Discussion

The objective of this study was to investigate the effects of F oral exposure on T [testosterone] biosynthesis and the neuroendocrine regulation of the HPT axis in male mice. T is one of the endpoint parameters inHTP axis. In this study, reduced serum T concentration replicates our prior report of F-induced decrease in T in male mice (Huanget al., 2007), suggesting that animal model for F-induced dysfunction of gonadal hormone secretion was established.

T, which is required for the normal morphology of seminiferous tubule and spermatogenesis (Amann, 2008), is considered to be a valuable indicator of testicular function. An earlier investigation in India showed that people exposed to 1.5–14.5 mg/L F in drinking water presented a significant decrease in serum T (Susheela and Jethanandani, 1996). The similar phenomena were also observed in men who receiving 3–27 mg F/day in Mexico (Ortiz-Pérez et al.,2003), and workers in Russia contacted with cryolite (Na3AlF6)(Tokar and Savchenko, 1977). During the recent years, a grow-ing number of animal experiments confirmed the above findings(Dvoráková-Hortová et al., 2008; Pushpalatha et al., 2005; Zhouet al., 2013). These findings demonstrated the link between high Fand low T. The maintenance of T level is highly regulated by the negative feedback control of the HPT axis, and the production ofT relies on the testicular steroidogenesis. In the present study, to clarify the mechanism of T reduction induced by F, we detected the expression levels of genes in hypothalamus, pituitary, and testis which are responsible for T regulation and biosynthesis.

In the feedback cycle of HPT axis, LH and FSH, two important gonadotropin hormones secreted from the pituitary, induce testicular steroidogenesis producing T biosynthesis (Sofikitis et al.,2008). In the present study, concentrations of serum LH and FSH were not affected by any level of F. In parallel with the stable gene expression levels of pituitary LH and FSH across all exposure groups, it is suggested that the mechanism underling F-induced low T in male mice appeared to be independent of LH and FSH. Moreover, serum GnRH released from the hypothalamus,which stimulates secretion of LH and FSH, was not affected by F either. To further clarify the less sensitivity of GnRH signaling toF exposure, mRNA expressions of genes involved in hypothalamicKiSS-1/GPR54 system were also measured. KiSS-1 is the natural ligand of the previously orphan G protein-coupled receptor, GPR54(Hameed and Dhillo, 2010). The hypothalamic KiSS-1/GPR54 system has been recognized as a gatekeeper of GnRH neurons (Silveira et al., 2010). In the current study, hypothalamic KiSS-1, GPR54,GnRH, and pituitary GnRH-R presented less change in gene expression, which further suggested that F does not disrupt hypothalamic and pituitary function. Taking all the above together, we inferred that the disturbed T biosynthesis in testis may be responsible for the reduction of T concentration in male mice exposed to F.

T biosynthesis requires the cooperation of many functional molecules, including StAR, P450scc, 3-HSD, CYP17, 17-HSD andCYP19 to complete the process of cholesterol transport and steroid biosynthesis in testicular Leydig cells (Jana and Samanta, 2006).StAR is responsible for cholesterol transport from the outer mitochondrial membrane to the inner mitochondrial membrane in Leydig cell and considered to be a rate-limiting factor for T biosynthesis in the testis (Stocco and Clark, 1996). In this study, the gene expression of StAR remained stable in F groups, indicating that F did not disrupt the cholesterol transport. After transported intothe inner mitochondrial, cholesterol is converted to pregnenolone by P450scc, which is the first step in steroidogenesis (Stocco andClark, 1996). Then pregnenolone is immediately converted to progesterone by 3-HSD. Followed by this, progesterone undergoes androstenedione concerted by CYP17. Finally, androstenedione is converted to T by 17-HSD. Here expressions of P450scc and 3-HSD, rather than CYP17 and 17-HSD, were inhibited, implying the production and conversion of pregnenolone were adversely infected by F. CYP19 is a key enzyme which catalyzes a conversion of androgen to estrogen, and therefore the increased transcription of CYP19 in this study could further reduce serum T level. Collectively, the down-regulated P450scc and 3-HSD, and up-regulatedCYP19 indicated that elevated F disturbed T biosynthesis in testis.

As mentioned above, the exact site of T biosynthesis is in mitochondrion. Notably, the morphological lesions in mitochondrion induced by F have been observed in different organs including thymus (Wang et al., 2009) and sperm (Sun et al., 2011). High F exposure in this study induced mitochondria swelling and crest broken. The ultra-structure observation provided the morphological evidence for the low production of T. Another evidence for supporting the disruption of testicular function by F exposure was the decreased LHR and FSHR mRNA expressions. Low LHR and FSHR transcription in testis along with the normal serum LH and FSH levels further demonstrated the testicular dysfunction rather than hypothalamus and pituitary.

Taking all these together, the present study suggested that high F exposure disrupted T biosynthesis in male mice. The inhibited testicular P450scc and 3-HSD and elevated CYP19 may be responsible for T suppression by F. Considering that hypothalamic KiSS-1,GPR54, GnRH, and pituitary GnRH-R, LH, and FSH which involved in the HPT axis remained unchanged, we inferred that hypothalamus and pituitary were not sensitive to F. In addition, the toxic effects of F on testicular mitochondrial function deserve further investigation.