AnnexinⅠ在PCOS 大鼠颗粒细胞中的表达及意义

目的:探讨膜联蛋白Ⅰ(AnnexinⅠ)在多囊卵巢综合征(PCOS)大鼠卵泡颗粒细胞的表达及生物学意义。方法:采用免疫组织化学方法及灰度值测定AnnexinⅠ在PCOS组和对照组的卵泡颗粒细胞中的表达。结果:AnnexinⅠ在两组中的各级卵泡颗粒细胞中均有表达,PCOS组AnnexinⅠ在窦状卵泡中表达显著高于对照组(P<0.05)。结论:PCOS组AnnexinⅠ在窦状卵泡颗粒细胞的表达上调,且PCOS中窦状卵泡颗粒细胞的凋亡增加,说明AnnexinⅠ参与了卵巢颗粒细胞的凋亡过程,并且发挥了重要作用。     

多囊卵巢综合征模型鼠颗粒细胞凋亡及TRAIL蛋白的表达

目的通过观察卵巢颗粒细胞凋亡及TRAIL(肿瘤坏死因子相关凋亡诱导配体)蛋白的表达情况,探讨颗粒细胞凋亡与PCOS发病的相关性及凋亡调控蛋白TRAIL在PCOS颗粒细胞凋亡中的作用。方法采用硫酸普拉睾酮钠诱导大鼠PCOS模型,3’-末端原位标记法(TUNEL)检测大鼠卵巢颗粒细胞凋亡情况,免疫组化染色及RT-PCR分析检测TRAIL蛋白及TRAIL mRNA在颗粒细胞的表达。结果PCOS组大鼠卵巢窦状卵泡颗粒细胞凋亡发生率及TRAIL蛋白的表达较对照组明显增强(P<0.01,P<0.05),窦前卵泡颗粒细胞凋亡发生率及TRAIL蛋白的表达两组无显著性差异(P>0.05),两组卵巢始基卵泡颗粒细胞未发现凋亡征象及TRAIL蛋白表达。PCOS组大鼠卵巢颗粒细胞TRAIL mRNA的表达较对照组明显增强(P<0.01)。结论PCOS大鼠卵巢窦状卵泡颗粒细胞凋亡明显增强,TRAIL在PCOS大鼠卵巢颗粒细胞凋亡调控中发挥了作用。     

D-半乳糖诱导建立的小鼠多囊卵巢综合征模型

目的研究D-半乳糖诱导ICR中年雌性小鼠多囊卵巢综合征(PCOS)动物模型的卵巢形态学、性激素以及胰岛素水平变化,并探讨D-半乳糖引致小鼠PCOS的意义。方法以D-半乳糖腹腔注射20周龄ICR雌性小鼠8周,观察卵巢形态的变化,检测血糖值及动情周期排卵情况,并采用ELISA法测定血清胰岛素、雌二醇(E2)、促卵泡生长激素(FSH)、睾酮(T)水平。结果 D-半乳糖处理组小鼠的卵巢重量显著高于对照组(P<0.05),有80%(10/12)的单侧或双侧卵巢呈现多囊性扩张,卵巢闭锁增多及颗粒细胞层数减少,并表现为紊乱的动情周期,提示无排卵;与对照组比较,D-半乳糖组小鼠血清T、E2和空腹血糖水平明显升高(P<0.001),FSH水平下降(P<0.0001),空腹血胰岛素水平显著高于对照组(P<0.01),胰岛素敏感指数显著低于对照组(P<0.05)。结论使用D-半乳糖诱导小鼠PCOS模型,无论在影响血清性激素还是卵巢局部形态学改变方面都与临床表现相似,并存在胰岛素抵抗现象,符合PCOS的典型特征,可作为动物模型用于科学研究。     

