中药六味地黄汤对衰老大鼠性激素与其受体表达的影响

目的探讨衰老雌性大鼠性激素及其受体的变化情况和六味地黄汤延缓卵巢组织衰老的作用机制。方法 D-半乳糖连续腹腔注射致亚急性衰老动物模型,造模后灌胃六味地黄汤连续60天后,大鼠断头取垂体、卵巢。采用全自动微粒子化学发光免疫分析方法检测各组大鼠血清LH,E2,FSH的含量;原位杂交方法检测各组大鼠卵巢细胞FSHR和LHR的变化;Western Bloting法检测各组大鼠卵巢ER的变化。结果同正常对照组相比,模型组大鼠垂体指数和卵巢指数均明显降低,血清E2含量明显降低、LH、FSH升高,ER、FSHR、LHR的表达明显降低,差异有统计学意义(P<0.05)。与模型组比较,六味地黄汤组垂体指数和卵巢指数均明显增高,血清E2含量明显升高、LH、FSH降低,ER、FSHR、LHR的表达明显升高,呈现一定的剂量依赖性。结论衰老时大鼠卵巢正常功能被破坏,六味地黄汤通过对性激素和其受体的调节而延缓了卵巢的衰老。     

被动吸烟下调大鼠卵泡刺激素、黄体生成素、促性腺激素释放激素及其受体的表达

目的 研究被动吸烟对Wistar大鼠卵巢结构、激素受体及血清中激素的影响. 方法 Wistar大鼠32只,分为实验组和对照组(各16只).实验组大鼠吸烟3个月,对照组不予吸烟,3个月后处死大鼠.光镜及电镜下观察各组大鼠卵巢结构的改变,免疫组织化学染色检测激素受体的改变,酶联免疫吸附试验检测血清中激素的改变情况. 结果 实验组大鼠卵巢髓质血管收缩、减少,间质疏松.卵巢颗粒细胞内线粒体水样变、空泡样变,线粒体嵴断裂、模糊;粗面内质网脱颗粒样变.血清卵泡刺激素(FSH)、黄体生成素(LH)、促性腺激素释放激素(GnRH)水平比对照组显著降低,其浓度值比较差异有显著性(P＜0.01,P＜0.05,P＜0.05).实验组大鼠卵巢组织中卵泡刺激素受体(FSHR)、黄体生成素受体(LHR)的表达显著低于对照组(P＜0.01,P＜0.05). 结论 被动吸烟可使大鼠血清中FSH、LH及GnRH水平明显降低,大鼠卵巢中FSHR及LHR的表达减少,提示被动吸烟可破坏卵巢的结构及功能,引起生殖内分泌失调.     

大鼠颌下腺黄体生成素及其受体的定位、核心片段的克隆及序列分析

目的 探讨大鼠颌下腺组织中是否表达黄体生成素(LH)及其受体(LHR),为进一步研究LH对颌下腺的功能调节提供理论依据.方法 采用免疫组织化学SABC和原位杂交方法进行定位研究;应用RT-PCR方法克隆获得LH及LHR基因的cDNA核心序列,并进行序列分析.结果 大鼠颌下腺浆液性腺泡上皮细胞呈LH和LHR免疫反应阳性.阳性物质分布于胞浆,胞核呈阴性反应.上述细胞同样含有LH和LHR mRNA杂交信号,信号物质亦分布于胞浆内,胞核呈阴性反应.从大鼠颌下腺组织中扩增的LH及LHR基因的特异性条带经序列分析发现,扩增产物的LH基因序列与文献报道的大鼠腺垂体的完全一致;扩增产物的LHR基因序列与大鼠睾丸组织的完全一致.结论 大鼠颌下腺浆液性腺泡上皮细胞既能产生LH,又能表达LHR,提示LH对颌下腺浆液性腺泡上皮细胞的作用可能是通过自分泌或旁分泌来实现的.     

大鼠下颌下腺卵泡刺激素及其受体的原位杂交及核心片段的序列分析

目的探讨大鼠下颌下腺组织中是否表达卵泡刺激素(FSH)及其受体(FSHR),为进一步研究FSH对下颌下腺的功能调节提供理论依据。方法正常SD大鼠20只,体质量(200±20)g,腹腔麻醉后切取下颌下腺,采用原位杂交方法对FSH及FSHR进行细胞定位,探讨大鼠下颌下腺是否存在FSH及其受体mRNA,从下颌下腺组织中分别提取总RNA,运用RT-PCR获得FSH及其受体基因的cDNA核心序列,并对其序列进行分析。结果大鼠下颌下腺浆液性腺泡及颗粒曲管上皮细胞含有FSH和FSHR mRNA杂交信号,信号物质分布于胞质内,胞核呈阴性反应。从大鼠下颌下腺组织中扩增的FSH及FSHR基因的特异性条带分别为193bp和413bp。结论大鼠下颌下腺浆液性腺泡及颗粒曲管上皮细胞能合成卵泡刺激素及其受体,进一步说明下颌下腺是FSH作用的靶器官。     

