牛促卵泡激素在毕赤酵母中的表达

目的:实现重组牛促卵泡激素在毕赤酵母中的表达。方法:依据毕赤酵母的密码子偏爱性设计并利用PCR方法合成了牛促卵泡激素的α亚基和带有6×his-tag的β亚基相应的DNA序列,构建表达载体pHIL-S-bFSH,转化毕赤酵母,表达蛋白进行ELISA定量和Western blot鉴定。结果:重组牛促卵泡激素的表达量为0.26mg/L,Western blot鉴定分子量为35kDa。结论:在毕赤酵母中实现了具有免疫源活性的重组牛促卵泡激素的表达。     

Hormonal Responses to Synthetic Luteinizing Hormone and Follicle Stimulating Hormone-Releasing Hormone in Man

The effects of the gonadotrophin-releasing hormone, synthetic decapeptide luteinizing hormone/follicle stimulating hormone-releasing hormone (LH/FSH-RH), have been studied in 18 normal men and five women in the follicular phase of their menstrual cycle. Rapid and dose-dependent (25 to 100 μg) increases in serum immunoreactive LH were seen, which reached a peak 20 to 30 minutes after a rapid intravenous injection. Similar but much smaller increases in serum immunoreactive FSH were seen. These conclusions have been validated by using two different immunoassay systems for each hormone. The LH/FSH-RH therefore causes both LH and FSH release in man as in animals but does not affect growth hormone, thyrotrophin, or ACTH. The gonadotrophin responses were the same in the women as in the men but were insufficient in the men to cause statistically significant changes in the serum levels of the gonadal steroid hormones, testosterone or oestradiol, or in their precursors 17 α-hydroxyprogesterone or progesterone. In the women, however, there was a rise in oestradiol after the 100-μg doses. The use of LH/FSH-RH will provide an important test to define the level of the lesion in hypogonadal patients and also should be valuable in the treatment of some types of male and female infertility. A simple and clinically useful LH/FSH-RH test of pituitary function is described (100 μg given intravenously), and the provisional normal responses of LH and FSH at 20 and 60 minutes are given.     

Luteinizing Hormone and Follicle Stimulating Hormone-Releasing Hormone Test in Patients with Hypothalamic Pituitary-Gonadal Dysfunction

A standard intravenous 100 μg luteinizing hormone/follicle stimulating hormone-releasing hormone (LH/FSH-RH) test was used to assess the pituitary gonadotrophin responses in 155 patients with a variety of diseases of the hypothalamic-pituitary-gonadal axis. In all but nine patients there was an increase in circulating levels of either LH or FSH in response to the releasing hormone though 137 (88%) were clinically hypogonadal. It was not possible with this test to distinguish between hypothalamic and pituitary causes of hypogonadotrophic hypogonadism, since a variety of LH and FSH responses emerged within the disease groups. However, primary gonadal failure characteristically resulted in exaggerated gonadotrophin response. The potential therapeutic use of the gonadotrophin releasing decapeptide is suggested in certain patients with hypogonadotrophic hypogonadism.     

Isolated Pituitary Gonadotrophin Deficiency: Gonadotrophin Secretion after Synthetic Luteinizing Hormone and Follicle Stimulating Hormone-releasing Hormone

The responses of serum immunoreactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) after intravenous injection of 100 μg of synthetic LH/FSH-RH have been studied in 14 patients with the syndrome of isolated pituitary gonadotrophin deficiency. Nine of the patients showed a rise of both hormones, two a small rise of FSH only, and three were unresponsive. In two of the unresponsive patients injection of a 500-μg dose produced a small rise of LH only. Of the patients who responded, four had LH and FSH responses within the normal adult range, while in the others the responses were smaller and delayed. It is suggested that this syndrome is due to a lack of the hypothalamic-releasing hormone itself, rather than to a pituitary deficiency. However, repeat assessment after prolonged administration of the releasing hormone will be necessary before a pituitary disorder can be excluded in all patients. The synthetic LH/FSH-RH, preferably as a depot preparation, may provide a means of treating these patients to induce the development of puberty and subsequent fertility.     

