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Objective To observe the morphological changes in in vitro growth of preantral follicle isolated from prepuberal mice and to assess impacts of gonadotropin (Gn), insulin transferrin selenium (ITS) and epidermal growth factor (EGF) on their development. Methods Early preantral mice follicles (90-130μm diameter) were mechanical isolated and selected from 2 weeks' old mice and then cultured in alpha-minimal essential medium (α-MEM) with or without Gn, ITS and EGF. The preantral follicles were cultured singly in 20 miovliters droplets for up to 14 d. The medium was replaced and the .follicles were observed everyday. Granulosa cells (GC) prolification, antrum formation and oocyte maturation were recorded. Results The medium with Gn supported preantral follicle culture in vitro, during which they retained a three-dimensional structure, maintained oocytes viability and increased in diameter and number of somatic cells'. Preantral follicles cultured in Gn medium grew obviously, while those without Gn grew slowly and after 6 d's culture began to shrink and blacken. Significant increase in survival rate and maturation rate of oocytes was observed in Gn group (P〈0.01), with 92.9% survived and 28.7%formed an antrum. Further supplementation of the Gn medium with ITS and rLH, resulted in the significant increase in survival and maturation of preantral follicle (P〈0.05) Conclusions α-MEM can be the medium for in vitro culture (IVC) of preantral follicles, but need to be added with rLH/rFSH, rHCG/rEGF to facilitate thecal cell attachment, GC proliferation and oocyte maturation.  相似文献   

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In order to investigate the mechanism of progestin and antiprogestin in the regula-tion of ovarian steroidogenesis, a dual-chamber culture system was prepared with the amnion membrane of human placenta. Isolated porcine granulosa and thecal cells from 4~6 mm-diameter follicles were grown on both sides of the amnion, respectively, and co-cultured with or without LNG and RU486. After 48 h incubation, the mRNAs of FSH receptor (FSH-R) and LH receptor (LH-R) of both cells were observed by in situ hybridization. The results showed that granulosa cells expressed both FSH-R mR-NA and LH-R mRNA, while thecal cells expressed LH-R mRNA only. Under the stimulation of FSH, both LNG and RU486 increased FSH-R mRNA expression of granulosa cells. Under the stimulation of LH, LNG enhanced LH-R mRNA expres-sion of thecal cells;while RU486 decreased its expression. When granulosa and thecal cells were exposed to FSH and LH both, the actions of LNG and RU 486 in thecal cells showed the same result as that stimulated by LH alone. In granulosa cells LNG de-creased LH-R mRNA expression, while RU486 increased its expression. These data suggest that; (1) granulosa cells expressed FSH-R mRNA significantly; (2) both the progestin and antiprogestin directly acted on the mRNA expression of gonadotropin re-ceptors of ovarian cells, but effects were different; (3) the response of granulosa or thecal cells to the action of LNG and RU486 was not the same. The mechanism needs to be further investigated.  相似文献   

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Objective To investigate whether acupuncture treatment during superovulation process improves ovarian response and increases egg production. Methods ICR female mice aged 12 -5 weeks were divided into control group, anaesthesia group and acupuncture group. Female mice were injected intraperitoneally with pregnant mare's serum gonadotropin (PMSG), followed by human chorionic gonadotropin (hCG) injection 56 h later. Anaesthesia group and acupuncture group were anaesthetized three times by injecting 10% nembutal solution according to 7.5-10.0μl/g weight. Acupuncture group was treated three times with puncture Sanyinjiao Points(SP6) under anaesthesia. After 17 h of hCG injection, eggs were recovered and ovaries were isolated. Matured eggs were counted, ovarian histology and expression of follicle stimulating hormone receptor(FSH-R) in ovary were analyzed. Results Acupuncture treatment statistically increased the number of ovulated eggs. Histological section showed that some matured follicles left in the ovaries of control and anaesthesia groups after ovulation. On the contrary, all matured follicles ruptured and converted into corpus lutea in Acupuncture group. Expression of FSH-R in ovary was decreased in acupuncture group compared with those of the two others. Conclusion Acupuncture treatment during superovulation process improves ovarian response so as to increase egg production. The positive effect of acupuncture may be associated with regulated FSH-R expression.  相似文献   

