羅愛月 楊書紅 沈 薇 丁 婷 賴志文 王 毅 盧運(yùn)萍 王世宣

(華中科技大學(xué)附屬同濟(jì)醫(yī)院婦產(chǎn)科,武漢430030)

卵巢催乳素受體mRNA在小鼠卵泡發(fā)育及排卵過程中的表達(dá)

羅愛月 楊書紅 沈 薇 丁 婷 賴志文 王 毅 盧運(yùn)萍 王世宣＊

(華中科技大學(xué)附屬同濟(jì)醫(yī)院婦產(chǎn)科,武漢430030)

目的 探討卵巢催乳素受體(Prolactin receptor,PRLR)mRNA在小鼠不同發(fā)育階段及排卵過程中的表達(dá)情況。方法 選擇不同發(fā)育時(shí)期的昆明小鼠,以及性未成熟昆明小鼠予以PMSG-HCG(Pregnancy Mare Serum Gonadotrophin- Humane chorionic gonadotrophin,孕馬血清促性腺激素-人絨毛膜促性腺激素)序貫處理,采用實(shí)時(shí)定量聚合酶鏈反應(yīng)(Real-Time Polymerase Chain Reaction,Real-Time PCR)檢測小鼠卵巢中PRLR mRNA的表達(dá)。結(jié)果 PRLR mRNA的表達(dá)水平隨小鼠的不斷發(fā)育而顯著性升高,PRLR在6周齡小鼠卵巢中的表達(dá)量是3周齡小鼠的3.86倍(P<0.01),11周齡與33周齡小鼠的表達(dá)水平分別是3周齡小鼠的19.67倍、19.81倍(P<0.01);PMSG處理后12h,PRLR表達(dá)水平是對(duì)照組的1.44倍(P<0.05),24h、48h后分別達(dá)到5.48倍和7.14倍(P<0.01),HCG處理后4h、8h、12h,PRLR表達(dá)水平有所下降,但仍高于對(duì)照組(P<0.01),24h、48h后其表達(dá)水平再次升高,分別是對(duì)照組的5.64倍和6.04倍。結(jié)論 PRLR對(duì)小鼠卵泡的生長、發(fā)育及其黃體形成與維持發(fā)揮重要作用。

催乳素受體;卵泡發(fā)育;排卵;Real-Time PCR

卵泡的募集、生長、成熟、排卵及排卵后黃體形成,以及絕大部分卵泡在不同發(fā)育階段的閉鎖等一系列過程,受到各種因素的復(fù)雜調(diào)控。下丘腦-垂體-卵巢軸(Hypothalamus-Pituitary-Ovary axis,H-P-O axis)分泌的激素是其中重要的調(diào)節(jié)因素之一,而卵巢本身表達(dá)的各種細(xì)胞因子對(duì)卵泡的正常發(fā)育不可或缺。催乳素受體(Prolactin Receptor,PRLR)是一個(gè)跨膜蛋白,屬于細(xì)胞因子受體超家族,因其不同的剪切方式,存在長形和短形兩種形式[1]。這兩種形式的受體其生物學(xué)功能在不同的組織中有所差別。如乳腺組織中主要為長形PRLR,其通過JA K/STAT信號(hào)轉(zhuǎn)導(dǎo)及轉(zhuǎn)錄激活途徑介導(dǎo)乳蛋白基因的表達(dá)[2-3];而肝臟組織中大量表達(dá)的短形PRLR可能與肝臟本身的受體調(diào)節(jié)及肝細(xì)胞功能調(diào)控有關(guān)[4]。在生殖系統(tǒng)中長短型受體均有表達(dá)。PRLR與大鼠的動(dòng)情周期及動(dòng)物的繁殖能力密切相關(guān)[5-7];其表達(dá)受到性腺激素的調(diào)節(jié)[8];且在卵巢早衰的發(fā)生發(fā)展中起重要作用[9]。本文在mRNA水平檢測PRLR前體在不同發(fā)育階段小鼠卵巢及排卵過程中的表達(dá)情況,旨在為PRLR在卵泡發(fā)育過程及卵巢功能中的進(jìn)一步研究奠定基礎(chǔ)。

