Nitric Oxide에 의한 흰쥐의 배란전 난포에서Apoptosis의 조절 = Nitric Oxide-Mediated Regulation in the Rat Preovulatory Follicular Apoptosis

다국어 초록 (Multilingual Abstract)

Nitric oxide (NO) has recently emerged as a potential regulator of follicular development because of its involvement in the regulation of several physiological functions of the ovary, including ovulation, oocyte meiotic maturation, and steroidogenesis. Moreover, NO influences apoptotic cell death of follicular cells as a follicle survival factor. It is reported that NO inhibits apoptosis by stimulating the induction of cytoprotective stress proteins in hepatocytes. However, little is known about the NO-mediated regulation mechanism in the inhibition of follicular apoptosis, especially the relationship between the effect of NO on ovarian cell death and the expression of HSP70 as well as p53 and Bax which are central players in the regulation of apoptosis.
The present study was conducted to investigate the protective effect of NO on spontaneously induced follicular apoptosis in serum-free condition and on the expression of HSP70, p53 (a tumor suppressor gene, an inducer of apoptosis), p21, and Bax (an inducer of apoptosis) mRNA and protein.
Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 h in serum-free medium with or without sodium nitroprusside (SNP), a NO generator. After culture, ovarian follicular apoptosis was assessed by DNA ladder analysis and concurrent expression of HSP70, p53, p21, and Bax mRNA and protein was measured. Granulosa cells within follicles incubated in medium alone exhibited extensive apoptosis after 24 h of incubation, and this onset of apoptosis was blocked by treatment with SNP. Both of mRNA and protein levels of HSP70 were highly increased but Bax were suppressed by treatment with NO donor. In addition, NO inhibited p53-independent p21 expression.
This study suggests that NO prevents rat preovulatory follicular apoptosis in vitro by stimulating HSP70 and suppressing Bax expression. Moreover, this NO-mediated regulation may be through a p53-independent p21- regulated mechanism.

국문 초록 (Abstract)

Nitric oxide (NO)는 난소의 여러 가지 생리적 기능인 배란, 호르몬 작용, 난자 성숙 등에 작용하는 중요한 조절자로서 알려져 있다. 특히 최근에는 난포 세포의 apoptosis를 억제하는 follicular survival factor로서 보고되었다. NO에 의한 apoptosis 억제는 hepatocytes 에서 cytoprotective stress proteins을 유도함으로써 일어난다고 알려졌으나 난포에 있어서 NO에 의한 apoptosis 억제 과정에서는 HSP70 유전자의 발현이나
apoptotic signal transduction에 대한 연구가 수행되고 있지 않다. 따라서 본 연구는 흰쥐의 배란전 난포에 NO의 유도 물질인 sodium nitroprusside (SNP)를 처리함으로써 난포의 apoptosis가 억제될 때, HSP70와 함께 apoptosis의 중요한 조절자로 알려진 p53, p21, Bax 등의 mRNA 또는 protein의 발현이 조절되는지 알아보았다.
생후 25일자의 SD 흰쥐에 PMSG 10 IU를 피하 주사하여 배란을 유도하여 48시간 후 배란 전 난포를 얻어 serum-free condition에서 24시간 체외배양하였다. 배양을 시작하기 전의 대조군, 24시간 배양군, NO의 유도 물질인 sodium nitroprusside (SNP)을 처리한 후 24시간 배양한 군으로 나누어 실험하였다. 배양 후 DNA ladder assay 결과, NO
donor 없이 배양한 군은 완전한 apoptosis가 유도된 데 비해서 SNP 처리군에서는 DNA ladder가 감소하는 경향을 보였다. 난포의 apoptosis억제와 함께 나타난 NO-mediated 유전자 발현양상에서는 SNP에 의해 유도된 NO에 의해 HSP70 발현이 현저하게 증가하였고 반면에 Bax의 발현은 유의적으로 감소하는 경향을 보였다. 또한 난포의 apoptosis는 p53-independent 하게 나타났으며 p21에 의해 조절되었다.
본 연구는 흰쥐의 배란 전 난포에서 NO에 의해 조절되는 apoptosis 억제에서 HSP70 및 p53, p21, Bax의 mRNA 또는 protein 발현을 연구한 최초의 보고로서 위의 연구 결과를 통해 난포의 apoptosis는 p53-independent한 p21에 의해 조절되는 것으로 보이며 또한 NO는 HSP70을 현저히 증가시키고 Bax의 발현을 감소시킴으로 follicular apoptosis를 억제한다고 사료된다.

목차 (Table of Contents)

• LIST OF TABLE = i
• LIST OF FIGURES = ii
• ABSTRACT - 1
• I. INTRODUCTION = 3
• II. MATERIALS AND AMETHODS = 6
• 1. Animal treatment = 6
• 2. Tissue sections = 6
• 3. Follicle culture = 6
• 4. DNA extraction and fragmentation analysis = 7
• 5. In situ localization of apoptotic cells = 8
• 6. Reverse Transcription - Polymerase Chain Reaction = 8
• (1) Total RNA isolation = 8
• (2) RT-PCR analysis = 9
• 7. Western blot analysis = 10
• (1) Preparation of cellular extracts = 10
• (2) Western blotting = 10
• 8. Data analysis = 11
• III. RESULTS = 12
• 1. NO protects follicles from spontaneously induced apoptosis = 12
• 2. NO induces HSP70 and inhibits Bax expression = 13
• (1) NO-mediated induction HSP70 = 14
• (2) NO-mediated inhibition of p53-independent p21 = 14
• (3) NO-mediated inhibition of Bax = 15
• IV. DISCUSSION = 28
• V. REFERENCES = 32
• ABSTRACT IN KOREAN = 40
• ACKOWLEDGEMENT = 42