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The protein expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in ovarian stimulated premature female rat ovary during a cycle of follicle development and corpus luteum formation was investigated. Animals were injected with 10 U pregnant Mare's serum gonadotropin (PMSG) and subsequently 10 U hCG 48 h later. Time-dependent immunohistochemistry and Western blotting experiments were performed before and 24, 48, 72 h after hCG treatment. The immunohistochemistry revealed that administration of PMSG stimulated the CFTR expression in thecal cell layer and granulosa cell layer of mature follicles 48 h post injection, coincident with the PMSG-induced peak in follicular estradiol. However, the expression of CFTR in the granulose lutein cell layer and thecal lutein cell layer was time-dependently reduced following hCG injection, in accordance with the gradually increased progestogen level during luteum corpus formation. Western blotting analysis demonstrated that rat ovarian tissue expressed the special CFTR band at 170 kD. It is concluded that cAMP-dependent Cl- channels are involved in regulation of follicle development and luteum formation.  相似文献   

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To investigate the expression of mRNA of leptin long-form receptor(OB-Rb) in lu-teinized granulosa cells of obese women with polycystic ovary syndrome(PCOS),and to determine the role of leptin in the physiopathology of PCOS,luteinized granulosa cells were collected from the follicle fluid of 10 obese women who met the diagnostic criteria for PCOS and their BMI was equal to or greater than 25 kg/m2,and at the same time,granulosa cells were collected from 10 normal women undergoing IVF-ET who served as the control group.Some luteinized granulosa cells were taken from normal women for in-vitro culture,into which human leptin of different concentrations was added(0,10,100 and 1000 ng/mL).After stimulation with leptin for 48 h,RT-PCR was em-ployed for the detection of the expression of OB-RLmRNA in the luteinized granulosa cells.Our re-sults showed that the level of OB-RLmRNA in luteinized granulosa cells of obese PCOS women was higher than those in the control(P<0.05).In luteinized granulosa cells cultured in vitro and stimulated by human leptin for 48 h,the level of OB-RLmRNA was higher than those without leptin stimulation(P<0.01),and when leptin concentration was at 100 ng/mL,and the level of OB-RLmRNA reached a peak.It is concluded that in obese PCOS women,the level of serum leptin is increased,which pro-motes the expression of OB-RL in luteinized granulosa cells and increases the sensitivity of the granulosa cells to leptin.Leptin may contribute to anovulation in obese women with PCOS  相似文献   

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Effects of luwinizing hormone releasing hormone ( LHRH), estradiot benzoate (EB), prostaglandin (PGF2α) and the age of animals on the development of ovarian follicles in immature and mature guinea pigs were studied. Administration of 5ng LHRH / hr, 10 times a day, starting from 20-30 days Of age induced the flrst vaginal opening within 4 days, and 5 of the 10 animals had first ovulation. Tire results also show that appropriate doses of EB facilitated the development of ovary. The granulosa cells of guinea pigs at different ages were cocuttured with the pituitart, glands of mature ones in vitro. Results indicate that the responsiveness of granulosa cells of 20-day-guinea prgs to gonadotropins was similar to that of the matured ones. Effects of gonadotrophin on ovulation in hysterectomized and PGF2α or saline-treated animals were tested. Results demonstrate that when PGF2α was given simultaneously with hCG or FSH, 80% of the guinea pigs ovulawd and had fresh, healthy looking corpus tuteum. It is concluded that increased LHRH release from hypothatamus may be the key factor for tire onset of puberty in guinea prgs. EB and PGF2α can influence the development of the ovary.  相似文献   

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In order to identify novel genes involved in spermatogenesis, testis cDNA samples from Balb/C mice of different postnatal days were hybridized with the whole mouse genome Affymetrix chip to screen the testis-specific genes. The characteristics of the selected genes were analyzed by RT-PCR as well as other bioinformatic tools. A novel differentially expressed testis-specific gene (GenBank Accession No: NM_029042) in the developmental stages of testes was identified, and named TSCPA. Cellular mapping prediction of TSCPA indicated that its protein was probably expressed in nuclei, and one putative domain (aa 332 377) was anchoring domain of cAMP-dependent type Ⅱ PK. The result of subcellular localization of GFP-TSCPA fusion protein in Cos-7 cells showed that TSCPA protein was expressed in nuclei. RT-PCR analysis revealed that TSCPA was expressed specifically in mouse and human testis. TSCPA gene was expressed weakly in 21-day-old mouse testis and the expression was increased gradually from 38th day to 6th month of mouse testes. No expression of hTSCPA was found in cryptorchidism and Sertoli-cell-only syndrome patients. It was concluded that the expression profile of TSCPA in human and mice indicated that TSCPA might play an important role in spermatogenesis.  相似文献   

