Our study has strengths and limitations We believe we are

Our study has strengths and limitations. We believe we are the 1st to report a novel model of secondary hypogonadism. Several animal models for hypogonadotropic hypogonadism (kisspeptin and the kisspeptin receptor knockout) exist but we are unaware of animal models of secondary hypogonadism with differential FSH and LH expression. Nitroso-redox imbalance in the Gsnor−/− animal model likely causes decreased LH and testosterone owing to nitrosylation of proteins involved in GnRH synthesis. Identifying the mechanism for impaired LH synthesis will enable us to identify causes for secondary hypogonadism and potentially identify therapeutic strategies for treatment of low testosterone other than exogenous testosterone therapy. Some limitations of the study include the small sample (limited by the breeding capabilities of Gsnor−/− mice) and variability in serum LH and testosterone levels in mice. Because of the lack of circulating sex hormone binding globulin, mice have highly fluctuating total testosterone serum concentrations and therefore LH levels are extremely variable. We accounted for this variability using an adequate sample size and drawing the blood in the morning (before 10 am). In addition, we did not investigate whether other pituitary hormones were altered in the Gsnor−/− mice. Moreover, we showed that administration of GnRH and hCG restores testosterone levels, although we did not assess for restored fertility. Future studies will include identifying markers of nitroso-redox imbalance in the hypothalamus such as 3-nitrotyrosine in Gsnor−/− mice. We also will evaluate whether exogenous agents such as GSNO when administered to WT mice can recapitulate the reproductive neuroendocrine phenotype of Gsnor−/− mice.
Conclusion GSNOR deficiency results in secondary hypogonadism and impaired fertility, and this might be mediated in part by increased nitrosative stress. Our results further suggest that nitrosative stress might be affecting the hypothalamic-pituitary-gonadal axis at the level of the hypothalamus because the function of the testis and pituitary remains intact in Gsnor−/− mice.
Statement of authorship
Introduction Nitric oxide (NO) acts as intracellular second messenger with regulatory roles in an array of physiological processes. Biological effects of NO are coordinated by systems which guarantee the appropriate balance between synthesis and degradation of adducts that NO is enable to form with chemical species present in cellular molecules and proteins. Nitric oxide-mediated “canonical” signal transduction takes place by its direct binding with iron located at the Fe-S cluster of Epoxomicin (e.g., aconitase) [1], or with Fe-heme group in heme-containing proteins, such as soluble guanylyl cyclase (sGC) [2] and cytochrome c oxidase [3] (Fig. 1). These modifications can activate or inhibit enzyme activities and are reversible, as iron moiety can both easily bind NO and release it as nitrite. Nevertheless, in the last decades it has become evident that the main mechanism by which NO exerts its signaling function goes through S-nitrosylation of cysteine residues [4]. S-nitrosylation is a redox-based posttranslational modification consisting of the covalent addition of an NO group to a reactive sulfhydryl (SH), this generating an S-nitrosothiol (SNO) [5] (Fig. 1). Similar to other redox (e.g., S-hydroxylation), and non-redox posttranslational modifications involved in signal transduction mechanisms (e.g., phosphorylation, acetylation), S-nitrosylation is reversible because of the activity of enzymatic systems aimed at reducing SNOs back to SH, reaction called denitrosylation (Fig. 2). However, at variance with de-phosphorylation and de-acetylation – in which modified residues are the direct substrates of specific classes of enzymes (i.e., phosphatases or deacetylases) – reduction of redox-modified cysteines is indirect and requires intermediate molecules, namely, glutathione (GSH) and thioredoxin (Trx) (Fig. 2, Fig. 3). By means of a non-enzymatically driven thiol exchange, GSH and Trx restore the reduced state of target cysteines, with their oxidized (disulfide- or nitroso-containing) forms generated upon reaction being the substrate of different NAD(P)H-dependent reductases (Fig. 2, Fig. 3). For what specifically concerns S-nitrosylation, Trx-mediated SNO-to-SH conversion involves the production of i) nitroxyl (HNO) or free NO groups, both representing still diffusible and reactive NO moieties and ii) Trx-disulfide, which is fully reduced by Trx reductase (TrxR) [6], [7], [8], [9] (Fig. 3). Otherwise, GSH-dependent protein denitrosylation generates GSNO, a relatively stable compound acting as the main nitroso reservoir upon massive NO production [4], [10]. Through this alternative mechanism, NO moiety is exchanged from protein-SNO to GSH (trans-nitrosylation), thus making it unable to freely diffuse (and react) in the surrounding environment. GSNO represents the elective substrate of S-nitrosoglutathione reductase (GSNOR) through which it is completely reduced to glutathione disulfide (GSSG) and ammonia (NH3) (Fig. 3). Cellular levels of GSNO are in equilibrium with a great number of PSNOs. Therefore, by directly catabolizing GSNO, GSNOR indirectly controls PSNOs amount and PSNO-associated signaling [11] (Fig. 2). Other enzymes have been shown being able to catalyze SNOs breakdown in vitro [12], [13], however none of them has been so far demonstrated to regulate the levels of endogenous SNOs in cells. By contrast, GSNOR has been frequently reported playing a crucial role in keeping GSNO/PSNOs balance in physiological contexts [14], [15], with downregulation, or lack of its expression deeply affecting cellular processes and healthy state of the whole organism.