两种SD大鼠多囊卵巢综合征模型的比较研究

目的比较两种SD大鼠多囊卵巢综合征动物模型的建模效果。方法建立两种PCOS动物模型。观察各组大鼠卵巢形态学、性激素及空腹血糖和胰岛素的变化。结果DHEA建模组大鼠的体重显著低于对照组（P〈0．05）;hCG建模组大鼠卵巢重量和体积均显著高于对照组（P〈0．05）。两建模组大鼠卵巢卵泡多呈囊性扩张,颗粒细胞层数减少,卵泡膜细胞、间质细胞增生。两建模组大鼠血清T、FINS、FBG和HOMA指数均显著高于对照组（P〈0．05）;DHEA建模组LH水平较对照组无显著性差异;hCG建模组大鼠血清LH、LH／FSH比值均显著高于对照组（P〈0．05）。DHEA建模组FSH水平显著高于对照组（P〈0．05）。结论利用hCG诱导SD幼年雌性大鼠出现了与PCOS患者极为相似的卵巢病理改变及LH、雄激素水平升高和胰岛素抵抗等典型内分泌变化,其建模效果优于DHEA。     

促卵泡激素受体结合抑制剂抑制小鼠卵巢发育

目的探讨不同浓度的促卵泡激素受体结合抑制剂(FRBI)对小鼠卵巢发育、生殖激素分泌及其蛋白表达的影响。方法给FRBI组小鼠分别肌肉注射20、30和40 mg/kg FRBI;促卵泡激素(FSH)组注射10 IU/kg FSH;对照组(CG)注射0.2 mL 0.9%氯化钠溶液,每组均连续注射5 d。精确称量每只小鼠体质量和双侧卵巢质量;测定卵巢组织切片中皮质厚度(OCT)和次级卵泡形态大小;ELISA测定血清FSH浓度;Western blot检测卵巢FSH受体(FSHR)表达。结果实验第15天起,FRBI组小鼠卵巢质量低于CG和FSH组(P0.05),以FRBI-30 mg组小鼠卵巢质量最低;FRBI组OCT、次级卵泡最大纵径(MLD)和最大横径(MTD)低于CG组和FSH组(P0.05),FRBI-40 mg组小鼠卵巢的MLD和MTD值最低;第15天时,FRBI-30 mg组小鼠血清FSH的浓度最低;随着FRBI注射剂量的增加,卵巢中FSHR表达水平逐渐降低,FRBI组的FSHR蛋白水平明显低于FSH组(P0.05)。结论 FRBI能抑制卵巢和卵泡发育,降低血清FSH浓度,下调卵巢FSHR蛋白表达水平。     

Alarelin免疫绵羊对垂体GnRHR、FSHR和LHR基因表达与生殖激素分泌的作用

 目的 探讨GnRHago主动免疫对绵羊腺垂体GnRHR、FSHR和LHR表达及生殖激素合成与释放作用,为深入研究GnRH-A调节生殖功能的机理及合理应用提供依据。方法 42只5-6月龄母绵羊（Ovis aries）随机分为6组（n =7）,EG-Ⅰ、EG-Ⅱ和EG-Ⅲ分别皮下注射阿拉瑞林抗原200μg、300μg和400μg,0d和14d各1次,共2次;EG-Ⅳ和EG-Ⅴ分别皮下注射阿拉瑞林抗原200μg、300μg,0d、7d、14d和21d各1次,共4次;对照组皮下注射药物的溶媒,0d和14d各1次,共2次。于70d在颈动脉放血处死绵羊,无菌切取腺垂体、子宫及卵巢。用ELISA测定血清GnRH抗体浓度,以激素检测试剂盒分别测定血清GnRH、FSH、LH和E2浓度。提取腺垂体总RNA,PCR扩增与反转录,实时荧光定量PCR（FQ-PCR）,分析GnRHR、FSHR和LHR mRNA的表达。 结果 绵羊腺垂体中有丰富的RNA。特异性条带扩分别为2150 bp,1100bp和180bp左右。实验组GnRHR、FSHR和LHR mRNA值均低于对照组,EG-Ⅳ和EG-Ⅴ的mRNA值低于EG-Ⅰ和EG-Ⅱ。实验组血清GnRH浓度逐渐降低值谷值,之后上升,70d 时达到免疫注射前水平。实验组血清FSH浓度始终高于对照组（P＜0.05）。35d时EG-Ⅳ和EG-Ⅴ低于EG-Ⅰ、EG-Ⅱ和EG-Ⅲ。实验组和对照组血清E2含量无显著差异。结论 阿拉瑞林主动免疫可抑制垂体GnRHR mRNA、FSHR mRNA和LHR mRNA的表达,促进垂体合成与释放FSH,增加血清FSH含量,降低GnRH和LH浓度,对E2浓度无明显影响。增加阿拉瑞林抗原的免疫剂量和注射次数,作用更加明显。     