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Summary: To investigate the expression and significance of CD28 and CTLA4 on T cells in bone marrow of aplastic anemia (AA) mice, in vitro bone marrow mononuclear cells (BMMNCs) were activated through being incubated with PHA (15 μg/mL). The expression of CD28 and CTLA4 on T cells incubated with or without PHA was detected by two-color flow cytometry. The expression of CD28 and CTLA4 was significantly increased after PHA stimulation. In the AA mice. the expression of CD28 with or without PHA stimulation was both higher than that in the normal mice (both P〈0.01), but the expression of CTLA4 with or without PHA stimulation showed no significant difference in comparison to that in the normal mice (both P〉0.05). In the AA mice, there were more activation and activated potential of T cells than the normal, and the abnormal expression of CD28 and CTLA4 may participate in immunological disorder mediated by T cells.  相似文献   

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大鼠卵巢发育过程中Smad4的表达   总被引:4,自引:0,他引:4  
OBJECTIVE: To clarify the signal transduction pathway of transforming growth factor-betasuperfamily (TGF-betas) in the regulation of follicle growth by investigating the expressions of Smad4 protein and mRNA in rat ovaries in different developmental stages. METHODS: Rat ovaries of different developmental stages were obtained to determine the expression of Smad4 protein by immunohistochemistry and image analysis system, with Smad4 mRNA measured by semi-quantitative RT-PCR. Specific primers of Smad4 and GAPDH (internal control) were used for amplification by RT-PCR, and the ratios of their integrated optical densities were calculated to estimate the relative quantity of Smad4 mRNA expression. RESULTS: Smad4 protein was widely expressed in the ovary, mainly in the follicles, and the location and intensity of Smad4 expression varied with the degree of maturation of the ovary. In the early developmental stages, Smad4 protein expressed mainly in the primordial and preantral follicles, but little in the stromal cells, and its expression intensity in the stroma increased gradually in the course of ovarian maturation. After sexual maturity, Smad4 expression intensity varied only insignificantly among the granulosa cells, theca cells and stromal cells of the antral and mature follicles (P>0.05). The staining intensity of Smad4 in the follicles also underwent changes in relation with their development, being less intense in the oocytes of the antral and matured follicles as compared to the preantral follicles (P<0.05 and P<0.01, respectively) but markedly greater in the theca cells of the antral and matured follicles than in the preantral follicles (P<0.01). No significant difference in Smad4 expression was found in the granulosa cells of different developmental stages (P>0.05). RT-PCR demonstrated that Smad4 mRNA was expressed in all the developmental stages of the rat ovary; and from the 3rd week on, the integrated optical density of Smad4 and GAPDH was significantly higher than that in 1-day-old neonatal rats. CONCLUSION: The expression patterns of Smad4 protein and mRNA in rat ovary in the course of its development indicate that Smad signal transduction may play a role in the folliculogenesis.  相似文献   

