The Expression Of NPY In The Estrous Cycle And Its Mechanism In Follicular Development And Ovulation

Research Purposes:In this work, the expression and distribution pattern of NPY during estrous cycle, as well as the effects of NPY on the maintenance of estrous cycle, the development of follicles and ovulation, should be investigated.Research Methods:The SPF C57BL/6 female mice were chosen as studied objects. The estrous cycle was ascertained by using daily vagina smear at a fixed time. The accuracy of estrous cycle was verified on the basis of the wet weight of uterus. Five identical ovarian tissues of the same estrous cycle were taken. After extracting the total RNA and total protein from ovarian tissue, the mRNA levels of NPY from different estrous cycles were determined by real-time PCR technology, the expression of protein was determined by using Western Blot method. After embedding ovary tissues with paraffin slice, the distribution of NPY in ovary during the estrous cycle was evaluated by immunohistochemistry. Next step, after intraperitoneal injection of BIIE0246 (NPY2R receptor antagonist) into mice, whether the estrous cycles would alter with the change of BIIE0246 were monitored consecutively for 15 days (3 period cycles). Fixed ovary and consecutively sections of HE dyeing then. The number of follicles was counted. Whether antagonistic NPY2R receptor would have effects on the development of follicles in different stages was observed. Meanwhile, the change of mRNA level genetically related to follicle development in ovary tissues was determined. In the ovulation inhibition experiment,10 IU PMSG were injected into each female mouse at the same time, and followed by injection of 10 IU HCG after 48 hours. The mice were executed 6 hours later, and the blood was taken by removalling the eyeballs according to hormone test, and the number of ovulation was recorded. For the treated group, BIIE0246 (0.2μg/g) was injected every 12 hours after the PMSG injection.Results and Conclusions:The mRNA level of NPY during the estrus cycle was three times higher than that of early estrus (p< 0.05) and higher than that of the metaoestrus and diestrus. Both methods of real-time PCR and Western Blot gave almost the same results, and its NPY was expressed with the highest level in late estrus (p< 0.05). The immunohistochemistry tests showed that, during estrus, NPY was expressed in the ovary interstitial tissues and was distributed in membrane cells and corpus as well, which indicated that the expression of NPY in ovary organization might be related to the estrous cycle. According to the aforementioned results, it can be speculated that NPY is likely to take part in the development of follicles and ovulation. The mice, treated with receptor antagonist BIIE0246, had the same estrous cycle as those of the control group, while the numbers of antral follicles (early stage), Graffin follicles, and corpora atretica of the treated mice were significantly decreased than those of the control group (p< 0.05), but the numbers of early follicles were not significantly influenced. The numbers of genes related to follicle development, i.e., c-kit, AMH, GDF-9, CD2, were significantly decreased (p< 0.05), and those of apoptosis genes, Bax, bax/bcl-2, and Fas, were also significantly decreased? (p< 0.05). The ovulation inhibition experiment indicated that the ovulation number of the treated group was much less than that of the control group (p< 0.05). The numbers of ovulation-related genes, Cox-2, HA-2, TSG-6, ADAMTS-1, were greatly decreased (p< 0.05). It can be concluded that:i) NPY is related to the development from antral follicles (early stage) to Graffin follicles and to the formation of antral follicles, ii) NPY takes part in ovulation process, iii) receptor antagonist of NPY neither changes the estrous cycle of female mice, nor takes part in the development of follicles before the antral follicles get ripe.