MicroRNAs And Check Point Kinases1/2(Chk1/2) Pathway On Mouse Ovaries

In mammals, the networks of follilular development and oocyte maturation are complex including cell proliferation, apoptosis, cell cycle and cell differentiation, which are regulated by many regulators and through different regulatory pathways. The regulatory of genes at transcriptional level(such as micro RNA) and translational level(such as sumoylation) play improtant roles in physiological process. Based on the deep sequencing results of micro RNA in mouse ovaries from birth to sexual maturity and superovulation, this topic focus on studying the following micro RNAs, mi R-15 a, mi R-16, mi R-410, mi R-485, mi R-495 and mi R-133 a, which predict to regulate checkpoint kinase 1(Chk1, the core component of the cell response to genotoxic stresses and cell cycle control), p53(the key regulator of apoptosis) and SUMO-1(post-translational modification). The primary objectives of this study were to study the expression pattern of micro RNAs in different developmental follicles, and assess the roles of FSH and LH on the expression pattern of the selected micro RNAs; the roles and regulatory mechanism of micro RNAs in in vitro culture follicles and granulosa cells; the roles of Chk1/2 during folliculogenesis, cellular proliferation/apoptosis and oocyte maturation; detection and verification the target genes(Chk1, p53, SUMO-1) of micro RNAs. The results of this study are as follows:1 The expression pattern of selected micro RNAs in follicles and the roles and regulatory mechanism of micro RNAs during folliculogenesis(1) The expression pattern of micro RNAs in different developmental follicles. Isolation of preantral follicles(100-130 μm and 200-280 μm) and antral follicles(450-550 μm and 500-600 μm), and Q-PCR was applied to detect the expression level of the six micro RNAs in different devolpmental follicles. Among them, mi R-410 and mi R-495 expression were lowest in prantral follicles(200-280 μm) compared with the other three samples(P<0.01; P<0.05); mi R-485 expression was lower in preantral follicles(200-280 μm) compared with the other three samples, and was higher in antral follicles than preantral follicles(P<0.01); mi R-15 a and mi R-133 a expression were peaked in preantral follicles(100-130 μm), and subsequently fell off in bigger preantral follicles(200-280 μm) and antral follicles(P<0.001; P<0.001); while mi R-16 expression was higher in antral follicles than preantral follicles(P<0.01).(2) The expression of the six micro RNAs were regulated by FSH and LH. Granulosa cells from 21 days old female mice ovaries(3w-GCs) were isolation and cultured with or without FSH, after cultured for 6 h, the expression of mi R-485, mi R-495, mi R-15 a and mi R-133 a in cells cultured with FSH were significantly lower compared with control(P<0.001); while mi R-410 and mi R-16 expression were significantly higher in FSH cultured granulosa cells compared with control(P<0.001; P<0.05). Granulosa cells from 21 days old female mice ovaries which injected with PMSG for 48 h(pre-GCs) were isolation and cultured with or without LH. At 3 h, the expression of mi R-410, mi R-485, and mi R-133 a were up-regulated in LH cultured cells compared with control(P<0.001), while mi R15 a and mi R-16 were down-regulated by LH(P<0.05); At cultured for 24 h, the expression of mi R-410, mi R-485, and mi R-133 a were lower compared with control. Furthermore, in order to confirm the role of FSH on the expression of mi R-485 and mi R-495, granulosa cells were transfected with mi R-485 or mi R-495 mimics and cultured with FSH, and Q-PCR results showed that FSH could also down-regulated mi R-485 and mi R-495 over-expression level in vitro.(3) The preliminary study of the roles of micro RNAs in follicular development. Preantral follicles(100-130 μm) from 12-14 days old female mice ovaries were isolated and cultured for 2 days and transfected with the selected micro RNAs mimics cultured for another 4 to 6 days, and recored the diameter every 2 days. Results showed that mi R-485 mimics could sifnificantly repress preantral follicles growth at cultured for 6 days(P<0.01); meanwhile the growth rate of 4 to 6 days was also lower in mi R-485 mimics transfection follicles than control(P<0.05). mi R-495 mimics tansfected for 4 days, there was no significantly difference between the transfection group and control, howerer, the growth rate of 2 to 4 days was lower in mi R-495 mimics transfection follicles than control(P<0.05). The results were consistant with the expression pattern of mi R-485 and mi R-495 in preantral follicles, and suggested that mi R-485 and mi R-495 may play important roles in follicular development. Follicles transfected with mi R-133 a, mi R-15 a and mi R-16 mimics showed no difference in follicular gowth and growth rate compared with control, however, the growth rate of 4 to 6 days were higher in transfection follilces than control.