In this study, the effect of 14 weeks of standard diet (controls) or folate and vitamin B12-free diet (VBD group) or vitamin D-free diet (VDD group) were assessed on mice testicular function, and sperm function. Vitamin D deprivation caused increased body weight with no effect from VBD confirming the calcium-independent role of vitamin D on body weight homeostasis. The two deprivations caused convergent damages including decreased testosterone, worsened Johnson scores, tubular differentiation index and spermatogenesis index, and serious worsening of sperm parameters and of sperm functional tests (DNA methylation, protamination, DNA damage and lipid peroxidation). From a metabolic point of view, the damage from both models converged on the one carbon cycle (methylations) and the transsulfuration pathway (GSH and antioxidant defences) and increased circulating homocysteine, although with different mechanisms: VBD appeared to hamper methylations due to lower ability to regenerate homocysteine to methionine whereas VDD appeared to interfere with homocysteine transsulfuration to cysteine and, thereafter, GSH. VDD also caused a huge paradox increase of vitamin B12, which was likely in a non-functional form and warrants further investigation. These findings strongly endorse the potential benefit of combined folate/B12 and vitamin D supplementation in infertile patients.

Vitamin D deficiency is a global health problem and has been linked to defective spermatogenesis and male infertility. In this study, we aimed to investigate the main enzymes are involved in transsulfuration pathway of 1-carbone metabolism, and spermatogenesis function. Therfore, sixteen male C57 mice were addressed to a control (standard diet) or vitamin D deficient (VDD) diet for 14 weeks. The results show that compared to the standard diet, VDD increased final body weight and reduced sperm quality, caused damage to the testicular structure, and decreased the serum levels of testosterone. In addition, serum concentrations of homocysteine, vitamin B12, and sperm oxidative stress markers increased. In testicular tissues, the CBS and CSE protein level were down-regulated whereas HO-1 was up-regulated at both mRNA and protein expression levels. Within a mice deprivation model, VDD deeply suppressed testosterone and impaired spermatogenesis with oxidative stress-mediated mechanisms. The effects of the deprivation appeared to be at least in part independent of genomic and receptor-mediated vitamin D actions and suggest a specific impairment of the alternative transsulfuration pathway.