大鼠胰腺卵泡刺激素(β)及其受体的免疫荧光定位及原位杂交

目的探讨大鼠胰腺组织中是否表达卵泡刺激素(FSH)及其受体(FSHR),为进一步研究FSH对胰腺的功能调节提供理论依据。方法采用单标记免疫荧光技术和原位杂交方法进行研究。结果大鼠胰岛大部分细胞及部分胰腺外分泌部细胞呈FSH蛋白和mRNA,FSHR蛋白和mRNA免疫荧光反应阳性,阳性物质分布于细胞质,细胞核呈阴性反应。结论大鼠胰腺细胞具有自身合成和分泌FSH及其受体的功能,说明FSH对胰腺细胞的作用可能通过旁分泌或自分泌的作用实现的。     

多囊卵巢综合征大鼠模型LHR、INSR和AR基因甲基化的变化

目的复制一种理想的多囊卵巢综合征(PCOS)动物模型,并检测LHR、INSR和AR基因DNA甲基化状态。方法给模型组24日龄大鼠皮下埋植左旋18-甲基炔诺酮硅胶棒3mm/只,3d后每日2次皮下注射人绒毛膜促性腺激素1.5IU。给对照组皮下注射等体积生理盐水。注射9d后观察大鼠卵巢形态学(HE染色),放射免疫法测定性激素和空腹胰岛素水平,己糖激酶法测定空腹血糖水平,计算胰岛素抵抗(HOMA)指数。用甲基化特异性PCR检测LHR、INSR和AR基因的DNA甲基化状态。结果模型组大鼠卵巢重量和体积均显著高于对照组(P<0.001)。模型组卵巢各级发育期卵泡及黄体少见,卵泡多呈囊性扩张。模型组大鼠血清孕激素、睾酮、促黄体生成激素(LH)、空腹胰岛素、空腹血糖水平均显著高于对照组(P<0.05);LH/促卵泡生长激素(FSH)比值和HOMA指数也均显著高于对照组(P<0.001)。模型组INSR基因甲基化率为76.7%,显著高于对照组(20.0%)(P<0.001)。结论DNA甲基化介导的胰岛素受体基因转录抑制是PCOS胰岛素抵抗发生机制之一。     

黄体生成素及其受体在大鼠肝脏的定位

目的观察黄体生成素(LH)及其受体(LHR)在大鼠肝脏的定位分布,为研究LH是否参与肝代谢功能的调节提供形态学依据。方法应用免疫荧光组织化学双标染色技术,在激光共聚焦扫描显微镜下观察染色结果。结果大鼠肝细胞既呈LH阳性又有LHR阳性,LH和LHR免疫反应阳性物质均分布在大鼠肝细胞的细胞质,细胞核为阴性。不同部位的肝细胞LH及其受体免疫反应强度存在差别。结论 LH及其受体存在于大鼠肝脏细胞,可能对肝脏的功能起调节作用。     

FSH-regulated gene expression profiles in ovarian tumours and normal ovaries

Development, growth and function of the ovary are controlled by endocrine and paracrine signals. These may also influence the development of ovarian cancer. The aim of this study was to identify the key molecular markers of the unregulated growth and hormone synthesis seen in ovarian tumours, particularly in granulosa cell tumours (GCT). Genes used in this study were chosen on the basis of our understanding of growth and differentiation in the normal ovary. We sought to define the patterns of gene expression in a panel of epithelial and stromal ovarian tumours. Expression was determined by RT-PCR using gene-specific primers for the FSH receptor (FSHR); the FSH early response genes: regulatory subunit of protein kinase A (RII-beta), cyclin D2 (cycD2) and sgk; and late response markers: cyclooxygenase-2 (COX-2) and the LH receptor (LHR). The GCT had high expression of FSHR compared with normal ovaries and the other tumours. cycD2 and RII-beta and COX-2 genes were also highly expressed in the GCT. sgk and LHR expression was lower in all of the tumours than in normal ovaries. Serous cystadenocarcinomas also had an unexpectedly high expression of COX-2. Comparison of the gene expression profiles between each tumour group suggests a molecular phenotype for GCT that is similar to that reported for FSH stimulated pre-ovulatory granulosa cells.     

