Aging in the Syrian hamster testis: Inflammatory-oxidative status and the impact of photoperiod.

Testicular aging is linked to histological, morphological and functional alterations. In the present study, we investigated whether aging affects the inflammatory and oxidative status in the testis by comparing young adult, middle-aged adult and aged hamsters. The Syrian hamster, a thoroughly studied seasonal breeder, was chosen as the experimental model since it allows further investigations on the role of photoperiod and melatonin in testicular aging with a minimal impact of the experimental intervention on the animal well-being and the subsequent results achieved. In testes of aged hamsters, we found a decrease in melatonin concentration, a thickening of the wall of the seminiferous tubules as well as a significant increase in IL-1ß, NLRP3 and cyclooxygenase 2 expression, PGD2 production, macrophages numbers, lipid peroxidation and anti-oxidant enzyme catalase levels. Interestingly, when aged hamsters were transferred from a long day (LD) to a short day (SD) photoperiod for 16 weeks, testicular melatonin concentration increased while local inflammatory processes and oxidative stress were clearly reduced. Overall, these results indicate that melatonin might display anti-inflammatory and anti-oxidant capacities in the aged testes.

Ageing and inflammation in the male reproductive tract.

Ageing is usually characterised by a mild chronic proinflammatory state. Despite the tight association between both processes, the phenomenon has recently been termed inflammageing. Inflammation in the male reproductive tract is frequently linked with bacterial or virus infections but also with a broad range of noninfectious processes. Prostatitis, epididymitis and orchitis, among others, can lead to infertility. However, in spite of the inflammation theory of disease, chronic inflammation in male urogenital system does not always cause symptoms. With advancing age, inflammatory processes are commonly observed in the male reproductive tract. Nevertheless, the incidence of inflammation in reproductive organs and ducts varies greatly among elderly men. Inflammageing is considered a predictor of pathogenesis and the development of age-related diseases. This article briefly summarises the current state of knowledge on inflammageing in the male reproductive tract. Yet, the precise aetiology of inflammageing in the male urogenital system, and its potential contribution not only to infertility but most importantly to adverse health outcomes remains almost unknown. Thus, further investigations are required to elucidate the precise cross-links between inflammation and male reproductive senescence, and to establish the impact of anti-inflammatory drug treatments on elder men's general health status.

Melatonin in testes of infertile men: evidence for anti-proliferative and anti-oxidant effects on local macrophage and mast cell populations.

Melatonin acting through the hypothalamus and pituitary regulates testicular function. In addition, direct actions of melatonin at the testicular level have been recently suggested. We have described that melatonin inhibits androgen production in hamster Leydig cells via melatonin subtype 1a (mel1a) receptors and the local corticotrophin-releasing hormone (CRH) system. The initial events of the melatonin/CRH signalling pathway have also been established. Melatonin and all components of the melatonergic/CRH system were also detected in Leydig cells of infertile men. This study attempted to search for additional targets of melatonin in the human testis, and to investigate the effects of melatonin on proliferation and the oxidative state in these novel target cells. To this aim, evaluation of human testicular biopsies of patients suffering from hypospermatogenesis or Sertoli cell only syndrome and cell culture studies were performed. Melatonergic receptors were found in macrophages (MACs) and mast cells (MCs) of the human testis. In biopsies of patients suffering idiopathic infertility, melatonin testicular concentrations were negatively correlated with MAC number per mm(2) and TNFα, IL1ß and COX2 expression, but positively correlated with the expression of the anti-oxidant enzymes SOD1, peroxiredoxin 1 and catalase. Melatonin inhibited proliferation and the expression of pro-inflammatory cytokines and cyclooxygenase 2 (COX2) in both the human non-testicular THP-1 MAC cell line and primary cell cultures of hamster testicular MACs. In the human HMC-1 MC line, melatonin increased the expression of anti-oxidant enzymes and decreased reactive oxygen species (ROS) generation. The results reveal new testicular targets of melatonin and describe anti-proliferative and anti-inflammatory effects of this hormone on testicular MACs. Furthermore, melatonin might provide protective effects against oxidative stress in testicular MCs.

