Neuropeptide Y acts within the rat testis to inhibit testosterone secretion
Introduction
The production of testosterone (T) by mammalian testes is under the regulation of complex factors that can be initiated both in the brain and within the male gonads themselves. In the brain, the hypothalamic peptide gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone (LH) production by the pituitary, which then acts on Leydig cells (Breen and Karsch, 2006, Kalra and Kalra, 1983, McCann et al., 1993). Within the gonads, an array of factors that include amines, prostaglandins, opiates and peptides {Refs. in (Rivier, 2008, Saez, 1994)} act locally to regulate the activity of the compounds essential for androgen synthesis, such as the steroidogenic acute regulatory (STAR) protein and the translocator protein (TSPO) previously known as the peripheral-type benzodiazepine receptor and the cytochrome P450 side-chain cleavage enzyme (P450scc) (Bose et al., 2002, Papadopoulos, 1993, Payne and Hales, 2004, Stocco and Clark, 1996).
Recently, neuropeptide Y (NPY) has been added to the list of secretagogues that influence reproductive functions (Pedrazzini et al., 2003). NPY was originally identified in the mammalian brain (Tatemoto et al., 1982) where it is one of the most abundant peptides, and exerts many influences upon endocrine functions (Pedrazzini et al., 2003). For example, it is involved in the seasonal regulation of GnRH and subsequent LH release in male sheep, and does so in a T-dependent manner during a long daylight photoperiod (Dobbins et al., 2004). The ability of this peptide to lower plasma LH levels (Pierroz et al., 1999) was recently corroborated by the finding that it also inhibits the neuronal activity of explanted GnRH neurons (Klenke et al., 2010). In addition, the intracerebroventricular (icv) injection of NPY in mice inhibits the gonadotropic axis by decreasing T secretion (Wahlestedt and Reis, 1993). This is due to either the occurrence of nonfunctional single LH peaks or the absence of LH peak “clusters” (Pierroz et al., 1999). In order to understand how NPY influences steroid hormone release, experiments have focused on the location and direct action sites of this peptide in the testes. Both NPY mRNA levels (Kanzaki et al., 1996) and immunoreactivity (Wang et al., 2004) have been reported in rat Leydig cells, as well as in nerve fibers around the testicular tubules and vessels. Interestingly, NPY gene expression increases with testicular development (Terado et al., 2006). While collectively these results suggested a potential influence of NPY on T production, to our knowledge this has not been reported.
NPY binds to several receptors characterized as Y1, Y2, Y4 and Y5 (Berglund et al., 2003, Larhammar, 1996, Lin et al., 2004, Pedrazzini, 2004, Pedrazzini et al., 2003). A study that examined the regulation of NPY Y1 receptors by testosterone found high levels of their immunoreactivity (ir) and mRNA in the smooth muscle cells of the rat testis (Kopp et al., 1997). As these receptors promote vasoconstriction (Collin et al., 1998, Kopp et al., 2008), it is possible that the potential influence exerted by NPY on Leydig cell activity might be due at least in part to decreased delivery of blood-borne secretagogues to these cells, and/or altered T release into the general circulation. The questions we therefore aimed to ask were: (a) does NPY act within the testes to alter T secretion; and (b) if so, does this involve the inhibition of the proteins that control cholesterol delivery into mitochondria, a rate-limiting step in steroidogenesis. Answering these two questions was accomplished by first examining the direct effects of intra-testicular (itt) injection of NPY on the T response to human chorionic gonadotropin (hCG) administration, a model that we have extensively validated for the investigation of testicular steroidogenic function (Ogilvie and Rivier, 1998, Selvage and Rivier, 2003, Turnbull and Rivier, 1997b). We then measured testicular levels of STAR and TSPO following itt NPY administration according to protocols that we had used in other models (Herman et al., 2006, Herman and Rivier, 2006, Ogilvie et al., 1999). These two proteins were chosen because they modulate essential steps in steroidogenesis (Bauer et al., 2000, Lin et al., 1995, Papadopoulos, 1993, Payne and Hales, 2004). Finally, using a morphological binding assay, in vitro NPY receptor autoradiography, we located the different NPY receptors within the testis to determine where NPY might bind following itt injection.
Section snippets
Animals
Sixty-five to 70 day old adult male Sprague–Dawley or Wistar rats were housed under controlled lighting conditions (lights on at 0600 h and lights off at 1800 h), with water and rat chow provided ad libitum. All protocols were approved by the Salk Institute and Bern University Institutional Animal Care and Use Committees (IACUCs).
Protocols and surgeries
For the experiments illustrated in Fig. 1, the animals were implanted with permanent intravenous (iv) cannulae under isoflurane anesthesia (Selvage and Rivier, 2003).
Effect of itt-injected NPY on T release
Preliminary experiments were carried out to determine the dose of NPY that would be used for all subsequent studies, as well as the optimum timing of its injection. Cumulative T release measured 15, 30, 60 and 90 min after hCG injection were: control (no NPY), 31.5 ± 4.68 ng/ml; 1 μg NPY/kg, 22.69 ± 3.15 ng/ml; 2 μg NPY/kg, 15.3 ± 2.3 ng/ml; 5 μg NPY/kg = 8.8 ± 1.8 ng/ml; 10 μg NPY/kg = 8.5 ± 1.7 ng/ml. On the basis of the results, a 5 μg/kg total dose administered 1 h prior to hCG was chosen as inducing a consistent
Discussion
The work presented here had three goals: to determine whether NPY was able to alter T release independently of LH secretion; to examine the potential role of decreased STAR and TSPO levels as mediators of the influence of NPY on T secretion; and to further support a role of this peptide in the rat testes, by localizing receptors in this organ. We report here that the injection of NPY directly into the testes blunted the T response to hCG. As these results were obtained regardless of whether the
Grant support
The project described was supported by Award Number 5 R01 AA012810 from the National Institute of Alcohol Abuse and Alcoholism. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Alcohol Abuse and Alcoholism or the National Institutes of Health.
Disclosure summary
C.D.A., B.W., M.K., J.C.R., S.L., and C.R. have nothing to disclose.
Acknowledgements
We would like to thank the following institutions and individuals for their generous gifts of reagents: Boehringer Ingelheim, Biberach, Germany (BIIE0246), as well as Dr. Jean Rivier, The Salk Institute, La Jolla (NPY and GnRH antagonist), D.B. Hales, Southern Illinois University School of Medicine at Carbondale, Chicago, IL (STAR antibody) and V. Papadopoulos, McGill University Health Center, Montreal, Quebec, Canada (TSPO antibody). We are also grateful for the excellent technical help of
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