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多囊卵巢综合征患者颗粒细胞AQP9的表达   总被引:1,自引:0,他引:1  
目的:研究多囊卵巢综合征(polycystic ovarian syndrome,PCOS)卵巢组织中窦状卵泡和在超促排卵(COH)周期黄素化颗粒细胞AQP9的表达,探讨颗粒细胞AQP9表达水平与COH周期卵泡液中甾体激素水平的关系。方法:行体外受精-胚胎移植的PCOS患者18例,对照组18例。收集卵泡(直径≥1.8 cm)液,测定甾体激素E2、P和T水平;取卵时分别收集大卵泡(直径>1.6 cm)和小卵泡(直径<1.4 cm)的黄素化颗粒细胞。用RT-PCR检测COH周期黄素化颗粒细胞AQP9mRNA的表达,免疫组化定位AQP9在PCOS卵巢内窦状卵泡和COH周期的黄素化颗粒细胞的表达。结果:RT-PCR检测证实黄素化颗粒细胞有AQP9 mRNA表达,PCOS组AQP9 mRNA表达水平与对照组比较差异无显著性(P>0.05),PCOS组大卵泡的颗粒细胞AQP9表达高于小卵泡,但无统计学差异。免疫组化显示PCOS患者卵巢的窦状卵泡内颗粒细胞和COH周期的黄素化颗粒细胞均有AQP9的表达,染色定位于胞浆和胞膜。在COH周期,颗粒细胞AQP9 mRNA表达水平与卵泡液中E2、P和T均无显著性相关。结论:PCOS患者卵巢组织中窦状卵泡的颗粒细胞及在COH周期黄素化颗粒细胞均有AQP9表达,推测AQP9可能通过水转运介导卵泡发育和窦卵泡形成。在COH周期,PCOS的大卵泡AQP9的表达有高于小卵泡的趋势,可能与卵泡发育有关。在COH周期,颗粒细胞AQP9 mRNA表达水平与甾体激素的分泌无相关性。  相似文献   

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大鼠卵巢发育过程中Smad4的表达   总被引:1,自引:0,他引:1  
目的 了解转化生长因子-β超家族(TGF-βs)在调节卵泡发育过程中的信号转导模式,以探讨在不同发育阶段大鼠卵巢中Smad4蛋白及mRNA的表达。方法 选择不同发育时期大鼠卵巢,运用免疫组化方法检测卵巢中Smad4蛋白表达,并进行图像分析;采用半定量逆转录聚合酶链反应(RT-PCR)方法检测Smad4mRNA在卵巢中的表达,以GAPDH作为内对照与特异性Smad4同时进行扩增,并计算Smad4和GAPDH的RT-PCR产物积分吸光度比值来表示Smad4mRNA的含量。结果 Smad4广泛表达于卵巢组织,但主要表达在卵泡中,其表达部位和强度随卵巢的成熟度不同而变化;在卵巢发育早期,Smad4主要在原始卵泡和窦前卵泡中表达,而在间质中的表达较弱;随着卵巢的发育成熟,Smad4在间质中的表达逐渐增强,性成熟后,在窦状及成熟卵泡颗粒细胞和卵泡膜的表达与间质细胞比较无显著差异(P>0.05)。Smad4在卵泡中的表达强度也发生了变化,即随着卵泡的发育,Smad4在窦状及成熟卵泡卵母细胞的表达明显减弱,与窦前卵泡卵母细胞比较有显著差异(P<0.05,P<0.01);与窦前卵泡比较,Smad4在卵泡膜细胞的表达逐渐增强(P<0.01),而在各级卵泡颗粒细胞中的表达无显著差异(P>0.05)。RT-PCR结果显示各阶段卵巢均有mRNA的表达,从第3周起Smad4mRNA的表达明显增强,Smad4和GAPDH积分吸光度比值与出生后1 d的差异均有统计学意义(P<0.05)。结论 卵巢内存在Smad4,提示TGF-β家族对卵泡发育的调节是通过Smad信号转导模式实现的。  相似文献   

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Cyclin G1在小鼠卵巢的表达及其与卵泡发育的关系   总被引:4,自引:1,他引:3  
目的观察细胞周期素G1(Cyclin G1)在小鼠卵巢中各级卵泡的表达及其与卵泡发育的关系。方法性成熟前小鼠用孕马血清促性腺激素(PMSG)刺激卵泡生长,用绒毛膜促性腺激素(hCG)刺激卵泡排卵及向黄体转化。于给药后不同时间取出卵巢,用免疫组化方法检测不同发育阶段卵泡Cyclin G1蛋白的表达和分布;用细胞免疫荧光方法检测不同成熟阶段的卵细胞中Cyclin G1蛋白表达。结果免疫组化染色结果显示,颗粒细胞中Cyclin G1的表达水平随卵泡发育成熟而逐渐增强,且一直定位在胞核;Cyclin G1在各个时期卵泡的卵母细胞中均有表达。该表达的定位与卵泡生长发育的不同阶段有关。随着卵泡的发育,Cyclin G1在卵母细胞的表达由胞核转至胞浆。至排卵前卵泡颗粒细胞,Cyclin G1表达水平减弱,当颗粒细胞分化为黄体细胞后表达水平进一步减弱。闭锁卵泡中Cyclin G1的表达水平很弱。细胞免疫荧光染色结果显示,处于生发泡时期(GV)卵母细胞仅有较弱的Cyclin G1表达,当卵母细胞生发泡破裂(GVBD)后,该表达增强;与处于第二次减数分裂中期(MⅡ)的卵母细胞相比,受精卵的Cyclin G1表达明显增强。结论Cyclin G1在小鼠卵泡发育及卵母细胞成熟过程中的表达具有时空特异性,可能作为细胞周期的正调节因子参与了卵泡生长发育和卵母细胞成熟过程的调控。  相似文献   