The Ca2+-activated, large conductance K+-channel (BKCa) is a player in the LH/hCG signaling cascade in testicular Leydig cells.

In Leydig cells, hormonal stimulation by LH/hCG entails increased intracellular Ca(2+) levels and steroid production, as well as hyperpolarization of the cell membrane. The large-conductance Ca(2+)-activated K(+)-channel (BK(Ca)) is activated by raised intracellular Ca(2+) and voltage and typically hyperpolarizes the cell membrane. Whether BK(Ca) is functionally involved in steroid production of Leydig cells is not known. In order to explore this point we first investigated the localization of BK(Ca) in human and hamster testes and then used a highly specific toxin, the BK(Ca) blocker iberiotoxin (IbTx), to experimentally dissect a role of BK(Ca). Immunohistochemistry and RT-PCR revealed that adult Leydig cells of both species are endowed with these channels. Ontogeny studies in hamsters indicated that BK(Ca) becomes strongly detectable in Leydig cells only after they acquire the ability to produce androgens. Using purified Leydig cells from adult hamsters, membrane potential changes in response to hCG were monitored. HCG hyperpolarized the cell membrane, which was prevented by the selective BK(Ca) blocker IbTx. Steroidogenic acute regulatory (StAR) mRNA expression and testosterone production were not affected by IbTx under basal conditions but markedly increased when hCG, in submaximal and maximal concentration or when db-cAMP was added to the incubation media. A blocker of K(V)4-channels, expressed by Leydig cells, namely phrixotoxin-2 (PhTx-2) was not effective. In summary, the data reveal BK(Ca) as a crucial part of the signaling cascade of LH/hCG in Leydig cells. The hyperpolarizing effect of BK(Ca) in the Leydig cell membrane appears to set in motion events limiting the production of testosterone evoked by stimulatory endocrine mechanisms.

Evidence for an adaptation in ROS scavenging systems in human testicular peritubular cells from infertility patients.

Fibrosis, increased amounts of immune cells and expression of COX-2 in the testes of infertility patients provide circumstantial evidence for a specific testicular milieu, in which reactive oxygen species (ROS) could be increased. If ROS level increase and/or ROS scavengers decrease, the resulting testicular oxidative stress may contribute to human male infertility. Primary peritubular cells of the human testis, from men with normal spermatogenesis (HTPCs) and infertile patients (HTPC-Fs), previously allowed us to identify an end product of COX-2 action, a prostaglandin derivative (15dPGJ2), which acts via ROS to alter the phenotype of peritubular cells, at least in vitro. Using testicular biopsies we now found 15dPGJ2 in patients and hence we started exploring the ROS scavenger systems of the human testis. This system includes catalase, DJ-1, peroxiredoxin 1, SOD 1 and 2, glutathione-S-transferase and HMOX-1, which were identified by RT-PCR/sequencing in HTPCs and HTPC-Fs and whole testes. Catalase, DJ-1, peroxiredoxin 1 and SOD 2 were also detected by Western blots and in part by immunohistochemistry in testicular samples. Western blots of cultured cells further revealed that catalase levels, but not peroxiredoxin 1, SOD 2 or DJ-1 levels, are significantly higher in HTPC-Fs than in HTPCs. This particular difference is correlated with the improved ability of HTPC-Fs to handle ROS, which became evident when cells were exposed to 100 µm H(2)O(2). H(2)O(2) induced stronger responses in HTPCs than in HTPC-Fs, which correlates with the lower level of the H(2)O(2)-degrading defence enzyme catalase in HTPCs. The results provide evidence for an adaptation to elevated ROS levels, which must have occurred in vivo and which persist in vitro in HTPC-Fs. Thus, in infertile men with impaired spermatogenesis elevated ROS levels likely exist, at least in the tubular wall.