Studies Of The Effect And Mechanism Of Hydrogen Sulfide Against Spermatogenic Failure And Sperm Dysfunction

Male infertility is a major problem of human reproductive disorders. Among those infertile male,85 percent can generate sperm, however, of low quality. Abnormal spermatogenesis impair the quality of sperm, which may cause male infertility. Apart from gene mutation and chromosomal abnormalities, one of the major causes suppressing impaired spermatogenesis and dysfunction of sperm is environmental and pathological factors, such as infection, the effect of which may be expressed by oxidative stress and inflammation. Male infertility, for now in clinical therapy, lacks effective or specific methods.Hydrogen sulfide (H2S) is the third biological active gasotransmitter got covered after nitric oxide and carbon monoxide. Two pyridoxal-5’-phosphate-dependent enzymes, cystathionine bete-synthase (CBS) and cystathionine gamma-lyase (CSE), have been shown to produce H2S in mammalian tissues with L-cysteine and/or homocysteine as the main substrate. According to previous reports, H2S of physiological concentration play significant role in various processes, such as scavenging oxygen free radicals, controlling the level of oxidative stress and fighting against inflammation. Under certain pathological conditions, supplementary exogenous H2S better protected body against pathological damage. In study related to female reproductive, it has been reported that H2S affect egg development and maturation, and helps the cardiovascular function of pregnant woman as well as birth-giving. H2S synthases also can be found in male reproductive systems, however, with the study about its function limited within male erectile dysfunction. H2S is expressed in testes and sperm, which may hold key effect in mediating spermatogenesis and sperm function. The effect of H2S on male fertility therapy and prevention, also on human sperm quality remain to be unknown.The main objective of this study was to study the effect of H2S on spermatogenesis, to find out whether H2S assists spermatogenesis and sperm function, and to explore underlying mechanism. Firstly, the H2S content in patient with low fertility and healthy controls were compared by electrochemical sensor, along with the endogenous H2S synthase expression by Western-blot. It was found that sperm samples with low vitality were more likely to be detected with low H2S level in seminal plasma, in which the CBS expression was also significantly decreased. It indicated that H2S is probably involved in male infertility. The animal experiments were carried out to further confirm that H2S serves as a key factor in male fertility. LPS-triggered inflammation and STZ-induced diabetes were used to establish a model for spermatogenesis disorder, which was used to observe the treating effect of two different H2S donors--GYY4137 or NaHS by intraperitioneal injection. Mice were killed after the last injection. Sperm quality was evaluated by the computer-assisted semen analysis system (CASA). Testes were collectedand stained with hematoxylin-eosin stains. The results showed thatquality decreasing of sperm and damage to testicular tissue caused by inflammation and diabetes can be significantly inhibited by H2S treatment. We tested mRNA level and protein expression of CSE and CBS in testis by Real-Time PCR and Western-blot. We found reduction of CBS was reversed by H2S treatment. Reactive oxygen species (ROS) level in testis was evaluated by histological Dihydroethidium (DHE) staining. The increased ROS level was significantly inhibited by H2S treatment.TUNEL staining showedH2S treatment after LPS stimulation largely relieved apoptosis of spermatogenic cells.Immunofluorescent analyses were performed on frozen testicular sections.H2S treatment after LPS stimulation maintained the integrity of BTB. The testosterone levels in the serum were measured using Enzyme-linked Immunoassay. Administration of H2S after LPS injection increased serum testosterone to values shown no significant change compared with those detected in controls. After further research the mechamisms of H2S anti-testiscular dysfunction effect, we found that H2S provided protection effect through suppression of NF-kB activation and proinflammatory factors expression of IL-1β、IL-6、TNF-α, and increase anti-inflammatory mediators such as IL-10. H2S inhibit nuclear translocation of Nrf2 and up-regulate the expression of anti-oxidants, thus reducing the testicular oxidative stress injury. H2S inhibited the activation of mitochondrial apoptosis signaling pathway and raise the relative ratio of Bcl2 to Bax, as well as consuming caspase-3 activity. Meanwhile H2S remarkably inhibit the stress-induced activation of MAPK signaling pathways. To further demonstrate the significance of endogenous H2S on the spermaiogenesis, we built a mice model with highly expression of CBS in right testis. After LPS injection, results of CSAS assay showed that sperm quality in right epididymis was significantly higher than that of the left epididymis. At the same time, HE staining and immunofluorescence staining results suggest that spermatogenic structure and the blood testis barrier integrity in right testis were significantly improved compared with left side. Moreover, the human sperm sample were used to test how H2S affect the sperm motility. AOAA, the inhibitor of endogenous H2S synthase CBS, significantly inhibited motility of normal human sperm cells and its ability of hyperactivation, which can be alleviated by exogenous supplement of H2S. Besides, H2S of proper concentration somehow increase the motility of weak sperm combined with seminal plasma with low H2S content.The results of current research can be concluded as follows: ①H2S is a significant mediator in male sperm function. Exogenous H2S supplement helps improve the sperm dysfunction caused due to lack of H2S. ②The animal experiments also indicated that H2S sustained blood-testis barrier integrity and improve testosterone synthesis abnormity, combined with protection for the structure of testicles and normal spermatogenesis.③H2S exogenous supplement can maintain H2S level in testis, thus inhibiting the MAPK signaling activated by inflammation. H2S exert the anti-inflammatory functions by surpressingNF-kB activation, anti-oxidative by promoting nuclear translocation of Nrf2and anti-apoptotic by inhibiting mitochondrial apoptosis signaling pathway. All these results, when taken together, provided novel thoughts for treatment and precaution for male infertility.