Effects Of Progesterone On Reproductive And Heat Shock Protein Gene Expression Of Brachionus Calyciflorus

Environmental hormones are also known as environmental endocrine disruptors (EDCs). EDCs affect the normal physiological function of organism, such as gender characteristics, reproductive behavior and development usually by involving the synthesis, secretion, transport, combination, function or decomposition of the hormones in organic. Water is the final destination of the most environmental hormones.The abuse of environmental hormones will directly interfere with the normal physiological and biochemical processes in endocrine system, and ultimately cause the loss of sexual characteristics of aquatic animals and low production of offspring. In recent years, it has gradually become a hot topic in the international environmental science and water ecology for aquatic animals as subjects to evaluate the toxic effects of drugs.Rotifers are the main groups of zooplankton and often used in ecotoxicology studies because of its propagation speed, short life cycle and high sensitivity to toxicants. This experiment studied the effects of progesterone which is one of typical vertebrate female hormones on the population growth in Brachionus calyciflorus (Pallas) with 2-day population growth rate (r value) as an index. In addition, the effects of different concentrations of progesterone on average life and all productive stages in B. calyciflorus. Based on this, we used Real-time quantitative PCR to determine the heat shock proteins mRNA expression of B. calyciflorus treated by different concentrations of progesterone.1. The effects of different concentrations of progesterone on the population growth in B. calyciflorusRotifers were treated by progesterone of 2mg/L,4mg/L,6mg/L,8mg/L and 10mg/L with a density of 3.0×106 cells/mL of Chlorella pyrenoidosa and then cultured at 25 ± 1 ℃. The experiment was run at 16L:8D and the illumination intensity was 4000Lx. The population growth rates of all treatments were respectively 1.50,0.81,1.14,0.23 and 0.29. The population growth rate at 2d treated by 2 mg/L progesterone was highest, obviously higher than the control groups (P=0.047). This indicated that low concentrations of progesterone was beneficial to B. calyciflorus population growth andhigh concentrations was toxic to B. calyciflorus.2. The effects of different concentrations of progesterone on average life and pre-production period in B. calyciflorusThe result showed the average lifespan in the control group was 137.3±6.6h. However, the average lifespan in all groups was 82.9±11.4,38.8±5.9,71.1±6.2,48.2±3.2 and 48.4±0.4 h, respectively and were significantly less than that in the control group(P<0.01). With increasing of concentration of progesterone, it was in decline in general.The result about the effects of progesterone on pre-production period showed that the average pre-reproduction period of amictic females in the 6mg/L and 8mg/L groups was 30.1±3.0 and 34.6±1.4. They were 1.5 times and 1.8 times longer than that in the control group (19.1±1.4h) (P<0.01). The pre-production period of amictic females in 2mg/L and 4mg/L treatment groups are similar with the control group. The pre-production period of amictic females in high concentration treatment groups are longer than the control group significantly. This indicated that the high concentrations have obvious effects on the extension of pre-production period. In the certain life span, the pre-production period is longer and the production period is shorter.the average production of B. calyciflorus was also counted. After the treatment of progesterone, the production was declined significantly. The offspring of the control was 10.6±1.lind and all treatments was 4.3±0.7、3.3±0.9、2.8±0.3、1.2±0.2 and 0.4±0.2ind, respectively. The result showed that the numbers of offsprings in progesterone treatments were less than the control.3. The effects of different concentrations of progesterone on the expression of hsps mRNA in B. calyciflorusRotifers with a initial density of 100 individual were respectively cultured in progesterone of 2mg/L,4mg/L,6mg/L,8mg/L and 10mg/L. Then we observed change of relative mRNA levels of hsps after 12h and 24h through RNA extraction, reverse transcription and real-time quantitative PCR. The results of Real-time quantitative PCR showed that the change of the expression of the four heat shock protein were different.The expression of hsp40 significantly increased exposed in 4 mg/L,8mg/L and 10mg/L progesterone treatment(P<0.05), but decreased in 6 mg/L progesterone treatment(P<0.05).The expression of hsp60 was regular. The expression showed a trend of increase had a similar trend to the the expression of hsp40, significantly increased exposed in 4 mg/L, 8mg/L and 10mg/L progesterone treatment(P<0.05), but decreased in 6 mg/L progesterone treatment(P<0.05).The expression of hsp90 significantly increased exposed in 2mg/L progesterone treatment after 12 hours (P<0.05), but significantly increased in lOmg/L progesterone treatment after 24 hours (P<0.05).The expression of the four heat shock protein under the stress is related with their function. The function of the four heat shock protein in the the process of rotifers endocrine disturbance still need further study.To sum up, progesterone which has effects of environmental female hormones has obvious effects of endocrine disruptors on the population growth, all aspects of reproduction and characteristics of the life cycle in B. calyciflorus. Excepted that, it has an significant impact on the expression of hsps mRNA. Therefore, rotifers have potential application value in monitoring endocrine disruptors of environmental hormones in water ecosystem.