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N-methyl pyrrolidone (NMP) is an FDA approved molecule used as an excipient in pharmaceutical industry. Besides having a central role in formulation of drugs, the most important function of any excipient is to guarantee the safety of the medicine during and after its administration. Several studies have shown that exposure to NMP and especially in rats produce a gonadotoxic effect leading to infertility. However, the mechanisms underlying the effect of NMP on male reproduction are unknown. The aim of this study was to assess the reproductive toxicity of NMP in male rats and to elucidate the underlying mechanism. Male Sprague Dawley rats were injected intraperitoneally, twice/ week, at a dose of 108 mg/ 100 g of body weight with NMP. Analysis of reproductive parameters revealed testicular atrophy in NMP treated animals compared to control animals. Germ cell composition within the seminiferous tubules was disturbed and manifested in an increase in number of cells with fragmented DNA. A subsequent decrease in number of spermatocytes and spermatids was observed. Alpha screen assay shows that NMP acts at the concentrations we applied in vivo as a low affinity inhibitor for BRDT (testis specific bromodomain protein). BRDT inhibition is mirrored by a significant decrease in the expression of early stage spermatocyte markers (lmna, aurkc and ccna1), during which BRDT expression predominates. A significant decrease in testosterone levels was also observed. Since NMP interferes with spermatogenesis on various levels, its use in humans must be carefully monitored.

Cobalt (Co) is an endocrine disruptor and a hypoxiamimicking agent. Its effect on spermatogenesis after prolonged administration remains unclear. The aim of the study was to assess chronic cobalt treatment on spermatogenesis. Pregnant mice were exposed to 75 mg (low dose) and/or 125 mg (high dose)/kg b.w. cobalt chloride (CoCl2×6H2O) with drinking water for 3 days before delivery and treatment continued until postnatal day 90. Age-matched control animals obtained regular tap water. At the end of the treatment, testes of control and Co-treated mice were excised and processed for histological and ICPMS analysis for metal content. Spermatozoa were isolated from both ductus deference and counted. Results showed a significant dose-dependent reduction in testicular index (testis weight to body weight ratio), up to 60% after treatment with 125 mg/kg and up to 34% after treatment with 75 mg/kg. Morphology of the testis was considerably affected in both doses of CoCl2 although in some tubules spermatogenesis was normal and completed. Diameter ofmany seminiferous tubules was significantly reduced and there was lack of lumen formation. We observed disorganization of seminiferous epithelium, islet of undifferentiated germ cells in luminal region of the tubules and presence of multinuclear germ cells. These findings corresponded to the significant reduction of sperm count by 2.15-fold for the low dose and 3.35-fold for the high dose. Chronic CoCl2 administration resulted in accumulation of the metal ions in the testes of the treated mice. The considerable impact of Co on male reproduction after chronic exposure shows negative correlation between metal concentration and male fertility.