龍詩韻，姜冬梅，陳咨余，管成，易治鑫，康波

（四川農(nóng)業(yè)大學(xué)動物科技學(xué)院/畜禽遺傳資源發(fā)掘與創(chuàng)新利用四川省重點實驗室，成都611130）

外源性亞精胺對鼠卵巢生殖激素受體基因表達的影響

龍詩韻，姜冬梅，陳咨余，管成，易治鑫，康波*

（四川農(nóng)業(yè)大學(xué)動物科技學(xué)院/畜禽遺傳資源發(fā)掘與創(chuàng)新利用四川省重點實驗室，成都611130）

為探討外源性亞精胺對鼠卵巢生殖激素受體基因表達的影響，采用不同質(zhì)量分數(shù)（0.05、0.10和0.15 mg/g）亞精胺腹腔注射6周齡昆明鼠，24 h后采集卵巢組織樣品，應(yīng)用實時熒光定量聚合酶鏈式反應(yīng)檢測卵巢組織中促卵泡激素受體、促黃體生成素受體、雌激素受體1、雌激素受體2和雄激素受體基因表達量。結(jié)果顯示：與對照組相比，0.05 mg/g亞精胺處理組卵巢促黃體生成素受體表達量顯著高于對照組（P＜0.05），0.15 mg/g組促黃體生成素受體表達量顯著低于對照組（P＜0.05）；隨亞精胺處理量的增加，卵巢組織中促卵泡激素受體、雌激素受體1、雌激素受體2和雄激素受體表達量均呈逐漸增加的趨勢，且均在0.15 mg/g處理組中的表達量顯著高于對照組（P＜0.05）。綜上表明，亞精胺可通過介導(dǎo)鼠卵巢組織生殖激素受體基因表達來參與調(diào)控鼠卵巢功能。

亞精胺；卵巢；激素受體；基因表達

SummaryThe polyamines spermidine and spermine and their precursor putrescine are a class of compounds containing two or more amino groups and participate in the regulation of various physiological and pathological processes by interacting with negatively-charged molecules,such as DNA,RNA and proteins.Recently,increasing studies suggest that polyamines also play important roles in follicular development,ovulation and steroidogenesis in female animals.The spermidine exists ubiquitously in species ranging from yeast to mammals.Recently,studies from our and other laboratories indicated that spermidine is involved in regulating animal reproduction through mediating spermatogenesis,oogenesis,follicular development and ovulation.However, to the best of our knowledge,the effect of the spermidine on the expression levels of hormone receptor genes associated with reproduction is unknown.

To demonstrate the effect of the spermidine on the transcription of reproductive hormone receptor genes in mouse ovaries, Kunming mice were administrated with 0.05,0.10 and 0.15 mg/g(body mass)spermidine by intraperitoneal injection.Ovarian tissue samples were collected after 24 h of spermidine administration.The levels of follicle-stimulating hormone receptor (FSHR),luteinizing hormone receptor(LHR),estrogen receptor 1(ER1),ER2and androgen receptor(AR)mRNA expression in mouse ovaries were measured using quantitative real-time polymerase chain reaction.

The results showed that the level ofLHRmRNA expression in ovaries of mice administrated with 0.05 mg/g spermidine was significantly higher than the untreated control group(P＜0.05).The amount ofLHRmRNA expression in ovaries of mice administrated with 0.15 mg/g spermidine was significantly lower than the untreated control group(P＜0.05).With increasing spermidine administration,a gradually increasing trend was observed in the change ofFSHR,ER1,ER2andARexpression levels.The amount ofFSHR,ER1,ER2andARgenes in ovaries of mice administrated with 0.15 mg/g spermidine was significantly high,compared with the untreated control group,respectively(P＜0.05).

In conclusion,our present results demonstrate that exogenous spermidine mediates the transcription of genes associated with reproductive hormone receptors in ovaries of mice.It is indicated that spermidine might play an important role in regulating the responsiveness of the ovary to reproductive hormones through an unknown mechanism.Further research is needed to elucidate the mechanism of action of spermidine mediating ovarian functions in animals.