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目的探讨垂体腺苷酸环化酶激活肽(pituitary adenylate cyclase—activating polypeptide,PACAP)mRNA在排卵过程中的表达情况与调节其表达的因素。方法采用RT-PCR对PACAP mRNA在使用外源性激素诱导的大鼠排卵过程中的表达变化进行研究,并在体外培养的排卵前卵泡颗粒细胞中分析了不同处理因素对其表达的影响。结果对预先用孕马血清(PMSG)处理的未成年大鼠给予hCG发现,给予hCG4~12h期间,PACAP转录水平持续升高,其表达水平在给予hCG4h后达高峰。对体外培养的排卵前卵泡颗粒细胞的研究结果分析显示,与对照组相比hCG和hCG+PAF能提高颗粒细胞PACAP mRNA水平。结论PACAP mRNA在排卵前卵泡呈瞬时性高表达,提示了PACAP可能在排卵过程中发挥重要作用。hCG能促进PACAP的转录,并且PAF可加强这种促进作用。  相似文献   

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目的 探讨他克莫司结合蛋白38(Fkbp38)在卵泡发育中的作用及其缺失导致早发性卵巢功能不全(POI)的机制。方法 采用Cre-loxp系统构建卵母细胞特异性敲除Fkbp38转基因小鼠,并应用PCR鉴定小鼠基因型,然后在蛋白水平上验证Fkbp38在卵母细胞中的敲除效果。通过HE染色在显微镜下计数敲除小鼠与同窝对照小鼠卵巢原始卵泡、初级卵泡、次级卵泡及窦卵泡数量分析卵母细胞缺失Fkbp38基因对小鼠卵泡发育的影响。通过繁殖实验及ELISA实验检测小鼠繁殖能力及血清性激素水平,明确卵母细胞敲除Fkbp38基因对卵巢功能的影响。TUNEL法检测敲除小鼠与同窝对照小鼠卵巢颗粒细胞凋亡情况。检测雷帕霉素靶蛋白(mTOR)信号通路下游靶蛋白磷酸化核糖体S6(PS6)活性验证Fkbp38基因对mTOR信号通路的影响。IF检测凋亡相关蛋白B细胞淋巴瘤/白血病-2(Bcl-2)及Bcl2-Associated X(BAX)表达明确Fkbp38基因敲除对卵母细胞发育的影响。结果 卵母细胞特异性敲除Fkbp38转基因小鼠模型构建成功。敲除小鼠较对照小鼠表现出生育力下降,性激素水平紊乱,卵巢内原始卵泡、初级卵泡和次级卵泡数均显著减少(P<0.05),引起POI样改变。卵母细胞特异性敲除Fkbp38基因后,mTOR信号通路激活,颗粒细胞凋亡增加,卵母细胞内BAX蛋白表达增强,Bcl-2蛋白表达减弱,BAX/Bcl-2蛋白比值显著升高(P<0.05)。结论 Fkbp38在卵泡发育中发挥重要作用,其缺失致POI发生的机制可能是通过激活mTOR信号通路诱导细胞凋亡而引起的。  相似文献   

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人们已在卵泡发育的不同阶段和不同细胞中均检测到小RNA(miRNAs)的存在,并在成熟卵泡的卵泡液中也发现外泌体miRNAs。miRNAs不仅参与调控正常的卵泡发育过程,其异常调控也可导致一些卵巢疾病的发生。为进一步系统阐明miRNAs对卵泡发育的调控机制,寻找可以预测卵母细胞发育质量的外泌体miRNAs,本文从颗粒细胞发育、卵母细胞发育和激素合成等方面总结了miRNAs对卵泡发育的调控作用。  相似文献   

