Toxic Effects Of Chronic And Low-dose Exposure To Microcystin (-LR) On Female Reproductive System And Its Mechanism

Microcystins (MCs) are a family of naturally cyclicptapeptide toxins produced by freshwater cyanobacteria. Among MCs, microcystin-LR (MC-LR) is the most prevalent and toxic detected in natural blooms. As the world’s increasing water pollution,the algae blooms outbreak has become increasingly common and MCs pollution has become a global environmental problems to be focused on. In recent years,it shows the tendency of rapid development in lake, reservoir eutrophication due to the ecosystem degradation. The concentration of MCs in the waters of lake Tai hu was detected as high as 54.897μg/L which is 55 times to the WHO 1998 safety limit 1μg/L in cyanobacteria peak season. Studies have shown that the MCs have toxic effects on liver, kidney, nervous system and gastrointestinal tract. We found the MCs have a strong toxic effects on the female reproductive system by acute animal experiments that cause the ovary weight lost, estrogen and progesterone levels dropped, estrus cycle disordered, all levels of follicle number changed, ovarian tissue structure damaged. For drinking water is the main exposure pathways in daily life, it is very necessary to study the effect of long-term low-dose exposure to MCs to simulate the toxicity effect of MCs in the natural environment. In addition, because the female reproductive system with complex anatomic structure and reproductive cycle, it is rarely reported about the toxic effect of MCs on female reproductive system. Therefore, this experiment study the effect of MC-LR on female reproductive function in mice with the MCs in the drinking water limit 1μg/L set up by WHO for reference to adopt the mode of chronic and low-dose exposure to MC-LR. In addition, the laboratory has found that MC-LR can enter the ovarian tissue and caused ovarian granulosa cell apoptosis which prompted that the target cells of MC-LR on female reproductive toxicity is ovarian granulosa cells. Based on this, we take the granulosa cells as the research object to further explore the mechanism of MC-LR on female reproductive toxicity in vitro.Part I Effects of chronic and low-dose exposure to MC-LR on the female reproductionObjectiveTo study the toxic effects of chronic and low-dose exposure to MC-LR on the female reproductive system in vivo.Methods1、Female specific pathogen free (SPF) mice (n=160) weighing about 10 g were randomly divided into two cohorts (3 months and 6 months)which had 4 groups in each cohort:one control and three experimental with 20 mice in each group. Mice were exposed in drinking water at 1μg/L,3.2μg/L and 10μg/LMC-LR for consecutive3 and 6 months in the experimental groups. The control animals got the same water only. After three-month and six-month treatment, blood samples were collected from carotid and took the organs such as ovarian, respectively.2、The weight of body,drinking water content and ovarian wet weight was weighed.The change of the estrus cycle of mice was observed though vaginal smear. Serum estradiol (E2), Progesterone (P4), Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) were measured by Enzyme-linked immunosorbent assay (ELISA) and Radioimmunoassay (RIA), respectively. The change of the follicles at different levels was detected by making ovarian paraffin section, serial section and H&E staining to take optical microscope photos. Tissue apoptosis of ovary was detected by TUNEL. Mice breed were observed though breeding experiment.Results1、In three-month treatment group, it was not significantly change in net weight, water content, LH and farrowing rate compared with the control group,while P4 level rise significantly (P<0.01) and there are time-dose dependent effect. The number of ovarian atresia follicles showed a marked increase (P<0.05). It showed a trend of decline in serum E2 level, ovarian coefficient decreased significantly (P<0.05) and FSH level significantly increased (P<0.05) in MC-LR 10μg/L mice. Mice breed mortality and the period length of diestrus were on the rise; the period length of estrum significantly reduced (P<0.05) and the period length of metaoestrus increased significantly (P<0.05) in 3.2μg/L and 10μg/L group. Ovarian tissue apoptosis increased slightly in high concentrations groups detected by TUNEL.2、In three-month treatment group, it was not significantly change in net weight, water content compared with the control group. Ovary coefficient, estrus duration and farrowing rate were decreased significantly (P<0.05); diestrus duration and atresia follicles rose significantly (P<0.05). FSH levels and the litter mortality had a marked increase (P<0.05) and P4 levels increased significantly (P<0.01) in 10μg/L group. LH levels increased significantly (P<0.05) in 3.2μg/L group.E2 levels decreased significantly (P<0.05) and ovarian tissue apoptosis increased significantly (P<0.05) in 3.2μg/L and 10μg/L group.Conclusion1、Chronic and low-dose exposure to MC-LR dosen’t have significantly