생쥐의 난포발달과 배란과정에서 PEA3 전사요소들의 발현에 관한 연구

Abstract

ETS 전사요소들은 다양한 생물학적 작용과 연관되어 있는데 ETS family 전사요소의 한 구성원인 ERM이 결핍된 수컷 생쥐는 정원세포의 자가증식을 유지하는 능력을 잃어버려 결국 불임이 된다. 그러나, 암컷 생식에서의 ERM의 기능에 대한 연구는 아직 진행되어 있지 않다. 본 연구에서는 생쥐 암컷에서 ERM의 기능을 밝히기 이전에 생쥐의 난소에서 PEA3 subfamily인 PEA3, ER81, ERM의 일시적인 발현 양상을 알아보았다. 난포발달과 배란의 각 단계별 난소 조직을 얻기 위해 암컷 생쥐에 PMSG와 hCG를 주사하여 난포발달과 배란을 유도하였다. 실험군은 PMSG만 주사한 것(0 hr)과 hCG를 주사한 후 3시간(3hr), 6시간(6 hr), 9시간(9 hr), 그리고 배란 이후인 15시간(15hr)이다. RT-PCR과 quantitative RT-PCR, 그리고 냉동 조직 절편에서 면역형광염색을 수행하였다. PEA3 subfamily의 구성원은 난포발달과 배란 동안 특별한 발현 양상을 보인다. PEA3의 발현은 3 hr에 증가하고 6 hr 이후에 감소한 후 배란시기에 다시 증가된다. ERM의 발현은 난포발달의 초기단계에 증가하여 배란 시까지 유지되고 배란 이후에 감소된다. ER81의 발현은 난포발달과 배란 동안 별다른 차이를 보이지 않는다. 면역형광으로 ERM과 PEA3의 단백질 존재부위를 실험한 결과 PEA3는 초기와 후기의 난포의 과립막세포층에서 나타났다. ERM은 과립막세포층과 난자에 흩어져있는 양상으로 나타났다. ETS 전사요소의 PEA3 Subfamily는 난포발달의 단계 동안 다른 발현과 존재부위에 나타난다. 생쥐의 난소에서 ERM과 PEA3의 특별한 발현 양상을 통해 이러한 전사요소가 난포발달과 배란에 포함되는 조절 유전자에 중요한 역할을 할 것으로 사료된다.; The PEA3 transcription factors are composed of three members: PEA3 (E1AF, ETV4), ER81 (ETV1), and ERM (ETV5). They share high sequence homology and are conserved in functional domains. Regulation of these factors at the transcription level is important for their availability in controlling target genes, while their activity may also be regulated by post-translational modification. PEA3 mutant mice are phenotypically normal except that males display sexual dysfunction possibly caused by neuronal defects. In contrast, ER81 deficient mice show severe motor discoordination due to reduced connections between sensory afferents and the corresponding motor neurons and die at age of 3-5 weeks. Male mice with targeted disruption of ERM lose the ability to maintain spermatogonial stem cell niche and to be infertility. However, potential roles for ERM in female reproduction in mice are yet to be investigated. The aim of this study is to assess the spatiotemporal expression profiles of PEA3 transcription factors, PEA3, ER81, and ERM, in the mouse ovary to gain insights into potential roles for these factors. Mice were stimulated with pregnant mare’s serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to obtain ovarian samples at each stage of follicular growth and ovulation. RT-PCR and quantitative RT-PCR were performed. Immunofluorescence staining on frozen tissue sections was performed to localize protein expression. Female mice with targeted disruption of ERM show poor response to gonadotropins, leading to low rates of mating and ovulation. Three members of PEA3 subfamily exhibit differential expression patterns during follicular growth and ovulation. The expression of PEA3 is increased at 3 hr-post hCG and decreased at 6 hr. PEA3 expression is upregulated again at ovulation. The expression of ERM is increased during early stages of follicular growth and this level is sustained until ovulation. Its expression is decreased after ovulation. The expression of ER81 is not notable during follicular growth and ovulation. Nuclear localization of PEA3 was noted in granulosa cell (GC) layers in early and late follicles. Immunoreactive ERM was localized in a scattered pattern in GC layers and also in oocytes. Collectively, the results show that PEA3 transcription factors exhibit differential expression and localization during stages of follicular growth. Specific spatiotemporal expression patterns of ERM and PEA3 in the mouse ovary suggest that these transcription factors may play important roles in regulating genes involved in events of follicular growth and ovulation.