材料和方法

1 實(shí)驗(yàn)動(dòng)物

昆明小鼠購自華中科技大學(xué)同濟(jì)醫(yī)學(xué)院動(dòng)物中心。選擇3、7、11、33周齡小鼠各10只。另3周齡小鼠50只予以 PMSG(杭州動(dòng)物藥品廠產(chǎn)品) 10U/只腹腔注射,其中25只小鼠在PMSG注射48h后再給予 HCG(麗珠制藥集團(tuán)產(chǎn)品)10U/只腹腔注射;設(shè) PMSG注射后4、8、12、24、48h組,及HCG注射后4、8、12、24、48h組,每組各5只;另取5只3周齡小鼠不予PMSG-HCG處理,設(shè)為對(duì)照組。頸椎脫臼法處死小鼠后,迅速取出雙側(cè)卵巢置入-80℃保存,以供提取總RNA。

2 實(shí)時(shí)定量聚合酶鏈反應(yīng)(Real-Time Polymerase Chain Reaction,Real-Time PCR)

2.1 RNA提取和檢測

采用 Trizol法提取總RNA。取出儲(chǔ)存于-80℃的卵巢組織加入 1ml Trizol研磨至碎;再加入200μl氯仿劇烈震蕩15s,室溫靜置10min,4℃離心(12000rpm ×8min),吸取上層水相;加入等體積預(yù)冷的異丙醇,置于-20℃30min以上,4℃離心(12000rpm×12min),留取沉淀;75%乙醇(無RNase三蒸水配制)漂洗,干燥后以20μl無RNase三蒸水溶解 RNA沉淀;并測定所提取RNA的濃度和純度。

2.2 Real-Time PCR反應(yīng)引物

在 NCBI(http://www.ncbi.nlm.nih.gov)中搜索小鼠PRLR核苷酸序列號(hào)NM_011169,委托 Takara公司設(shè)計(jì)Sub Green熒光嵌合法的Real-Time PCR反應(yīng)引物,由上海 Sangon公司合成。PRLR引物序列(5’-3’):上游引物AACATGCCATCTGCACTTGCTTAC,下 游 引 物 TCCATCTGACCCAGGA TTCCA。同時(shí)以3-磷酸-甘油脫氧酶(GAPDH)作為內(nèi)參。GAPDH引物序列(5’-3’):上游引物 TGTGTCCGTCGTGGATCTGA,下游引物TTGCTGTTGAAGTCGCAGGAG。

2.3 Real-Time PCR反應(yīng)體系、條件及mRNA表達(dá)量計(jì)算

反應(yīng)體系含2×SYBR Premix Ex Taq混合物10μl,引物前后鏈各0.5μl,cDNA 1μl,加滅菌雙蒸水至20μl。置DNA Engine Opticon 2連續(xù)熒光檢測系統(tǒng)擴(kuò)增,條件為95℃2min,95℃10s,退火溫度20s,72℃20 s,共40個(gè)循環(huán)。反應(yīng)完畢后從95℃-55℃,每下降0.4℃記錄一次熒光強(qiáng)度作溶解曲線。同一樣本在同一批反應(yīng)中包括GAPDH和目的基因,每一反應(yīng)設(shè)3個(gè)復(fù)孔。每一樣本重復(fù)檢測2次。檢測樣本中目的基因mRNA的相對(duì)表達(dá)量:待測基因cDNA進(jìn)入PCR指數(shù)增長期的起始點(diǎn)即為循環(huán)閾值(Cycle threshold,Ct)。以每個(gè)樣本在同一批反應(yīng)中的 GAPDH為內(nèi)參照,計(jì)算樣本△Ct值,再以對(duì)照組的△Ct值為參照,計(jì)算實(shí)驗(yàn)組△△Ct值,根據(jù)公式計(jì)算mRNA相對(duì)表達(dá)量[10]:

