Although fluoride is safe and even healthy at low concentrations, sustained consumption of large amounts of soluble fluoride salts is dangerous. It was well known that toxic levels of fluoride exposure over a long period of time can adversely cause skeletal and tooth fluorosis induced by oxidative stress of osteoblasts and osteoclasts34,35,36. It also can lead to some adverse effects on a number of physiological functions, for example, thyroid dysfunction37, nephrotoxicity35,38, cardiometabolic risk39,40, neurodevelopmental disorder in juvenile stage38,41,42 and even male reproductive endocrine disruption7,8.

However, the mechanisms of reproduction injury induced by taking in excess fluoride were still inconclusive. Attempting to address the root cause, this experiment was the first time using the transcriptome sequencing in the testicle of experimental fluorosis mice to explore the relative gene expression levels in mouse testis and interpret the effect of fluoride poisoning in the male reproductive system. Different from earlier studies, our study considered the damages of fluoride on the male reproductive system holistically, including a variety of pathways and genes, rather than just a single factor.

Generally, the testis and the capacity of sperm were of the important indices for evaluating the reproductive system. The testis comprises mostly seminiferous tubules and interstitial cells, localized between seminiferous tubules, to produce and secrete testosterone43. The epithelium of the tubule consists of a type of sustentacular cells known as Sertoli cells, which differentiate through meiosis into sperm cells. During spermatogenesis, the main function of Sertoli cells is to nourish the developing sperm cells and also act as phagocytes, consuming the residual cytoplasm and secreting the inhibin, activins and androgen binding protein44. While our previous studies reported that the pathologic and morphological changes of chronic fluorosis in testicles and sperm were observed. The cavitation of seminiferous tubules, cellular atrophy and other structural damages can result in the reduction of androgen binding protein synthesis and the inadequate amounts of testosterone, which, in turn, can cause spermatogenesis to be blocked and spermatid developed abnormally with different morphology. Song Ke qin et al.45 also found the distention and vesiculization of smooth endoplasmic reticulum and the deposition of large lipid droplets appearing in the Sertoli cells under the ultrastructural observations of rat testes. So what happened in these cells?

Mendoza-Schulz A. et al.46 said that fluoride had significantly effect on hormone secretion and protein synthesis in the endocrine cells. They found the changes in phosphorylation status of both cytoskeletal and cytosolic protein fractions, as well as in actin cytoskeletal arrangements were observed. Similarly, in our research of the 50 mg/l NaF-treated group, actin nucleation and actin cytoskeleton signal pathways were stimulated with the significant expression of growth factors. In response to these microenvironmental and functional alterations, immune cells often represent dramatically change their functional activities to reprogram their cellular metabolism and release the metabolic stresses47. A lymphocyte, such as T cells, transforms from a relatively inert cell to a cell engaging in robust growth and proliferation, often producing large amounts of effector molecules, including cytokines47. Yet despite all that, there is emerging evidence that metabolic enzymes and regulators can also have a direct role in controlling immune cell functions47. For instance, in CD4 T cells, GAPDH has been described to bind to IL2 mRNA and inhibit translation. Accordingly, the testicular immunological efficiency was enhanced along with the increase of doses of fluoride.

Although the testis is an immune privileged organ and the most important spaces of spermatogenesis and steroidgenesis, toxic agents and inflammation may overwhelm immune suppressor mechanisms inducing autoimmune reactions against spermatic antigens which result in aspermatogenesis and infertility18. The cytokine interleukin-17, ukin-17 (IL-17 or IL-17A) and the pathology associated with aberrant IL-17 signaling played an important role in maintaining the testicular immune including cell immunity, mucosal immunity and cytokines, especially in experimental autoimmune orchitis (EAO)18,48,49. Jacobo P. et al.49 reported that in EAO testis developed by active immunization with spermatic antigens, testis-infiltrating cells revealed an increased number of macrophages, dendritic cells and T cell subsets including Th17 cells so that TNF-α, IL-17 and other immune cells secreted pro-inflammatory cytokines, which disrupted the normal testicular immune suppressor microenvironment. And they said IL-17 cells in EAO testis have a mature immunogenic status and are able to induce immune responses to testicular antigens.