亞精胺是廣泛存在于動物、植物和微生物體內(nèi)的一種多胺，可通過介導(dǎo)雄性動物精子發(fā)生，以及雌性動物卵泡發(fā)育、卵子發(fā)生和排卵活動，進而參與調(diào)控動物的繁殖過程[1-2]。多胺能通過與多種生殖激素建立復(fù)雜的反饋聯(lián)系，進而參與調(diào)控動物繁殖[3]。多胺在維持卵巢類固醇激素水平和生成過程中具有重要作用[4]。雄性動物體內(nèi)的多胺合成受到雄激素的調(diào)節(jié)[5]。FASHE等[6]研究發(fā)現(xiàn)，多胺合成受阻時，卵巢性激素含量顯著降低。趙越超[7]研究發(fā)現(xiàn)，性激素可通過其受體途徑影響鼠子宮內(nèi)多胺代謝。大量研究證實，雄激素在治療男性不育癥上具有重要作用，亞精胺在不育男性精漿中的含量明顯比正常男性低[8-10]，推測亞精胺可能通過作用于雄激素及其受體來參與調(diào)控雄性動物的繁殖功能。而在雌性動物中，亞精胺是否與雄激素受體（androgen receptor,AR）存在直接或間接的作用還未有研究報道。

促卵泡激素（follicle-stimulating hormone,FSH）和促黃體激素（luteinizing hormone,LH）分別與促卵泡激素受體（follicle-stimulating hormone receptor, FSHR）和促黃體生成素受體（luteinizing hormone receptor,LHR）作用，共同參與調(diào)控動物卵泡發(fā)育和卵泡顆粒細胞增殖[11]。LH和FSH還可參與調(diào)控卵巢組織中雌激素的合成，而雌激素通過負反饋調(diào)節(jié)機制影響垂體LH和FSH的分泌[12-13]。雌激素受體（estrogen receptor,ER）包含ER1和ER2這2種亞型，雙敲除ER1和ER2的雌性鼠不能排卵，說明ER對雌性動物排卵具有至關(guān)重要的調(diào)控作用[14]。研究表明，多胺可通過介導(dǎo)哺乳動物生殖激素合成與分泌來參與調(diào)控動物繁殖功能[4]。然而，目前有關(guān)亞精胺調(diào)控雌性動物卵巢功能的研究相對較少，而且尚未見有關(guān)亞精胺調(diào)控卵巢生殖激素受體基因表達影響的報道。因此，本試驗研究了不同質(zhì)量分數(shù)亞精胺對鼠卵巢組織中FSHR、LHR、ER1、ER2和AR基因表達的影響，以期為闡明多胺調(diào)控哺乳動物卵巢功能的作用機制提供理論依據(jù)。

1 材料與方法

1.1 試驗動物及樣品采集

選取雌性健康同胞昆明鼠（SPF級試驗用昆明系鼠，購于成都達碩公司），常規(guī)分籠飼養(yǎng)，鼠自由采食；在試驗過程中對動物處置符合動物倫理學(xué)標準。6周齡時，將鼠隨機分為4組，每組8只，分別腹腔注射生理鹽水和0.05、0.10和0.15 mg/g（體質(zhì)量）亞精胺。注射24 h后頸部脫臼處死，迅速采集鼠卵巢組織，用生理鹽水洗凈，濾紙吸干后，置于－80℃冰箱保存，備用。

1.2 引物設(shè)計和實時熒光定量聚合酶鏈式反應(yīng)

按照RNA提取試劑盒（RNAiso Plus kit，TaKaRa公司，大連）說明書提取鼠卵巢組織總RNA，然后參照反轉(zhuǎn)錄試劑盒說明書（TaKaRa公司，大連）將總RNA樣品反轉(zhuǎn)錄成cDNA模板，置于－20℃冰箱中保存，備用?；贕enBank數(shù)據(jù)庫，采用Primer Premier 5.0和Oligo 6.0軟件設(shè)計特異性引物，并委托華大基因有限公司合成。引物序列信息見表1。

實時熒光定量反應(yīng)體系（10 μL）：實時熒光定量聚合酶鏈式反應(yīng)試劑盒（iQTMSYBR Green Supermix kit，Bio-Rad公司，北京）5.0 μL，上、下游引物（10 μmol/L）各0.2 μL，cDNA模板0.5 μL，用無RNA酶水補足至10 μL。反應(yīng)條件：95℃預(yù)變性3 min；95℃變性10 s，57～63℃退火30 s，72℃延伸30 s（采集熒光），40個循環(huán)；95℃保持10 s，繪制溶解曲線。用3-磷酸甘油醛脫氫酶（glyceraldehyde-3-phosphate dehydrogenase,GAPDH）基因作為內(nèi)參基因。每個樣品設(shè)3個重復(fù)。