3 統(tǒng)計(jì)學(xué)處理

數(shù)據(jù)分析采用SPSS13.0計(jì)算機(jī)統(tǒng)計(jì)軟件,進(jìn)行T-檢驗(yàn)分析。以 P值小于0.05為有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 PRLR在不同周齡小鼠卵巢中的表達(dá)情況

PRLR mRNA的表達(dá)水平與小鼠的周齡密切相關(guān)。以3周齡組為參照,其余各組與之相比較,計(jì)算各組mRNA相對(duì)表達(dá)水平。6周齡組與3周齡組相比較其表達(dá)水平明顯增高(P<0.01);11周齡組和33周齡組均分別與3周齡組、6周齡組相比,其表達(dá)水平均具有顯著性差異(P<0.01),但11周齡組與33周齡組相比表達(dá)水平無統(tǒng)計(jì)學(xué)差異(P>0.05),見圖1。

2 PRLR在小鼠排卵過程中的表達(dá)情況

采用PMSG-HCG處理性成熟前小鼠,建立小鼠排卵模型。檢測排卵過程中小鼠卵巢 PRLR mRNA的表達(dá)。在PMSG處理后12h,PRLR表達(dá)水平升高,是對(duì)照組(未處理組)的1.44倍,有顯著性差異(P<0.05),24h、48h后其表達(dá)水平分別為對(duì)照組的5.48倍和7.14倍(P<0.01);HCG處理后4h、8h、12h,PRLR表達(dá)水平有所下降,但與對(duì)照組相比仍有顯著性差異(P<0.01),在HCG處理后24h、48h,其表達(dá)水平再次升高,分別達(dá)5.64倍和6.04倍(P<0.01),見圖2。

討 論

多項(xiàng)研究結(jié)果認(rèn)為,PRLR在生殖系統(tǒng)中扮演重要角色。PRLR缺失的雌性小鼠其繁殖能力下降,且可能出現(xiàn)植入前胚胎發(fā)育的完全停滯[11]。Kang B[7]與Liu W[6]等分別報(bào)道 PRLR與家禽的產(chǎn)卵數(shù)和哺乳動(dòng)物的繁殖性能直接相關(guān)。本研究顯示PRLR的表達(dá)水平隨小鼠卵巢的發(fā)育而不斷增高。在性成熟期前(3周齡)的小鼠卵巢中PRLR呈相對(duì)低水平表達(dá),進(jìn)入性成熟期(6周齡)后其表達(dá)量明顯增加,而在繁殖適齡期(11周齡、33周齡)的小鼠其表達(dá)量更高,是性成熟前的近20倍,性成熟期的5倍之多。這一結(jié)果從另一角度說明了PRLR與卵巢功能及生殖能力的關(guān)系。

Fig.1 PRLR expression of the mouse ovary in different developmental stages注:＊與3周齡組相比有顯著性差異(compared with 3Wand 6W)P<0.01。△分別與3周齡組、6周齡組相比,有顯著性差異(compared with 3W)P<0.01。

Fig.2 PRLR expression during the process of mouse ovulation注:＊與對(duì)照組相比有顯著性差異(compared with the control),P<0.05?！髋c對(duì)照組相比(compared with the control)P<0.01。