In many cases, an excess of IL-17 is associated with abnormal inflammation, implicated in rheumatoid arthritis, asthma, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus and autoimmune encephalomyelopathy, which, not surprisingly, have become a major therapeutic target for these diseases. It have been recently found Th17 cells are a subset of T helper cells and play important functions in host defense and the pathogenesis of various human autoimmune and inflammatory diseases50,51,52. Th 17 cells could produce IL-17A, who would mediate many of the downstream pathologic functions in the cells. IL-17A utilizes IL-17RA and IL-17RC as its receptors that are mainly expressed on tissue epithelial cells and fibroblasts. While IL-17A is important for host defense against many extracellular pathogens, they can also cause excessive tissue damage and exacerbate proinflammatory responses during autoimmunity52. Therefore, as for our study, the IL-17 signal pathway and its proinflammatory cytokines were expressed in higher levels in high fluoride-exposed testis (100 mg/l NaF). It was the response to the invasion of the immune system by extracellular fluoride and involve in the maintenance of testicular immune privilege and spermatogenesis53. And the activation of toll-like receptors IL-17RA and IL-17RC suggested that these cells played important roles in protecting the seminiferous epithelium from invading fluoride.

During this study we also found the transforming growth factor beta (TGF-β) signaling pathway has the most important research value in the toxicology field. TGF-β signaling pathway is involved in many cellular processes in both the adult organism and the developing embryo including cell growth, cell differentiation, apoptosis, cellular homeostasis and other cellular functions54,55,56. TGF-βs belong to a family of the immunosuppressive and anti-inflammatory TGF superfamily and widely distributed in embryonic and adult tissues18. Most TGF-βs are present in the testis as the latent inactive precursor form and are expressed constitutively at high levels being produced mainly by Sertoli cells, Leydig cells, and peritubular, but in post pubertal testis, cytokines of the TGF-β family are also expressed by early spermatids and spermatocytes. Once activated at its site of action by local proteases, TGF-βs would contribute to the immunological privileged site of the testis through their strong immunosuppressive ability18,52. Thus, what we think is that the gene activation of TGF-β family have help to sustain the immune exemption of testicle in fluorosis.

In addition, based on the expression of MKK3/6, MKK1/2 and PI3K in RNA-seq and the intracellular metabolic processes of IL-17 signaling pathway, we could infer that the IL-17 family members took part in the activation of the Mitogen-activated protein (MAP) kinase pathway and PI3 Kinase-AKT pathway, which are involved in the regulation of a variety of growth and differentiation pathways through several phosphorylation cascades52,57. The MAP signaling cascade is activated by a number of receptors: the extracellular mitogen binds to the membrane receptor, then this allows Ras (a GTPase) to swap its GDP for a GTP, and activate MAP3K, which activates MAP2K, which activates MAPK, finally MAPK can activate a transcription factor58,59. MAPK-ERK1/2 played an important role in the regulation of cell growth and cell cycle progression. PI3-kinase and its downstream kinase AKT are potent inhibitors of apoptosis in many cell types. AKT is phosphorylated upon IL-17stimulation and also adds to the possible involvement of PI3-kinase in the propagation of signal through the IL-17R52. Together, these results indicated that PI3-kinase/AKT and MAPKs serves as the upstream arbitrator of the IL-17 pathway activation and had contributed to the increased binding of the inflammatory transcription factor in IL-17 pathways.

Anyway, all the found helped us to better understand the molecular basis of reproduction and sperm metabolism disorder and deeply identify the mechanisms involved in reproductive toxicity and other pathological disorders associated with fluoride. At first, the aim we were pursuing was to find really reliable molecules and genes associated with reproduction by RNA direct sequencing of testis. However, what we got was quite surprising: a plenty of other metabolic pathways and classic genes of the systemic and comprehensive responses were dig out. Peeping a spot to see overall picture: local delicate change was packed with the complex issues of the whole organism. But for the further verification and exploration, researches on the cellular level and the significant expression of proteins during the spermatogenesis should be carried out.