The Adverse Effects of Triptolide on the Reproductive System of Caenorhabditis elegans: Oogenesis Impairment and Decreased Oocyte Quality

Abstract

Previous studies have revealed that Triptolide damages female reproductive capacity, but the mechanism is unclear. In this study, we used Caenorhabditis elegans to investigate the effects of Triptolide on the germline and explore its possible mechanisms. Our data show that exposure for 4 h to 50 and 100 mg/L Triptolide reduced C. elegans fertility, led to depletion and inactivation of spermatids with the changes in the expression levels of related genes, and increased the number of unfertilized oocytes through damaging chromosomes and DNA damage repair mechanisms. After 24 and 48 h of the 4 h exposure to 50 and 100 mg/L Triptolide, we observed shrink in distal tip cells, an increase in the number of apoptotic cells, a decrease in the number of mitotic germ cells and oocytes in diakinesis stage, and chromatin aggregates in -1 oocytes. Moreover, expression patterns of the genes associated with mitotic germ cell proliferation, apoptosis, and oocyte quality were altered after Triptolide exposure. Therefore, Triptolide may damage fertility of nematodes by hampering the development of oocytes at different developmental stages. Alterations in the expression patterns of genes involved in oocyte development may explain the corresponding changes in oocyte development in nematodes exposed to Triptolide.

Keywords: Caenorhabditis elegans; Triptolide; apoptosis; mitosis; oogenesis.

Figure 1

Survival of nematodes exposed to TP for 4 and 8 h. TP, Triptolide. Note: There are no error bars for the control and 10 mg/L Triptolide groups because there were no dead nematodes in the three repeated experiments.

Figure 2

Comparison of body length in nematodes exposed to TP for 4 and 8 h. TP, Triptolide. Bars represent means ± SEM. * p < 0.05 vs. the control group.

Figure 3

Effects of exposure to TP on the fertility of nematodes: (A) effects of 4 h TP exposure on brood size; and (B) effects of 4 h TP exposure on brood size in each spawning day. TP, Triptolide. Bars represent means ± SEM. * p < 0.05 vs. the control group.

Figure 4

The number of unfertilized oocytes per nematode exposed to TP for 4 h. TP, Triptolide. Bars represent means ± SEM. * p < 0.05 vs. the control group.

Figure 5

The number of spermatids per nematode exposed to TP for 4 h. TP, Triptolide. Bars represent means ± SEM. * p < 0.05 vs. the control group.

Figure 6

Effects of TP on the size of spermatids: (A) the size of spermatids per nematode exposed to TP for 4 h; (B) the spermatids morphology of the control group after 6 h of exposure; and (C) the spermatids morphology of nematodes exposed to 100 mg/L TP after 6 h of exposure. Scale bar is 15 µm. TP, Triptolide. Bars represent means ± SEM. * p < 0.05 vs. the control group.

Figure 7

The rate of spermatids activation (%) of nematodes exposed to TP for 4 h. TP, Triptolide. Bars represent means ± SEM. * p < 0.05 vs. the control group.

Figure 8

Effects of exposure to TP for 4 h on the oocytes at different developmental stages: (A) comparison of the effects of TP on the area of DTCs after 24, 48, and 72 h of exposure; (B) comparison of the effects of TP on the number of mitotic germ cells per gonad arm of nematodes after 24, 48, and 72 h of exposure; (C) comparison of effects of TP on the number of apoptotic cells per gonad arm of nematodes after 24, 48, and 72 h of exposure; (D) comparison of effects of TP on the number of oocytes in diakinesis per gonad arm of nematodes after 24, 48, and 72 h of exposure; (E) effects of TP on the morphology of DTCs after 24 h of exposure (scale bar, 50 µm); (F) effects of TP on mitotic germ cells after 24 h of exposure, where the left area beside the white line contains mitotic cells (scale bar, 10 µm); (G) effects of TP on apoptosis after 24 h of exposure, where the arrow indicates apoptotic cells (scale bar, 50 µm); and (H) effects of TP on oocytes in diakinesis after 24 h of exposure (scale bar, 50 µm). TP, Triptolide. Bars represent means ± SEM. * p < 0.05 vs. the control group.

Figure 8

Effects of exposure to TP for 4 h on the oocytes at different developmental stages: (A) comparison of the effects of TP on the area of DTCs after 24, 48, and 72 h of exposure; (B) comparison of the effects of TP on the number of mitotic germ cells per gonad arm of nematodes after 24, 48, and 72 h of exposure; (C) comparison of effects of TP on the number of apoptotic cells per gonad arm of nematodes after 24, 48, and 72 h of exposure; (D) comparison of effects of TP on the number of oocytes in diakinesis per gonad arm of nematodes after 24, 48, and 72 h of exposure; (E) effects of TP on the morphology of DTCs after 24 h of exposure (scale bar, 50 µm); (F) effects of TP on mitotic germ cells after 24 h of exposure, where the left area beside the white line contains mitotic cells (scale bar, 10 µm); (G) effects of TP on apoptosis after 24 h of exposure, where the arrow indicates apoptotic cells (scale bar, 50 µm); and (H) effects of TP on oocytes in diakinesis after 24 h of exposure (scale bar, 50 µm). TP, Triptolide. Bars represent means ± SEM. * p < 0.05 vs. the control group.

Figure 9

Effects of exposure to TP for 4 h on the chromosome of −1 oocytes in diakinesis: (A) percent of −1 oocytes with chromosome morphology defects; (B,C) the chromosome of the same −1 oocyte in diakinesis of the control group on different focal planes, with six intact DAPI-stained bodies distributed dispersedly in the nucleus; and (D,E) the chromosome of the same −1 oocyte in diakinesis of 100 mg/L TP group on different focal planes. A chromatin aggregate was observed in the nucleus. White arrows point to the −1 oocytes. Grey arrows point to the chromatin aggregates. Scale bar is 10 µm. TP, Triptolide. Bars represent means ± SEM.

Figure 10

Relative mRNA expression levels of genes involved in gametogenesis of L4 larval nematodes exposed to TP. Gene expression was analyzed at 24 h after the exposure: (A,B) relative mRNA expression levels of genes involved in mitotic germ cell proliferation and oocyte quality; (C) relative mRNA expression levels of genes involved in apoptosis; and (D) relative mRNA expression levels of genes involved in spermatogenesis. TP, Triptolide. Bars represent means ± SEM. * p < 0.05 vs. the control group.

Figure 10

Relative mRNA expression levels of genes involved in gametogenesis of L4 larval nematodes exposed to TP. Gene expression was analyzed at 24 h after the exposure: (A,B) relative mRNA expression levels of genes involved in mitotic germ cell proliferation and oocyte quality; (C) relative mRNA expression levels of genes involved in apoptosis; and (D) relative mRNA expression levels of genes involved in spermatogenesis. TP, Triptolide. Bars represent means ± SEM. * p < 0.05 vs. the control group.