此外,PRLR表達(dá)水平隨大鼠的不同動(dòng)情周期而改變。在動(dòng)情前期其表達(dá)量最高,在動(dòng)情期隨即下降,而在動(dòng)情后期又恢復(fù)高水平表達(dá)[5]。本文采用尚未建立動(dòng)情周期且不含有成熟卵泡的性成熟期前小鼠進(jìn)行PMSG-HCG序貫注射,建立小鼠排卵模型,使各組內(nèi)實(shí)驗(yàn)標(biāo)本的同質(zhì)性更高;同時(shí)模擬成熟小鼠的動(dòng)情周期,并且能夠更精確的掌握動(dòng)情周期的每個(gè)時(shí)間點(diǎn)。PMSG、HCG分別模擬體內(nèi)FSH(Follicle-Stimulating Hormone,促卵泡激素)、LH(Luteinizing Hormone,促黃體激素)在排卵過程中的表達(dá)及效應(yīng)發(fā)揮。PMSG促進(jìn)卵泡的發(fā)育和募集,本實(shí)驗(yàn)中 PMSG注射后12h, PRLR表達(dá)出現(xiàn)上升趨勢,有學(xué)者報(bào)道 PRLR能夠通過促進(jìn)卵泡細(xì)胞的增殖影響卵泡的發(fā)育和成熟[12],本研究結(jié)果與之相吻合。而在 PMSG注射后24h直至48h后,相當(dāng)于動(dòng)情前期階段即優(yōu)勢卵泡募集階段,PRLR呈高水平表達(dá)。優(yōu)勢卵泡的募集必然伴隨FSH的高表達(dá),而顆粒細(xì)胞中的FSH能夠誘導(dǎo) PRLR的合成[12]。序貫的 HCG表達(dá)高峰使得排卵成為可能,通常在 HCG注射后10-12h發(fā)生排卵。本研究中,HCG注射后PRLR表達(dá)水平顯著下降,直到24h才恢復(fù)高水平表達(dá)并持續(xù)到注射后的48h。在排卵時(shí)間點(diǎn) PRLR表達(dá)量降低,與Clarke等[5]的 PRLR在動(dòng)情期低表達(dá)這一研究結(jié)果相一致。排卵后的黃體形成時(shí)期,本研究中PRLR再度升高,新近研究表明 PRLR的表達(dá)能夠直接導(dǎo)致LH受體的生成,從而滿足黃體形成及維持的需要,并且協(xié)同LH調(diào)控卵巢黃體的功能[13]。

綜上,PRLR在卵泡的募集、發(fā)育、成熟以致排卵及排卵后的黃體形成等一系列過程中發(fā)揮著重要作用,但該作用究竟通過何種信號(hào)途徑,其中有哪些相關(guān)因子參與,值得我們更進(jìn)一步的研究。

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EXPRESSION OF OVARIAN PROLACTIN RECEPTOR mRNA DURING FOLLICULARDEVELOPMENT AND OVULATION IN THE MOUSE

Luo Aiyue,Yang Shuhong,Shen Wei,Ding Ting,Lai Zhiwen,Wang Yi,Lu Yunping,Wang Shixuan
(Department of Obstetrics and Gynecology,Tongji Hospital,Huazhong University of Science and Technology,Wuhan430030,China)

Objective To explore the expression of prolactin receptor(PRLR)mRNA in the different developmental stages of the mouse ovary and the procedure of ovulation.Methods Mouse ovaries of different developmental stages were collected.The immature mice were chosen to be treated with PMSG and HCG sequentially and the ovaries were collected at various time.The expression of PRLR was detected by real time polymerase chain reaction(Real-Time PCR).Results The expression of PRLR was increased with the development of the mouse.PRLR expression of 6-week-old mice was 3.84 fold of 3-weekolds(P<0.01).The expression levels of 11 and 33-week-olds were 19.67 and 19.81 fold respectively of 3-week-olds(P<0.01).The expression of PRLR was increased after 12h of PMSG treatment(P<0.05) and 5.48 and 7.14 fold respectively of the control after 24h and 48h(P<0.01).After the sequential treatment of HCG,the expression level was significantly decreased but still higher than that of the control until 24h(P<0.01).PRLR expression at 24h and 48h post-HCG treatment was 5.48 and 7.14 fold of the control respectively(P<0.01).Conclusion PRLR may be involved in folliculogenesis and play an important role in the formation and maintenance of corpus luteum in the mouse ovary.

Prolactin receptor;Folliculogenesis;Ovulation;Real-time PCR

R711.7

A

10.3870/zgzzhx.2010.04.017

2010-03-08

2010-04-26

羅愛月,女(1978年),漢族,博士研究生

＊通訊作者(To whom correspondence should be addressed)