雌性哺乳动物促卵泡素受体与促黄体素受体的结构与表达

本文从雌性哺乳动物促卵泡素受体(FSHR)和促黄体素受体(LHR)的结构及在卵泡发育过程中的表达和功能加以综述,指出FSHR及LHR在卵泡的生长发育、优势化及排卵等方面具有重要的功能。     

山羊卵泡刺激素α亚基cDNA的分子克隆与序列分析

从新屠宰的雌山羊脑垂体中提取总RNA ,反转录获得cDNA .以此cDNA为模板用PCR法扩增目的片段 ,获得长为 380bp的山羊卵泡刺激素α亚基cDNA片段 .将它克隆至pMD 18 T Verctor.随机挑选 3个阳性重组子进行测序 ,将测序结果与绵羊、牛、猪等多种哺乳动物该基因的核苷酸序列及相应氨基酸序列进行比较 .结果表明 ,山羊卵泡刺激素α亚基基因氨基酸序列与绵羊、水牛的同源性最高 ,达 96 % ,与牛的同源性达 95 % ,与人的同源性较低 ,为 74 % .山羊卵泡刺激素α亚基基因编码区的核苷酸与绵羊的同源性最高 ,达 95 % ,与水牛、牛的同源性达 94 % ,与马和大鼠的同源性较低 ,为 85 % .总体来看 ,在哺乳类动物中FSHα亚基基因同源性还是很高的 .     

改变大鼠促性腺激素细胞内cAMP含量对卵泡刺激素分泌的影响

目的:分析大鼠卵泡刺激素(FSH)分泌的受体后信号转导机制。方法:将促性腺激素(GTH)细胞用毛喉素(FSK)或腺苷酸环化酶抑制剂SQ22536处理后,用促性腺激素释放激素脉冲刺激,再用酶联免疫吸附法检测其FSH分泌量,并与空白对照组比较。结果:FSK能显著提高GTH细胞中环磷酸腺苷(cAMP)含量,SQ22536能显著降低GTH细胞中的cAMP含量,FSK和SQ22536都不会影响GTH细胞的蛋白激酶C活性,GTH细胞cAMP含量的变化对FSH分泌的影响不显著。结论:cAMP-PKA(蛋白激酶A)不是FSHβ亚基分泌的受体后信号转导途径。     

双峰驼血浆促卵泡素的放射免疫分析法

用单峰驼促卵泡素标准品（ＣａｍＦＳＨ）,ｈＦＳＨ抗血清和＾１２５Ｉ－ｈＦＳＨ建立了测定双峰驼血浆ＦＳＨ的放射免疫分析方法,并通过一系列实验证明,该方法可以用于测定双峰驼血浆ＦＳＨ,是研究双峰驼生殖内分泌学的可靠手段之一。     

Follicle-Stimulating Hormone Increases the Risk of Postmenopausal Osteoporosis by Stimulating Osteoclast Differentiation

Objective

The objectives of this study were to observe the changes in follicle-stimulating hormone (FSH) and bone mineral density (BMD) in postmenopausal women, to research the relationship between FSH and postmenopausal osteoporosis, and to observe the effects of FSH on osteoclast differentiation in RAW264.7 cells.

Methods

We analyzed 248 postmenopausal women with normal bone metabolism. A radioimmunoassay (RIA) was used to detect serum FSH, luteinizing hormone (LH), and estradiol (E₂). Dual-energy X-ray absorptiometry was used to measure forearm BMD. Then, we analyzed the age-related changes in serum FSH, LH and E₂. Additionally, FSH serum concentrations were compared between a group of postmenopausal women with osteoporosis and a control group. Osteoclasts were induced from RAW264.7 cells in vitro by receptor activator of nuclear factor kappa B ligand (RANKL), and these cells were treated with 0, 5, 10, and 20 ng/ml FSH. After the osteoclasts matured, tartrate-resistant acid phosphatase (TRAP) staining was used to identify osteoclasts, and the mRNA expression levels of genes involved in osteoclastic phenotypes and function, such as receptor activator of NF-κB (Rank), Trap, matrix metalloproteinase-9 (Mmp-9) and Cathepsin K, were detected in different groups using real-time PCR (polymerase chain reaction).

