dose that does not induce neuronal damage nor leak to the periphery), rapidly decreased StAR and PBR, but not P450scc protein levels.  We knew that the unstable gas nitric oxide (NO) is found in the testes, where it is thought to exert inhibitory effects on steroidogenic enzymes.   A125 kDa isoform alternate promoter of the 150 kDa human neuronal NOS, the constitutive enzyme responsible for NO formation, was recently found in mouse Leydig cells and called T neuronal (n)NOS. We found that ISO, CRF and EtOH significantly upregulated TnNOS in Leydig cells over the time-course of altered StAR/PBR concentrations. However, pretreatment of the rats with Nwnitro-arginine methylester (L-NAME), which blocked ISO-induced increases in TnNOS, neither restored the T response to hCG nor prevented the decreases in StAR and PBR. These results indicate that activation of a neural brain-testicular pathway rapidly decreases concentrations of  StAR and PBR. While they also increased testicular NO production, this phenomenon does not appear to play a physiological role in our model.

Role of CRF-related peptides in the testes.  CRF has previously been reported in rat testes where it inhibits Leydig cells activity.  However, recent studies in our laboratory have suggested that some of the effects originally attributed to CRF were instead due to the related peptide Urocortin 1 (Ucn 1) and that this latter hormone, not CRF, was detectable in Leydig cells.  We show here that Ucn 1 [a mixed CRF receptors type 1 (CRFR1) and CRFR2 agonist] and the CRFR1-selective peptide stressin 1, but not Ucn 2 or Ucn 3 (both considered selective CRFR2 ligands), significantly blunt the testosterone (T) response to human chorionic gonadotropin (hCG).  The effect of Ucn 1 is observed regardless of whether this peptide is injected intravenously or directly into the testes, and it is reversed by the mixed CRFR1/R2 antagonist Astressin B.  Blockade of gonadotropin-releasing hormone receptors with the antagonist Azaline B does not interfere with the influence of Ucn 1, thereby demonstrating that pituitary luteinizing hormone does not appear to be involved in this model.  Collectively, these results suggest that Ucn 1, not CRF, is present in the rat testes and interferes with Leydig cells activity.  However, while we previously reported that alcohol upregulated gonadal Ucn 1 gene expression, CRF receptor antagonists were unable to reverse the inhibitory effect exerted by alcohol on hCG-induced T release.  The functional role played by testicular Ucn 1 in stress models characterized by blunted androgen levels, therefore needs to be further investigated.

Conclusion. We hypothesize that the brain-testicular connection that we have uncovered may be relevant in pathologies, such as brain injury or conditions that induce catecholamine synthesis in the CNS, that are associated with impaired testicular function. In addition, this pathway may play a role in androgen-dependent functions that are unrelated to fertility, such as cardiovascular, renal and immunological activity, muscle mass, behavior and cognitive abilities. These are usually considered genomic effects of androgens. In addition, testosterone exerts many very rapid, non-genomic effects as varied as airway smooth muscles reactivity, as well as reward, learning and analgesia. Because of their rapidity, these effects would be good candidates for regulation by an equally rapid and fast responsive neural pathway. The activation of the hitherto unknown circuitry that we recently described may thus help explain the mechanisms through which the brain monitors environmental stress and alters testosterone secretion accordingly.

References

Rivest S and Rivier C. 1995 The role of corticotropin-releasing factor and interleukin-1b in the regulation of neurons controlling reproductive functions. Endocr Rev 16:177-199.

Turnbull AV and Rivier C. 1997 Inhibition of gonadotropin-induced testosterone secretion by the intracerebroventricular injection of interleukin-1b in the male rat. Endocrinology 138:1008-1013.

Ogilvie K and Rivier C. 1998 The intracerebroventricular injection of interleukin-1b blunts the testosterone response to human chorionic gonadotropin: Role of corticotropin-releasing factor and adrenergic-dependent pathways. Endocrinology 139:3088-3095.

Rivier C. 1999 Alcohol rapidly lowers plasma testosterone levels in the rat: Evidence that a neural brain-gonadal pathway may be important for decreased testicular responsiveness to gonadotropin. Alcoholism: Clin Exp Res 23:38-45.

Lee S, Miselis R and Rivier C. 2002 Anatomical and functional evidence for a neural hypothalamic-testicular pathway that is independent of the pituitary. Endocrinology 143:4447-4454.

Rivier C. 2002 Inhibitory effect of neurogenic and immune stressors on testosterone secretion in rats. NeuroImmunoModulation 10:17-29.

Selvage DJ and Rivier C. 2003 Importance of the paraventricular nucleus of the hypothalamus as a component of a neural pathway between the brain and the testes that modulates testosterone secretion independently of the pituitary. Endocrinology 144:594-598.

Selvage D, Lee S, Parsons L, Seo D and Rivier C. 2004 A hypothalamic-testicular neural pathway is influenced by brain catecholamines, but not testicular blood flow. Endocrinology 145:1750-1759.

Selvage DJ and Rivier C. 2004 Leydig cell activity is regulated by a neural pathway between the hypothalamus and the testes that does not include the pituitary. In: Recent Research Developments in Endocrinology. Transworld Research Network, Kerala, India, pp 97-114.

Selvage D, Hales D and Rivier C. 2004 Comparison between the influence of the systemic and central injection of alcohol on Leydig cell activity. Alcoholism: Clin Exp Res 28:480-48.

Rivier C. 2004 Role of pro-inflammatory cytokines in regulating the hypothalamic-pituitary-gonadal axis of the male rat, in Geenen V, Chrousos G (eds): Immunoendocrinology in Health and Disease. New York, Marcel Dekkker, Inc., pp 107-126.

Herman M and Rivier C. 2006 Activation of a neural brain-testicular pathways lowers Leydic cell levels of the steroidogenic acute regulatory protein and the peripheral-type benzodiazepine receptor while increasing levels of neural nitric oxide synthase. Endocrinology 147:624-633.

Herman M, Kang SS, Lee S, James P and Rivier C. 2006 Systemic administration of alcohol to adult rats inhibits Leydig cell activity: Time-course of effect and role of nitric oxide. Alcoholism: Clin Exp Res 30:1-13.

Selvage DJ, Parsons L and Rivier C. 2006 Role played by brain stem neurons in retulating testosterone secretion via a direct neural pathway between the hypothalamus and the testes. Endocrinology 147:3070-3075.

Lee SO, Braden B, Kang S and Rivier CL. 2007 Alcohol increases urocortin gene expression in rat testes. 37th Annual Meeting of the Society for Neuroscience: San Diego, CA.

James PJ, Rivier C and Lee S. 2008 Presence of corticotropin-releasing factor and/or tyrosine hydroxylase in cells of a neural brain-testicular pathway that are labelled by a transganglionic tracer. J Neuroendocrinol 20:173-181.

Rivier CL. 2008 Urocortin 1 inhibits rat leydig cell function. Endocrinology 149:6425-6432.  PMCID: PMC2613056

Allen CD, Waser B, Körner M, Reubi JC, Lee S and Rivier C. 2011 Neuropeptide Y acts within the rat testis to inhibit testosterone secretion. Neuropeptides 45:55-61. PMCID: PMC3053052.