表1 實時熒光定量聚合酶鏈式反應(yīng)引物序列Table 1 Primer sequences used in quantitative real-time polymerase chain reaction(PCR)

1.3 數(shù)據(jù)處理及統(tǒng)計分析

采用2－ΔΔCT法處理數(shù)據(jù)[7]，用GAPDH作為內(nèi)參基因，以空白組基因的表達量作為對照組，計算不同質(zhì)量分數(shù)亞精胺處理組卵巢組織FSHR、LHR、ER1、ER2、AR基因的相對表達量。應(yīng)用SAS 9.2統(tǒng)計分析軟件中的MEANS過程進行描述性統(tǒng)計分析，并進行方差分析和鄧肯多重比較，結(jié)果用平均值±標準差表示，P＜0.05表示差異有統(tǒng)計學(xué)意義。

2 結(jié)果

2.1 亞精胺對鼠LHR和FSHR基因表達的影響

由圖1可知：腹腔注射0.05 mg/g亞精胺時，鼠卵巢組織中LHR相對表達量顯著高于其他各組，是對照組的2.07倍（P＜0.05）；腹腔注射0.15 mg/g亞精胺組LHR表達量顯著低于對照組，為對照組的9%（P＜0.05）。腹腔注射0.15 mg/g亞精胺時，F(xiàn)SHR表達量顯著高于對照組，是對照組的31.67倍（P＜0.05）；而腹腔注射0.05和0.10 mg/g亞精胺時，F(xiàn)SHR基因表達量與對照組相比差異無統(tǒng)計學(xué)意義（P＞0.05）。

圖1 亞精胺對鼠卵巢LHR和FSHR表達的影響Fig.1 Effect of spermidine onLHRandFSHRmRNA expression in mouse ovaries

2.2 亞精胺對ERER1 1和ERER2 2基因表達的影響

腹腔注射0.15 mg/g亞精胺處理組中，ER1和ER2表達量均顯著高于對照組，分別為對照組的1.95倍和6.43倍（P＜0.05）；而注射0.05和0.10 mg/g組中，ER1和ER2與對照組間差異均無統(tǒng)計學(xué)意義（P＞0.05）（圖2）。

圖2 亞精胺對鼠卵巢ER ER1 1和ER ER2 2表達的影響Fig.2 Effect of spermidine onER1andER2mRNA expression in mouse ovaries

2.3 亞精胺對AR基因表達的影響

如圖3所示：當腹腔注射0.15 mg/g亞精胺時，卵巢組織中AR相對表達量顯著高于對照組和其他處理組，是對照組的4.31倍（P＜0.05）；而注射0.05和0.10 mg/g組中，AR表達量與對照組間差異無統(tǒng)計學(xué)意義（P＞0.05）。

圖3 亞精胺對鼠卵巢AR表達的影響Fig.3 Effect of spermidine onARmRNA expression in mouse ovaries

3 討論

生殖激素及其特異性受體在調(diào)控動物繁殖過程中具有關(guān)鍵作用，而FSH和LH在調(diào)控動物繁殖過程中具有核心作用[15-16]。在卵巢顆粒細胞中LHR表達量降低會抑制排卵功能[17]；而FSHR表達量升高能增強卵巢對外源性促性腺激素的應(yīng)答能力。THYSSEN等[18]研究發(fā)現(xiàn)，外源性腐胺和亞精胺能抑制下丘腦和垂體組織中LH和FSH的分泌；用二氟甲基鳥氨酸（一種腐胺生物合成的不可逆抑制劑）處理后FSH分泌增加：表明多胺可參與調(diào)控垂體LH和FSH分泌。本研究發(fā)現(xiàn)，腹腔注射亞精胺顯著提高了卵巢組織中FSHR和LHR表達量：說明亞精胺也可通過介導(dǎo)卵巢FSHR和LHR表達來發(fā)揮其對雌性動物繁殖功能的調(diào)控作用。表皮生長因子（epidermal growth factor,EGF）作為一種卵巢旁分泌因子，通過介導(dǎo)卵泡發(fā)育和卵母細胞生成來調(diào)節(jié)卵巢功能。研究表明，EGF可下調(diào)哺乳動物卵巢FSHR表達[19]。BLACHOWSKI等[20]發(fā)現(xiàn)，多胺能參與EGF信號轉(zhuǎn)導(dǎo)通路，提示多胺與EGF參與的生殖調(diào)控具有相關(guān)性。本研究發(fā)現(xiàn)，注射0.15 mg/g亞精胺顯著增加了卵巢組織中FSHR表達量，推測亞精胺促進FSHR表達的作用可能與EGF有關(guān)，有待進一步研究證實。此外，本研究還發(fā)現(xiàn)，低質(zhì)量分數(shù)亞精胺（0.05 mg/g）能促進卵巢LHR表達，但對FSHR表達影響不顯著；而高質(zhì)量分數(shù)亞精胺（0.15 mg/g）抑制LHR表達，但促進了FSHR表達：表明亞精胺影響FSHR和LHR表達的效應(yīng)具有劑量差異。

