Estrogen On The C-type Natriuretic Peptideinduced Meiotic Arrest Of Goat Oocytes

In female germ cells, oocytes meiosis is initiated in follicular during fetal life, but subsequently arrested for a prolonged period at the diplotene stage of the first meiotic prophase. The primary oocytes also form a germinal vesicle(GV) structure, so the oocytes of this period is also known as germinal vesicle stage oocytes. C-type natriuretic peptide(CNP) is a member of natriuretic peptide family. Recent studies have shown that CNP is naturally occurring in the follicle of mouse, and it is the key material to maintain meiotic arrest in mouse oocytes, which is generated by MGCs, whereas the transmembrane guanylyl cyclase natriuretic peptide receptor 2(NPR2) is expressed predominantly by cumulus cells. Further studies suggest that endocrine hormones, such as oocyte-derived paracrine factors(ODPFs), 17β-estradiol(17β-E2), follicle-stimulating hormone(FSH), and luteinizing hormone(LH) may involve in oocyte meiotic arrest by mediating the expression of CNP/NPR2. Our laboratory also has indicated that estrogen act on CNP-induced meiotic arrest of goat oocytes. However, the effect of different concentrations of estrogen is disparity in oocyte meiotic arrest, so we speculate that this may be due to the different estrogen receptors, the results are as follows:(1) We studied the effects of different concentrations of estrogen on the CNP-induced meiotic arrest of goat oocytes after culture for 6 h by hochest33342 dyeing method. GV oocytes number was analyzed using descriptive statistics. Incubation of COCs with 17β-E2(1 μg/m L) blocked the inhibitory effects of CNP on meiotic arrest, whereas 17β-E2(0.27 ng/m L) promoted the maintenance of CNP-induced meiotic arrest.(2) Based on the experiments, we using agonist and antagonist of estrogen receptor to discuss the different effects of different concentrations of estrogen and to investigate which receptor participated in CNP-induced meiotic arrest of goat oocytes. The results in 1 μg/m L 17β-E2 showed that incubation of COCs with 1 μg/m L 17β-E2 or G1 significantly promoted spontaneous meiotic maturation of oocytes, which was reversed by co-treatmented with G15(P>0.05). However, incubation with ICI182780 was ineffective. On the other hand, the results in 0.27 ng/m L 17β-E2 indicated ICI182780 impeding the increasing effects of estrogen on meiotic arrest. This showed that 1 μg/m L 17β-E2 suppressed the effect of CNP on goat oocytes meiotic arrest through GPR30, while 0.27 ng/m L 17β-E2 enhanced the effect of CNP on goat oocytes meiotic arrest through ER.(3) The different effects of different concentrations of estrogen on CNP-induced meiotic arrest might be mediated by the expression of Npr2 m RNA. The results showed that 0.27 ng/m L 17β-E2 increased the expression of Npr2 m RNA in culture for 2 h. Conversely, 17β-E2(1 μg/m L) treatment caused a decline in Npr2 m RNA levels. However, compared to CNP groupe, 1 μg/m L 17β-E2 and 0.27 ng/m L 17β-E2 all promoted the expression of Npr2 m RNA in COCs for 4 h and 6 h. But as time goes on, Npr2 m RNA levels in all treatment groups were declining. In addition, 0.27 ng/m L 17β-E2 treatment groups had no significant differences in Npr2 m RNA expression levels both at 4 h and 6 h compared with 2 h CNP group(P>0.05). This showed that 0.27 ng/m L 17β-E2 maintained the expression of Npr2 m RNA at least for 6 h.(4) Using agonists and antagonists of estrogen receptor to further explore high concentration of estrogen on Npr2 m RNA expression in 2 h. Results showed that COCs cultured in 17β-E2(1 μg/m L)+G15 reversed the effect of 17β-E2(1 μg/m L) on Npr2 m RNA levels, whereas ICI182780 was ineffective. Moreover, COCs incubated with G1 further illustrated that 1 μg/m L 17β-E2 declined the expression of Npr2 m RNA through GPR30.(5) COCs was incubated with CNP, CNP+E2(0.27 ng/m L) or CNP+E2(1 μg/m L) for 6 h and then in OM for 18 h. The rate of oocyte maturation in CNP, CNP+E2(0.27 ng/m L) were 70.00±5.93%, 73.73±6.28%, respectively(P>0.05). Nevertheless, CNP+E2(1 μg/m L) group was only 52.83±8.11%(P<0.05). After 24 h culture in vitro, cumulus expansion of goat COCs were also different. All groups in 0 grade、Ⅰgrade and Ⅲgrade had no significant differences(P>0.05). But theⅡgrade numbers in CNP+E2(1 μg/m L) group were significantly lower than those in CNP group and CNP+E2(0.27 ng/m L) group(P<0.05). However, Ⅳgrade numbers in CNP+E2(1 μg/m L) group were significantly higher than those in CNP group and CNP+E2(0.27 ng/m L) group(P<0.05).In conclusion, different concentrations of estrogen played a diverse role in the CNP-induced meiotic arrest of goat oocytes. 1 μg/m L 17β-E2 blocked the effect of CNP on meiotic arrest through GPR30, whereas 0.27 ng/m L 17β-E2 elevated the effect of CNP on meiotic arrest through ER. In addition, 1 μg/m L 17β-E2 decreased the expression of Npr2 m RNA through GPR30, while 0.27 ng/m L 17β-E2 could elevate the expression of Npr2 m RNA. Goat COCs cultured in medium containing 100 ng/m L CNP and 1 μg/m L 17β-E2 for 6 h and then cultured in OM for 18 h can significantly promoted the expansion of cumulus, but reduced the maturation rate.