Results

1. FSH serum concentrations in postmenopausal women with osteoporosis increased notably compared with the control group. 2. RANKL induced RAW264.7 cell differentiation into mature osteoclasts in vitro. 3. FSH increased mRNA expression of genes involved in osteoclastic phenotypes and function, such as Rank, Trap, Mmp-9 and Cathepsin K, in a dose-dependent manner.

Conclusions

The circulating concentration of FSH may play an important role in the acceleration of bone loss in postmenopausal women. FSH increases osteoclastogenesis in vitro.     

Inferences from the ADMET analysis of predicted inhibitors to Follicle Stimulating Hormone in the context of infertility

Follicle stimulating hormone (FSH) is a glycoprotein secreted by gonadotrophs of the anterior pituitary gland that regulatesreproduction in mammals. FSH targets its receptor (FSHR) expressed only on grannulosa cells and induce the maturation ofovarian follicles in females. The levels of both FSH and FSHR rise until the middle of estrus cycle and then falls on level at the timeof ovulation. It is associated with stimulated sertoli cell proliferation in testes and supports spermatogenesis in males. Theinteraction between the polypeptide FSH hormone and its corresponding receptor is highly selective. Therefore, it is of interest toinhibit FSH in the context of infertility. The structure of FSH (PDB ID: 1XWD) is screened using molecular docking techniquesagainst the ZINC database (a database of 2.7 million compounds) with reference to known standard compounds. This exerciseidentifies compounds with better binding and ADMET (Absorption, Digestion, Metabolism, Excretion and Toxicity) propertiescompared to known standard compounds. These observations find application for the consideration of such compounds for furthervalidation towards inhibiting the FSH.     

Gonadotrope-Specific Expression and Regulation of Ovine Follicle Stimulating Hormone Beta: Transgenic and Adenoviral Approaches Using Primary Murine Gonadotropes

The beta subunit of follicle stimulating hormone (FSHB) is expressed specifically in pituitary gonadotropes in vertebrates. Transgenic mouse studies have shown that enhancers in the proximal promoter between −172/−1 bp of the ovine FSHB gene are required for gonadotrope expression of ovine FSHB. These enhancers are associated with regulation by activins and gonadotropin releasing hormone (GnRH). Additional distal promoter sequence between −4741/−750 bp is also required for expression. New transgenic studies presented here focus on this distal region and narrow it to 1116 bp between −1866/−750 bp. In addition, adenoviral constructs were produced to identify these critical distal sequences using purified primary mouse gonadotropes as an in vitro model system. The adenoviral constructs contained −2871 bp, −750 bp or −232 bp of the ovine FSHB promoter. They all showed gonadotrope-specific regulation since they were induced only in purified primary gonadotropes by activin A (50 ng/ml) and inhibited by GnRH (100 nM) in the presence of activin (except −232FSHBLuc). However, basal expression of all three viral constructs (in the presence of follistatin to block cellular induction by activin) was relatively high in pituitary non-gonadotropes as well as gonadotropes. Thus, gonadotrope-specific regulation associated with the proximal promoter was observed as expected, but the model was blind to distal promoter elements between −2871/−750 necessary for gonadotrope-specific expression of ovine FSHB in vivo. The new adenoviral-based in vitro technique did detect, however, a novel GnRH response element between −750 bp and −232 bp of the ovine FSHB promoter. We conclude that adenoviral-based studies in primary gonadotropes can adequately recognize regulatory elements on the ovine FSHB promoter associated with gonadotrope-specific regulation/expression, but that more physiologically based techniques, such as transgenic studies, will be needed to identify sequences between −1866/−750 bp of the ovine FSHB promoter that are also required for tissue/cell specific expression in vivo.     