雌激素受體基因包括ER1和ER2，在鼠卵巢組織中均有表達，但其表達量和生理作用各不相同。在未成熟和成熟的嚙齒類動物和人卵巢中，ER2表達量遠高于ER1[21]。研究表明，在卵巢組織中ER2的表達和分布與鼠的生殖周期存在密切聯(lián)系，尤其是在發(fā)情期的鼠卵巢組織中有高度表達，其表達規(guī)律與小鼠體內(nèi)雌激素水平相適應(yīng)[22]。本研究發(fā)現(xiàn)，0.15 mg/g亞精胺處理可顯著增加鼠卵巢組織中ER1和ER2表達，提示高質(zhì)量分數(shù)亞精胺可通過提高ER表達進而參與調(diào)控鼠卵巢功能。

AR在調(diào)節(jié)卵巢功能和維持女性生育能力方面具有重要作用[23]。KIMURA等[24]發(fā)現(xiàn)，在卵泡發(fā)育早期即有AR的表達，AR缺陷型鼠易出現(xiàn)卵泡發(fā)育障礙和卵巢早衰。AR和雄激素水平與哺乳動物在卵巢發(fā)育過程中FSHR表達密切相關(guān)[25]。此外，雄激素還參與調(diào)控卵巢癌的發(fā)生和發(fā)展過程，AR水平及其活性增強會使卵巢癌細胞對雄激素的敏感性增大，從而促進卵巢癌變[26]。本研究結(jié)果表明，0.15 mg/g亞精胺處理組的鼠卵巢組織中AR表達顯著高于對照組：表明高質(zhì)量分數(shù)亞精胺可通過促進AR表達進而參與調(diào)控鼠卵巢功能。

綜上所述，在本試驗中，腹腔注射不同質(zhì)量分數(shù)亞精胺后，鼠卵巢組織中LHR、FSHR、ER1、ER2和AR表達量相較于對照組均發(fā)生了顯著變化。低質(zhì)量分數(shù)的亞精胺促進LHR表達；高質(zhì)量分數(shù)的亞精胺促進FSHR、ER1、ER2和AR表達，同時抑制LHR的表達，且具有劑量依賴性。說明亞精胺可通過介導(dǎo)鼠卵巢組織生殖激素受體基因表達來參與調(diào)控鼠卵巢功能。

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Effect of exogenous spermidine on expression profile of reproductive hormone receptors in mouse ovaries.

LONG Shiyun,JIANG Dongmei,CHEN Ziyu,GUAN Cheng,YI Zhixin,KANG Bo*(Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province/College of Animal Science and Technology,Sichuan Agricultural University, Chengdu 611130,China)

spermidine;ovary;hormone receptor;gene expression

Q 492

A

10.3785/j.issn.1008-9209.2016.09.191

Journal of Zhejiang University(Agric.&Life Sci.),2017,43(2):247-252

國家自然科學(xué)基金（31201798）；高等學(xué)校博士學(xué)科點專項科研基金（20105103120003）。

康波（http://orcid.org/0000-0003-2811-0642），E-mail:bokang@sicau.edu.cn

（First author）：龍詩韻（http://orcid.org/0000-0002-5735-2731），E-mail:longsy210@163.com

2016-09-19；接受日期(Accepted):2016-12-06