Differential regulation of leucine-rich primary response gene 1 (LRPR1) mRNA expression in rat testis and ovary

In immature rat Sertoli cells, leucine-rich primary response gene 1 (LRPR1) represents a follicle stimulating hormone (FSH)-responsive gene; the function of the encoded protein is not yet known. LRPR1 mRNA expression is up-regulated very rapidly and specifically by FSH, both in cultured Sertoli cells and in vivo in regulation in more detail, in testis and ovary of fetal, immature, and adult rats. In addition, we have studied the expression of FSH receptor (FSHR) mRNA in relation to LRPR1 mRNA expression. In rat testis, LRPR1 mRNA and FSHR mRNA followed a similar expression pattern, during postnatal development and also at different stages of the spermatogenic cycle in the adult rat. Furthermore, after short-term challenge of the FSH signal transduction pathway in intact immature rats by injection with a relatively high dose of FSH, an inverse relationship between LRPR1 mRNA (up-regulation) and FSHR mRNA expression (down-regulation) was observed. Similar studies in the ovary provided completely different results. LRPR1 mRNA in the postnatal ovary is present well before expression of FSHR mRNA can be first detected. In addition, incubation of ovaries of immature rats with FSH or dibutyryl cyclic AMP (dbcAMP) did not result in up- regulation of LRPR1 mRNA expression. During fetal development, the LRPR1 mRNA expression pattern involved many more tissues, in contrast to the relatively tissue-specific expression of LRPR1 mRNA in gonads of 21 day old and adult rats. Moreover, LRPR1 mRNA expression could be detected as early as 12.5 days post-coitum, whereas FSHR mRNA is absent at this stage of fetal development. We concluded that the pronounced regulation of LRPR1 by FSH observed in the immature rat testis does not occur in the ovary. Furthermore, in the ovary LRPR1 mRNA expression does not appear to be dependent on FSH action. Finally, the LRPR1 gene product may play a general role during fetal development.      

The Extracts of Pacific Oyster (Crassostrea Gigas) Alleviate Ovarian Functional Disorders of Female Rats with Exposure to Bisphenol a Through Decreasing FSHR Expression in Ovarian Tissues

Background

Bisphenol-A (BPA) is one of the widespread industrial compounds, which has adverse effects on animal and human health. The study was aimed to explore the effects of Crassostrea gigas extracts (CGE) in alleviating ovarian functional disorders of female rats with exposure to BPA and the underlying possible mechanism.

Materials and methods

Eighteen four-week-old female Sprague-Dawley (SD) rats were randomly divided into BPA group (50mg/kg BPA), BPA+CGE group (50mg/kg BPA+50mg/kg CGE), and control group (equivalent dosage of vehicle) with 6 rats in each group. After a 6-week treatment ended, the serum levels of estradiol (E₂), follicle stimulating hormone (FSH), luteinizing hormone (LH) were measured by using commercial standard assay kits. The expression levels of FSH receptor (FSHR) in the rat ovarian tissues were respectively detected by immunohistochemistry and Real-time PCR.

Results

CGE treatment markedly increased E₂ levels and decreased FSH levels in the serum (P<0.05), however, the alterations of serum LH levels were not significant (P>0.05). The protein and mRNA expression levels of FSHR were the lowest in the ovaries of control rats and the highest in BPA rats (P<0.05). CGE treatment markedly decreased the expression levels of FSHR in the ovarian tissues (P<0.05).

Conclusions

Crassostrea gigas successfully alleviates ovarian functional disorders of female rats with exposure to BPA partly through decreasing FSHR expression levels in the ovarian tissues.     