人促卵泡激素体外生物学活性测定方法的建立

目的:建立人促卵泡激素(FSH)体外生物学活性检测方法,并对方法进行验证。方法:以人卵巢颗粒细胞KGN为基质细胞,用不同浓度的FSH与KGN细胞表面的FSH受体结合,刺激细胞产生不同浓度的孕酮,通过测定细胞培养上清中的孕酮含量来评价FSH的生物学活性,结果用国际标准品进行校正;对方法的专属性、线性与范围、回收率、精密度、耐用性进行验证;对市售FSH产品进行检测,并与传统动物体内活性检测结果进行比较。结果:KGN细胞对FSH有很好的剂量反应关系,方法专属性符合要求;线性范围为0.1~200 ng/m L,相关系数R2≥0.99;回收率为80%~120%;经3次重复测定,3批市售样品和3批自制样品的活性分别为(13.4±0.4)~(13.5±0.8)和(12.9±0.7)~(14.3±1.2)IU/μg,变异系数在15%之内;结果显示该方法有很好的耐用性。测定3批市售重组人FSH产品和1批尿源FSH国家标准品,其体外活性测定结果与传统动物体内活性测定结果一致。结论:建立并验证了一种利用KGN细胞体外测定FSH生物学活性的方法,有望代替传统的动物体内活性测定方法,可用于FSH的生物学活性评价和质量控制。     

药学角度探讨和分析不同来源卵泡刺激素对体外受精-胚胎移植结局的影响

目的:比较基因重组卵泡刺激素(r-FSH)及高纯度尿源性卵泡刺激素(HP-u FSH)对体外受精-胚胎移植(IVF-ET)妊娠结局的影响,结合临床表现从药学角度探讨和分析。方法:回顾分析具IVF/单精子卵细胞浆内显微注射授精(ICSI)指诊并首次接受IVF/ICSI助孕的不孕症患者235例,以给予的FSH药物种类不同分为2组:HP-u FSH组(n=135)和r-FSH组(n=100)。主要研究指标为出生率,次要研究指标为临床妊娠率,流产率及着床率。结果:HP-u FSH组外源性促性腺激素(Gn)总量和FSH量均多于r-FSH组(P0.01),获卵数和可移植胚胎数较r-FSH组少(P0.01),受精率较r-FSH组低(P0.05)。两组平均Gn刺激天数,妊娠结局及中重度卵巢过度刺激综合征(OHSS)发生率均无统计学差异(P0.05)。对患者年龄35岁和≥35岁分别进行分组发现,年龄35岁患者中的HP-u FSH组较r-FSH组平均年龄更大,不孕年限更长,基础窦卵泡数(AFC)更少(P0.01),≥35岁患者中的HP-u FSH组与r-FSH组的基本特征(年龄,不孕年限,AFC)无统计学差异(P0.05);无论年龄35岁或年龄≥35岁,HP-u FSH组的Gn量和FSH量较r-FSH组均更大,获卵数及可移植胚胎数更少(P0.01)。结论:在IVF/ICSI周期治疗中,国产HP-u FSH与r-FSH进行COS具有等同的IVF临床妊娠率及出生率。     

不同波段电磁辐射对大鼠Sertoli细胞雄激素受体和卵泡刺激素受体表达的影响

探讨不同波段电磁辐射对大鼠睾丸Sertoli细胞雄激素受体(androgen receptor,AR)和卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)表达的影响。原代培养的Sertoli细胞分别经场强6×104 V/m的电磁脉冲(electromagnetic pulse,EMP)、平均功率密度为100 mW/cm2的S波段微波(S-band high power microwave,S-HPM)和X波段微波(X-bandhigh power microwave,X-HPM)辐射4 min。应用real-time RT-PCR和Western blot方法检测Sertoli细胞AR的表达,结果显示具有不同程度的降低(P<0.05,P<0.01);Wistar大鼠分别经S-HPM、X-HPM和EMP照射20 min,应用免疫组化方法检测睾丸组织AR的表达,发现辐射后7 d有显著降低(P<0.05,P<0.01)。三者比较,AR的表达总体呈EMP>X-HPM>S-HPM的趋势,而FSHR的表达无明显变化。Sertoli细胞AR表达的变化,可能参与了电磁辐射致...     