effect on the basic physiological index of female mice.2、MC-LR can reduce the ovarian tissue weight;MC-LR may affect the growth and development of ovarian tissue3、MC-LR can cause follicular atresia increased significantly at different levels in ovarian tissue4、MC-LR can interfere with the synthesis of estrogen and progesterone, reduce serum E2 level, increased P4 level.5、MC-LR can cause female mice oestrous cycle disorder, shorten estrus time and lengthen metaoestrus and diestrus time to extend the estrus cycle length.6、MC-LR can cause the female farrowing rate reduced and pups mortality increased.7、MC-LR can destroy ovarian tissue structure damage,cause an increased cell apoptosis.Part Ⅱ The toxic effect of MC-LR on primary cultured ovarian granulosa cells in mice in vitroObjectiveTo explore the toxic effects of MC-LR on ovarian granulosa cells in vitro and the toxic mechanisms of MC-LR on the female reproductive system in vivo.Methods1、Improvement of the extraction of ovarian granulosa cells:take out the ovaries of 21 day-BALB/c mouse under a dissecting microscope and puncture the follicles mechanically with lml syringe. Next gently pipetting repeatedly and isolate ovarian granulosa cells through filter. After incubation for 24h, the medium was changed and get higher purity of ovarian granulosa cells after continuous cultivation for 48h.2、Observation of morphology. The growth behavior and morphology of granulosa cells were observed using an inverted phase contrast microscope.3、Proliferation curve plotting of granulosa cells using CCK-8 assay.4、The purity identification of ovarian granulosa cells by immunofluorescence.5、The ovarian granulosa cells were divided into five groups different levels of MC-LR concentrations of 0,0.05,0.5,5 and 50μM and then to detect the indicators.6、The access detection of MC-LR in ovarian granulosa cells by immunofluorescence.7、The cell viability was detected by MTT assay, and the effects of MC-LR oxidative stress on cells was assessed by MDA, CAT, and SOD detection kit MC-LR,the cell membrane integrity was determined by LDH kit8、The apoptosis analysis of MC-LR on ovarian granulosa cells was detected by Annexin V/PI and TUNEL assay.Results1、An inverted phase contrast microscope observations showed that ovarian granulosa cells were spherical, uniform size after freshly isolation. Cells began to be adherent after 12h but still small. After 24h the cells were gathered growing with irregular, fusiform or polygonal morphology. The granulosa cells completely covered the bottom of dish and form a monolayer after 72h.2、The granulosa cells proliferated with the extension of the initial time and the OD values continue to increase after 24 h and 48h, while cell growth is extremely strong in 72 h and the OD value was in peak.3、High purity of ovarian granulosa cells were detected by immunofluorescence and the MC-LR can enter the ovarian granulosa cells.4、After exposure to MC-LR of 5μM for 24h, cell viability was significantly lower (P<0.05) and decreased significantly in 50μM (P<0.01) that detected by MTT cell viability assay; after exposure to MC-LR in 5μM and 50μM for 48h, granulosa cells viability were both significantly decreased (P<0.01).5、After exposure to MC-LR for 24h, MDA levels were significantly higher in the experimental group (0.05,0.5,5,50μM) (P<0.05); after exposure to MC-LR for 48h, MDA levels in 0.5μM significantly increased (P<0.05) and significantly elevated in 5μand 50μM (P<0.01). After exposure to MC-LR for 24h, SOD levels were increased in 0.05μM (P<0.05)but decreased subsequently, decreased significantly in 50μM (P<0.01); After exposure to MC-LR for 48h, SOD levels in 0.5μM significantly lower (P<0.05), decreased significantly in 5μM and 50μM (P<0.01). After exposure to MC-LR for 24h, CAT significantly lower in 50μM levels (P<0.05); after exposure to MC-LR for 48 h, CAT levels 5μM and 50μM significantly lower (P<0.05).6、After exposure to MC-LR for 24h, LDH release rate tended to increase, however there is no significant difference compared with the control; when exposed to MC-LR for 48 h, LDH release rate in the experimental group (0.05,0.5,5,50μM) were significantly increased (P<0.05).7、After exposure to MC-LR for 24h, cell apoptosis rate(early apoptosis+late apoptosis) have increased trend and increased significantly in 50μM (P<0.05) by Annexin V/PI detection.8、TUNEL assay showed that after exposure to MC-LR for 24h, apoptosis rate was significantly higher in 0.5μM (P<0.05), and there were significantly elevation in 5μM and 50μM (P<0.01); after exposure to MC-LR for 48 h, cell apoptosis rate were significantly increased in the experimental group (0.05,0.5,5,50μM) were significantly increased (P<0.01).Conclusion1、High purity of ovarian granulosa cells can be obtained in vitro.2、MC-LR can enter ovarian granulosa cells.3、MC-LR can cause the granulosa cell viability decreased,oxidative stress rose,led to oxidative damage, the integrity of ovarian granular cell membrane destroyed and induce ovarian granulosa cell apoptosis.