Serum levels of sex hormones and expression of their receptors in thyroid tissue in female patients with various types of thyroid neoplasms

Previous studies have demonstrated the expression of estrogen receptor (ER) and progesterone receptor (PR) in thyroid cancer; however, little is known regarding the levels of estrogen, progesterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in serum and the expression of ER, PR, FSH receptor (FSHR), and LH receptor (LHR) in thyroid tissues of patients with different types of thyroid neoplasms. Serum levels of estrogen, progesterone, FSH, and LH were measured by chemiluminescence, and expression of ER, PR, FSHR, and LHR in thyroid tissue was detected by immunohistochemistry in female patients with thyroid adenoma (n = 70), nodular goiter (n = 73), thyroid papillary cancer (n = 149), poorly differentiated thyroid carcinoma (n = 12), or undifferentiated thyroid carcinoma (n = 8) and in normal controls (n = 60). The positive rates of serum estrogen level and ERα expression were significantly greater in patients with various types of thyroid neoplasms than in normal controls. The positive rates of ERβ expression were significantly less in various types of thyroid neoplasms than in normal thyroid tissues, especially in poorly differentiated carcinoma and undifferentiated carcinoma. The negative rates of serum progesterone level and positive rates of PR expression in thyroid tissue were significantly greater in patients with thyroid adenoma, nodular goiter, or thyroid papillary cancer than in normal controls. The positive rates of serum FSH and LH levels and FSHR and LHR expression were significantly greater in the thyroid adenoma group than in other groups. Our findings suggest that thyroid neoplasms might be sex hormone-dependent. The positive expression of ERα and PR often indicates thyroid papillary carcinoma, and the ERβ expression status is important for the diagnosis of poorly differentiated carcinoma and undifferentiated carcinoma. In addition, thyroid adenoma is often accompanied by an increase in serum FSH and LH levels, as well as FSHR and LHR expression. Thus, the combined detection of serum levels of sex hormones and expression of their receptors allows for a differential diagnosis and evaluation of the degree of differentiation among various types of thyroid neoplasms.     

Follicle stimulating hormone receptor protein is expressed in ovine uterus during the estrous cycle and utero-placenta during early pregnancy: An immunohistochemical study

Follicle stimulating hormone (FSH) is a well characterized gonadotropin that controls primarily development and functions of ovarian follicles in mammalian species. FSH binds to a specific G protein-coupled receptor (FSHR) belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although the primary location of FSHR is in the gonads (mainly in ovarian follicles), FSHR protein and/or mRNA have also been detected in extragonadal female reproductive tissues including embryo, placenta, endometrium, cervix, ovarian cancer tissues, and/or endometriotic lesions in several species. To determine the pattern of FSHR expression in the uterus and placenta, uterine tissues were collected at the early, mid- and/or late luteal phases of the estrous cycle from non-treated or FSH-treated ewes, and utero-placental tissues were collected during early pregnancy followed by immunohistochemistry and image generation. FSHR was immunolocalized to several uterine and utero-placental compartments including luminal epithelium, endometrial glands and surrounding stroma, myometrium, and endothelium and vascular smooth muscle cells in endometrium, myometrium and mesometrium. Intensity of staining and distribution of FSHR in selected compartments differed and seems to depend on the stage of the estrous cycle or pregnancy, and FSH-treatment. These novel data demonstrate differential expression of FSHR protein indicating that FSH plays a specific role in regulation of uterine and utero-placenta functions in sheep.     

缝隙连接蛋白43和黄体生成素受体在小鼠卵巢中的表达

目的 观察小鼠卵泡发育过程中缝隙连接蛋白43（Cx43）和黄体生成素受体（LHR）在小鼠卵巢中的表达以及黄体生成素(LH)对卵巢Cx43表达的影响,为这两种分子与卵泡发育的关系提供实验依据。 方法 选择出生后1d、4d、7d、2周、3周、4周、6周、8周龄雌性C57小鼠共8组,每组10只,取卵巢。HE染色观察卵巢形态、卵泡发育并测量颗粒细胞体积分数。免疫组织化学和Western blotting观察卵巢组织中Cx43及LHR的表达,Western blotting检测不同浓度黄体生成素（LH）孵育后对卵巢Cx43表达的影响。 结果 HE染色显示,出生1d小鼠卵巢中见原始卵泡,出生7d卵巢内出现初级卵泡,2周时出现窦前卵泡和少量窦卵泡,3周、4周时,出现较多大的窦卵泡,6～8周时出现黄体。卵泡颗粒细胞体积分数在2周后的卵巢中明显增加。Cx43表达在各阶段卵泡的颗粒细胞胞膜上,LHR表达在卵泡膜细胞、颗粒细胞及卵母细胞胞质中。Cx43和LHR出生后2周卵巢中的表达开始明显增加,并随卵泡发育表达逐渐增强,在3周、4周、6周和8周维持相对较高水平。LH体外干预可增加卵巢组织Cx43的表达。 结论 Cx43和LHR在卵泡发育中发挥重要作用,LH可能通过LHR促进卵巢卵泡颗粒细胞Cx43的表达。