(4) The roles of micro RNAs in the proliferation and apoptosis of granulosa cells. Pre-GCs were tansfected with micro RNA mimics or inhibitor for 48 h, then cells were collected for the analysis of proliferation and apoptosis. Results showed that mi R-485 could promote cell proliferation(P<0.001) and suppress cell apoptosis(P<0.01); while mi R-495 could promote cell apoptosis(P<0.01) and suppress cell proliferation(P<0.01), and arrested the cell cycle at S stage(P<0.01); mi R-133 a mimics could suppress the proliferation(P<0.01) and arrest the cell cycle at G1 stage(P<0.01), meanwhile mi R-133 a inhibitor could significantly promote the proliferation of granulosa cells(P<0.001); mi R-15 a mimics could significantly promote cell proliferation(P<0.001).(5) The role of mi R-495 in estrogen secretion. Pre-GCs transfected with micro RNAs mimics for 48 h and the medium were collected for the detection of estrogen level. Results showed that mi R-495 could significantly suppress the expression level of estrogen(P<0.001), and Q-PCR results showed that mi R-495 could down-regulate the m RNA exoression level of genes related to steroid, such as CYP11A1, CYP19A1, and St AR. The results indicated that mi R-495 may play an important role in follicular and granulosa cells development through regulatory the steroid related genes expression and estrogen synthesis and secretion.(6) Three target genes were confirmed. Through the use of luciferase reporter assays and the culture model of granulosa cells, three target genes were confirmed. mi R-485 could bind to the 3’ UTR of p53, and both the m RNA and protein level were down-regulated by mi R-485; both mi R-15 a and mi R-16 could bind to the 3’ UTR of Chk1, and mi R-16 could down-regulated the expression level of both m RNA and protein of Chk1, while mi R-15 a could only suppress the Chk1 protein expression. This result indicated that mi R-16 directly induced Chk1 m RNA destabilization, while mi R-15 a regulated Chk1 expression through translational repression; mi R-133 a could bind to the 3’ UTR of SUMO-1, and down-regulate the SUMO-1 protein expression, which indicated that mi R-133 a regulated SUMO-1 expression through translational repression.2 The roles and regulatory mechanism of Chk1/2 duting folliculogenesis, cellular proliferation/apoptosis and oocyte meiotic maturation.(1) Chk1/2 played crucial roles during preantral follicular development. Preantral follicles cultured with or without Chk1/2 inhibitor for 4 days, and follicles showed no growth even negative growth in inhibitor cultured follicles compared with control; follicles cultured with inhibitor showed atresia under the microscope.(2) Inhibition of Chk1/2 blocked the growth of granulosa cells. Granulosa cells were cultured with different concentration of Chk1/2 inhibitor for 3 days, and results showed that Chk1/2 inhibitor could significantly suppress the proliferation of granulosa cells(P<0.001), and in time and dose dependent manner; meanwhile the cell cycle were arrested at S and G2 stages(P<0.001). Granulosa cells cultured with Chk1/2 inhibitor for 2 days showed apoptosis compared with control(P<0.05). Furthermore, the m RNA and protein expression level of PCNA(a marker gene of proliferation) were significantly down-regulated by Chk1/2 inhibitor(P<0.01; P<0.01); and m RNA and protein expression level of Bax(a marker gene of apoptosis) were significantly up-regulated by Chk1/2 inhibitor(P<0.001; P<0.05)(3) Inhibition of Chk1/2 could block the mature of oocytes. Oocytes cultured with Chk1/2 inhibitor showed no effect on GVBD, but significantly suppressed the extrusion of PB1(P<0.05); through adding the inhibitor in the culture medium at different developmental time points, we found that inhibition of Chk1/2 mainly functioned before MI stage.(4) Chk1/2 played important role in spindle formation and chromosome alignment through a new signaling pathways/molecules, including α-tubulin, γ-tubulin, Securin, CREST, and phospho-P38-MAPK.(5) Inhibition of Chk1/2 could destroy the spindle morphology and inhibit the activity ratio of MII stage oocytes. Mature oocytes were collected from ampulla of uterine tube of female mice after superovulation, then cultured with Chk1/2 inhibtior for 1 h or 2 h. Results showed that the spindle morphology of mature oocytes showed no difference at cultured for 1 h, while the spindle morphology was destroyed when cultured with inhibitor for 2 h. Meanwhile, after activation the second polar body extrusion and pronuclear formation were significantly suppressed in oocytes cultured with Chk1/2 inhibitor(P<0.01; P<0.001).In conclusion, our results revealed the expression patter of the six micro RNAs in different developmental follicles, and the regulatory of FSH and LH on micro RNAs expression; we showed preliminary study of the six micro RNAs during folliculogenesis, granulosa cells proliferation, apoptosis, cell cycle, and the synthesis/ecretion of estrogen; we detected and proved three target genes(Chk1, p53, SUMO-1) of selected micro RNAs; and we studied the roles and regulatory mechanism of Chk1/2 duting folliculogenesis, cellular proliferation/apoptosis and oocyte meiotic maturation. The results of this study could establish a theoretical foundation for micro RNAs in the regulation of follicular growth/atresia, and granulosa cells proliferation/apoptosis/differentiation through target Chk1, p53, and SUMO-1; and provide new understanding about the functions and regulatory mechanism of micro RNAs during folliculogenesis/oogenesis.