Xing Duan1, Xiao-Xin Dai1, Teng Wang1, Hong-Lin Liu1, Shao-Chen Sun2. 1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. 2. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China sunsc@njau.edu.cn.
Abstract
STUDY QUESTION: Does melamine have a toxic effect on oocyte development and fertility in vivo? SUMMARY ANSWER: Melamine had toxic effects on oocyte quality and fertility due to its effects on the oocyte cytoskeleton, apoptosis and autophagy induction, and epigenetic modifications in an in vivo mouse model. WHAT IS KNOWN ALREADY: Melamine is a chemical compound that is widely used during the manufacture of amino resins and plastics. In 2008, melamine was reported to adulterate milk and infant formulas in China, which sparked food safety concerns worldwide. Ingesting melamine may result in reproductive damage, and bladder or kidney stones, which can lead to bladder cancer. STUDY DESIGN, SIZE, DURATION: Mice were randomly assigned to three groups and fed a diet that included melamine (0, 10 and 50 mg/kg/day) for 8 weeks. The in vivo effect of melamine on female reproduction was examined. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used immunofluorescent staining, western blotting and qRT-PCR to examine the effect of melamine on oocyte quality. MAIN RESULTS AND THE ROLE OF CHANCE: Our results showed the following effects of this melamine-containing diet. (i) Ovary weights were reduced in melamine fed mice. Oocyte developmental competence was also reduced, as shown by reduced polar body extrusion rates. (ii) Melamine feeding resulted in abnormal oocyte cytoskeletons, as shown by increased rates of aberrant spindles and reduced actin microfilament expression. (iii) Melamine exposed oocytes had higher rates of abnormal mitochondrial distributions and early stage apoptosis/autophagy, which were shown by increased microtubule-associated protein 1 light chain 3 (LC3) protein expression level and caspase 9, autophagy-related protein 14 (atg14), and lc3 mRNA levels. (iv) Fluorescence intensity analysis showed that DNA methylation levels were reduced in the oocytes of melamine fed mice. Histone methylation levels were also altered, as Di-methyl-Histone H3 (Lys4) (H3K4me2) level was increased and Tri-methyl-Histone H3 (Lys9) (H3K9me3), Di-methyl-Histone H3 (Lys9) (H3K9me2), and Tri-methyl-Histone H3 (Lys27) (H3K27me3) levels were reduced in oocytes from melamine fed mice. (v) The litter sizes of melamine fed mice were significantly reduced when compared with those of controls. LIMITATIONS, REASONS FOR CAUTION: Although we examined the possible effects of melamine on oocyte quality and fertility, we did not determine the effect of melamine on offspring development. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that melamine plays a major role in oocyte quality and fertility. This information could contribute to a better understanding of melamine toxicity in female reproduction. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Basic Research Program of China (2014CB138503) and the Natural Science Foundation of Jiangsu Province (BK20140030). The authors have no conflict of interest to disclose.
STUDY QUESTION: Does melamine have a toxic effect on oocyte development and fertility in vivo? SUMMARY ANSWER: Melamine had toxic effects on oocyte quality and fertility due to its effects on the oocyte cytoskeleton, apoptosis and autophagy induction, and epigenetic modifications in an in vivo mouse model. WHAT IS KNOWN ALREADY: Melamine is a chemical compound that is widely used during the manufacture of amino resins and plastics. In 2008, melamine was reported to adulterate milk and infant formulas in China, which sparked food safety concerns worldwide. Ingesting melamine may result in reproductive damage, and bladder or kidney stones, which can lead to bladder cancer. STUDY DESIGN, SIZE, DURATION: Mice were randomly assigned to three groups and fed a diet that included melamine (0, 10 and 50 mg/kg/day) for 8 weeks. The in vivo effect of melamine on female reproduction was examined. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used immunofluorescent staining, western blotting and qRT-PCR to examine the effect of melamine on oocyte quality. MAIN RESULTS AND THE ROLE OF CHANCE: Our results showed the following effects of this melamine-containing diet. (i) Ovary weights were reduced in melamine fed mice. Oocyte developmental competence was also reduced, as shown by reduced polar body extrusion rates. (ii) Melamine feeding resulted in abnormal oocyte cytoskeletons, as shown by increased rates of aberrant spindles and reduced actin microfilament expression. (iii) Melamine exposed oocytes had higher rates of abnormal mitochondrial distributions and early stage apoptosis/autophagy, which were shown by increased microtubule-associated protein 1 light chain 3 (LC3) protein expression level and caspase 9, autophagy-related protein 14 (atg14), and lc3 mRNA levels. (iv) Fluorescence intensity analysis showed that DNA methylation levels were reduced in the oocytes of melamine fed mice. Histone methylation levels were also altered, as Di-methyl-Histone H3 (Lys4) (H3K4me2) level was increased and Tri-methyl-Histone H3 (Lys9) (H3K9me3), Di-methyl-Histone H3 (Lys9) (H3K9me2), and Tri-methyl-Histone H3 (Lys27) (H3K27me3) levels were reduced in oocytes from melamine fed mice. (v) The litter sizes of melamine fed mice were significantly reduced when compared with those of controls. LIMITATIONS, REASONS FOR CAUTION: Although we examined the possible effects of melamine on oocyte quality and fertility, we did not determine the effect of melamine on offspring development. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that melamine plays a major role in oocyte quality and fertility. This information could contribute to a better understanding of melaminetoxicity in female reproduction. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Basic Research Program of China (2014CB138503) and the Natural Science Foundation of Jiangsu Province (BK20140030). The authors have no conflict of interest to disclose.