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目的观察人工合成的非甾体化合物己烯雌酚(DEs)对性成熟前小鼠和大鼠卵巢中卵泡发育的影响,以及出现有卵泡同步化机理。方法取性成熟前雌性小鼠和大鼠,腹腔注射一定剂量的DES,然后取卵巢经连续切片和HE染色后,统计整个卵巢内三级卵泡数量。同时,取卵巢利用免疫组织化学和免疫印迹方法,分别检测泛素末端水解酶LI(UCH-L1),c-Jun结合蛋白l(Jabl)和细胞周期素依赖激酶抑制因子p27KipI在小鼠卵巢中的定位和含量。结果UCH-LI定位在卵巢中各级卵泡的卵母细胞中,Jab1分布于次级和三级卵泡的卵母细胞核中,而p27KipI广泛分布于卵巢各类细胞中。免疫组化结果显示,仅就卵母细胞而言,由于DES的处理,UCH—L1和Jab1的表达信号增强,而p27KipZ的信号变弱,与肿瘤细胞中的情况类似。然而,免疫印迹分析结果表明,以上3种蛋白在卵巢中相对b-actin的含量因DES处理而均呈现一定程度的上升变化趋势。青春前期的小鼠,在1-4周龄期间,卵巢中UCH.L1和p27KiPI的蛋白量随着周龄增加的趋势,而Jab1的变化趋势不明显。结论腹腔注射DES或导致卵巢内卵泡发育同步化,其中涉及的机理可能是,UCH-L1及其关联蛋白Jab1导致p27KiPI在卵母细胞核中的含量下降,进而导致了多个卵泡的卵母细胞同时发育启动。  相似文献   

19.
目的 探讨化疗药物对卵巢的损伤及其可能机制.方法 将成年SD大鼠分为两组:生理盐水组和环磷酰胺组.大鼠腹腔注射环磷酰胺;8周后处死,固定一侧卵巢,连续切片,观察卵巢形态学改变并计数卵泡数目.免疫组化方法检测干细胞因子(SCF)蛋白在卵巢的表达,RT-PCR半定量法检测对侧卵巢SCFmRNA的表达.结果 环磷酰胺可引起卵巢间质的严重损害和局部纤维化的发生.SCF可表达于大鼠卵巢原始卵泡和初级卵泡的卵母细胞,次级卵泡和早期窦卵泡的颗粒细胞也可见SCF的表达.环磷酰胺组注射后8周卵泡颗粒细胞SCF蛋白显著升高,卵母细胞SCF蛋白显著减少(P<0.05);卵巢SCF mRNA显著升高(P<0.05).结论 SCF在化疗卵巢表达的变化有助于进一步阐释化疗药物损伤卵巢的机制.  相似文献   

20.
目的:对多囊卵巢综合征(PCOS)大鼠卵巢中kisspeptin/kiss1r系统的表达变化及对卵泡发育的影响进行研究。方法采用腹腔注射来曲唑构建PCOS大鼠模型,酶联免疫吸附(ELISA)法测定血清中性激素水平,苏木精‐伊红(H‐E)染色检测卵巢的形态学改变,免疫组化检测卵巢中kisspeptin/kiss1r、卵泡刺激素受体(FSHR)的蛋白水平变化。结果从灌胃后第10天开始,PCOS组大鼠体重明显增加,血清中睾酮(T)、黄体生成素(LH)的水平明显升高,卵泡刺激素(FSH)水平无明显改变;大鼠卵巢皮质中含有较多的小卵泡及闭锁卵泡,多数卵泡成囊性扩张,颗粒细胞层数明显减少;卵巢中kisspeptin/kiss1r、FSHR均在细胞质表达,kisspeptin/kiss1r主要在颗粒细胞、膜细胞和黄体中表达,而FSHR仅表达在颗粒细胞。PCOS组卵巢中kisspeptin/kiss1r、FSHR表达明显减少。结论在PCOS卵泡发育中,卵巢表达的kisspeptin/kiss1r系统发挥重要的作用,为PCOS的临床治疗和基础研究提供新思路。  相似文献   

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