FSH促进牦牛输卵管上皮细胞分泌输卵管特异性糖蛋白的研究

为在胚胎共培养过程中添加相关激素提高哺乳动物胚胎发育的研究提供理论依据,本研究探讨了促卵泡生成素(FSH)对牦牛输卵管上皮细胞分泌特异性糖蛋白的影响。体外分离培养牦牛输卵管上皮细胞,并在细胞中添加不同浓度的FSH,作用6 h后运用荧光定量PCR分析输卵管特异性糖蛋白mRNA的表达水平,并用细胞免疫标记对其分泌输卵管蛋白的部位进行分析。结果显示,FSH的浓度为0.5-5.0μg/m L时,输卵管蛋白的表达量随着FSH浓度的上升而增加,在5.0μg/m L的FSH作用后,输卵管蛋白的mRNA表达量最高;浓度超过10.0μg/m L时,输卵管蛋白基因的表达量降低。结果表明,FSH具有促进输卵管上皮细胞分泌输卵管特异性糖蛋白的作用,且具有剂量依赖性,其最佳作用浓度为5.0μg/m L,输卵管蛋白主要由细胞质分泌。     

OCT4 介导卵泡刺激素对永生化人卵巢上皮细胞株增殖、 凋亡和侵袭能力的影响

目的:探讨OCT4基因对卵泡刺激素作用下的永生化人卵巢上皮细胞株(Moody细胞)增殖、凋亡和侵袭能力影响。方法:将不同浓度的FSH(0、25、50、100mIU/ml)作用于Moody细胞48小时,应用Western-blot技术检测OCT4表达情况。采用慢病毒介导将重组质粒OCT4稳定转染至人卵巢上皮细胞株中,应用Western-blot法鉴定OCT4蛋白表达情况。FSH以50 mIU/ml作为工作浓度,实验对象分为4组:①siCon组,转染空载体的阴性对照组;②OCT4组:稳定转染OCT4基因的Moody细胞组;③FSH+siCon组:以FSH处理的siCon组;④FSH+OCT4组:以FSH处理的OCT4组。采用MTT比色法检测各组细胞的增殖情况,流式细胞仪检测各组细胞凋亡情况,Transwell侵袭实验检测各组细胞侵袭能力的变化。结果:(1)随着FSH浓度的增加,Moody细胞中OCT4蛋白表达逐渐增高,在FSH浓度为50 mIU/ml时达最高;(2)OCT4基因成功转染至Moody细胞中,经Western-blot检测该基因在细胞中进行蛋白高表达;(3)FSH+OCT4组细胞增殖活性明显增高,同时凋亡率降低,与另外三组相比差异具有统计学意义(P<0.05);(4)在FSH作用下,转染OCT4后明显增强了细胞的侵袭能力,与另外三组相比差异具有统计学意义(P<0.05)。结论:OCT4介导了FSH对人卵巢上皮细胞增殖、凋亡、侵袭活性的调控。     

Bioassay of Thyroid Stimulating Hormone Using the Goldfish

A new method for the bioassay of thyroid stimulating hormone using goldfish is described. The technique can detect thyroid stimulating substances in euthyroid unconcentrated plasma. Early results show that the method compares well with other techniques. Some evidence has been obtained which suggests that in primary myxoedema there may be some impairment of the thyropituitary “servo-mechanism” in older patients.     

去促甲状腺激素血清的研制

采用亲和层析法.经过固体硫酸铵预处理的人促甲状腺激素抗血清与CNBr-Sepharose 4B在碱性条件下偶联,偶联率大于90%.亲和柱规格:1.2cm×40cm.淋洗流速:3ml/min.正常人血清上样流速:0.25ml/min.淋洗液用0.01mol/L PBS(pH7.4).柱子再生液用6mol/L盐酸肌溶液.此法处理过的血清符合TSH免放药盒的要求.     

Palmitoylation Regulates Epidermal Homeostasis and Hair Follicle Differentiation

Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs). Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep) is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE) and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear β-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis.