| Ovary, an important female reproductive organ, determines animal breeding potential. Although many follicles is in the mammalian ovary, few can develop the ovulatory follicles in life and the vast majority (about 99.9%) are atresia or degradation in preantral phase among them. This is a great waste. Existing research has shown that Wnt/β-catenin pathway closely relative to follicular development and ovarian function. In this study, firstly, we confirmed vaginal cytology-based estrous cycle stage classification through the real-time Q-PCR method, then detected mRNA expression of the Frizzled family genes in the mice ovary during estrous cycle by real-time Q-PCR, at last, using ISH, IHC and Western blotting methods, studied the temporal and spatial expression patterns of Frizzled 2 and 3 in the estrous ovary in mice. These works should have great theoretical and practical significance, help to clarify specific function and mechanism of Wnt/Frizzled signaling pathway during the development of female germ cells, to improve the fertility of female animals, a more reasonable utilization of follicles in the ovary, as well as to study the etiology and treatment of the reproductive disease.1. Estrous cycle stage classification and validation in miceIn order to choose a reasonable and accurate estrous cycle, the author observed vaginal cytology changes within estrous cycle by vaginal smears and dividing into four periods (proestrus, estrus, metestrus, diestrus). To confirm vaginal cytology-based estrous cycle stage classification, uterine wet weights and histone H3.2 mRNA expression were analyzed. Uterine weight varies with the estrous cycle. Uteri obtained from mice are the heaviest in the proestrus and estrus (p< 0.05). The relative expression level of H3.2 mRNA parallels wet weight and consistent with known changes in uterine cellular proliferation (p< 0.05). This suggest that sampled mice hav the reasonable and accurate estrous cycle in this study and lay the foundation for our follow-up study. 2. The relative expression levels of Frizzled family genes mRNA in the estrous ovary in miceTen Frizzled genes encoding Frizzleds have been identified in mammals. They can be activated as a receptor binding to Wnt ligands (a class of glycoproteins) to affect cell fate, including the development of embryonic ovary, but little known about the expression and function of the receptor molecules in the adult ovary. Therefore, we used real-time Q-PCR to analyze the relative expression levels of Frizzled 1-10 mRNAs in estrous ovary in mice. The results showed that The others had a different expression level except Frizzled 8(A-I). Expressions of Frizzled 1 and 2 were highest in proestrus and both were about 4 times higher than that in diestrus (P<0.05) (A, B); Frizzled 3 was 1.5 fold higher in proestrus and diestrus than those in the other two periods (P<0.05) (C); Frizzled 4 was found significantly high expression in estrus, which 2-3 fold than ones of the other three stages (P <0.05) (D); Frizzled 5 had marked higher signal in diestrus than those in proestrus and estrus, being approximately 5 times higher than that in estrus (P<0.05) (E), Frizzled 7 was similar to Frizzled 5, about 4 fold high than the level of proestrus (P<0.05) (G); Frizzled 10 expression in proestrus was significantly higher than those in estrus and diestrus and about more than 3 times higher than that in estrus(P<0.05) (I); but Frizzled 6 and 9 were no significant difference during estrous cycle(P<0.05) (F,H). These results suggested that Frizzled 1-4 may play an more important role in the development of follicle during estrous cycle stage. As previously studied, Frizzled 1 and 4 have been confirmed, but Frizzled 2 and 3 with higher expression levels have been little reported in the adult ovary. Therefore, the next work is that study the temporal and spatial expression patterns of Frizzled 2 and 3 in the estrus mouse ovary.3. The temporal and spatial expression patterns of Frizzled 2 mRNA and protein in estrous ovary in miceThe Wnt/β-catenin pathway is a conserved signalling pathway that regulates gene expression and controls diverse developmental processes such as cell fate specification, cell proliferation and cell differentiation. To our knowledge, the potential role of this pathway in the adult ovary has been poorly addressed. To this end, we investigated the expression pattern of Frizzled 2 in the mouse ovary during estrous cycle by real-time Q-PCR, in situ hybridization, Western blotting and immunohistochemistry. In this study, We found that during estrous cycle, Frizzled 2 mRNA and protein exhibited the highest level in the proestrus stage and rapidly decreased from estrus to diestrus. In situ hybridization results showed that the positive signals for Frizzled 2 were highly detected in the oocyte and stroma in proestrus stage, while moderate or weak Frizzled 2 mRNAs were localized in the oocyte, granulose cells, stroma and corpus luteum from estrus to diestrus. The localization pattern of Frizzled 2 protein was mostly consistent with its mRNA, but stronger Frizzled 2 proteins were present in the granulosa cells and membrane of oocyte in proestrus and estrus. Our data suggested that Frizzled 2 may be involved in regulating follicle growth, oocyte maturation during estrous cycle.4. Expression and localization of Frizzled 3 in the mice ovary during estrous cycleDuring the early period and adult, Frizzled genes regulate embryonic development, tissue and cell polarity, the formation of neural synapses, the cell proliferation, etc. To date, Frizzled 3 have not been investigated in adult ovary. Here, we detected the expression and localization of Frizzled 3 mRNA and protein in the mice ovary during estrous cycle through real-time Q-PCR, in situ hybridization and immunohistochemistry. It was found that the relative expression level of Frizzled 3 was highest in proestrus and diestrus, significantly decreased in estrus and metestrus (P<0.05). In situ hybridization results showed that in proestrus, high expression signals were found in the granulosa cell and stroma, weak level in corpus luteum. In estrous, stroma displayed high level, but granulosa cells and corpus luteum were revealed weak almost invisible levels. In metestrus, the moderate expression level was found in stroma, luteal cells were weak and almost no expression signals at all follicles. In diestrus, high expression level was in stroma, weak in granulosa cells and corpus luteum. Overall, the strongest hybridization signals were in proestrus and diestrus, second level in the other stages. This was mostly consistent with real-time Q-PCR. Expression patterns of Frizzled 3 protein were almost in accord with mRNA, the strongest signal was in the stroma, but moderate level were displayed in granulosa cells of estrus and diestrus, as well as low expression level in the oocyte cell membrane during proestrus and estrus. These results show that Frizzled 3 may regulate the developement of follicle, oocyte maturation in estrous cycle. |
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