性激素反馈调节下丘脑ARC和AVPV中kisspeptin-GPR54信号通路的核团差异

下丘脑-垂体-性腺（HPG）轴是调控生殖系统的发育和功能的重要内分泌系统。下丘脑中促性腺激素释放激素（GnRH）神经元,能够接收各种神经传导物质和神经调节物质的信号输入,引起HPG轴的级联反应。下丘脑弓状核(ARC）和前腹侧脑室周围核团(AVPV）中的kisspeptin-GPR54信号通路,可以调控GnRH的分泌和释放,影响性腺激素的分泌。近年来研究发现,性激素能够对下丘脑kisspeptin-GPR54信号通路产生反馈调节,且具有核团差异性。本文就性激素在下丘脑ARC和AVPV中对kisspeptin-GPR54信号通路反馈调节的差异性进行了综述,探讨下丘脑中不同核团对性激素刺激作用产生的不同反应。     

重复制动应激对雌性大鼠下丘脑-垂体-卵巢轴的影响

目的: 探索重复制动应激对雌性大鼠下丘脑-垂体-卵巢轴的影响。方法: 40只SD雌鼠随机分为两组(n=20),对照组和实验组,一组正常饲养,一组采取递增负荷束缚应激,每天置于束缚器内制动应激一次(从上午9:00开始),第1日制动2 h,以后采用递增负荷,每日增加0.5 h,持续两周,通过检测体重、脏器系数、动情周期、性激素、病理和相关基因的表达探索其对下丘脑-垂体-卵巢轴的危害。结果: 重复制动应激使雌性大鼠体重下降、动情周期延长,卵巢和子宫的脏器系数和形态发生改变,利用qPCR技术对其相关基因检测,发现下丘脑促性腺激素释放激素、垂体促性腺激素释放激素受体、促卵泡生成素和促黄体生成素mRNA的表达显著下降,卵巢促卵泡生成素和黄体生成素受体 mRNA的表达显著上升,卵巢和子宫雌激素受体mRNA的表达显著下降。结论: 重复制动应激可能通过干扰下丘脑-垂体-卵巢轴的内分泌调节作用,使动情周期紊乱,从而损伤雌性动物的性腺和生殖内分泌功能。     

新生小鼠卵巢组织的玻璃化冻存、移植及分离卵泡的体外成熟培养初步研究

目的:系统地探索新生小鼠卵巢组织的玻璃化冻存建立卵巢库,移植和分离卵泡以及体外成熟培养的实验方法。方法:对1日龄小鼠卵巢组织进行冻存,解冻复苏和同种肾包膜下移植,从卵巢移植物种中进行卵泡分离和体外成熟培养。结果:①采用平衡液(ES)处理25min,玻璃化液(VS)处理3min方案冻存的卵巢组织具有更高比例的形态完整卵泡,其完整原始卵泡率均值达96.6%,显著性高于实验中其它四组方案(P0.05);②在分别移植2周和4周后回收卵巢移植物,发现二者的卵巢回收率无显著差异(P0.05),但移植4周组的卵泡回收数目要明显高于2周组(P0.05);③在培养基中添加适量的自体血清(10%,V/V)能显著提高卵子的体外成熟率,培养12h后对照组中生发泡破裂(GVBD)发生率为(34.74±4.26)%,添加血清后提高至(54.60±3.37)%,成熟的MⅡ期卵子获得率从(43.17±1.31)%升高至(57.75±5.31)%,有显著性差异(P0.05)。结论:通过该实验较好地建立了卵巢组织的玻璃化冻存、移植和卵泡分离以及体外成熟培养的实验方法。     

Cd24a和前列腺素代谢相关酶在PCOS小鼠模型卵巢颗粒细胞中表达水平的实验研究

目的:探讨Cd24a分子和前列腺素代谢相关酶在多囊卵巢综合征(polycystic ovary syndrome, PCOS)小鼠模型卵巢颗粒细胞中的表达水平。方法:取20只雌性C57BL/6小鼠,随机分为对照组(正常小鼠)和实验组(应用脱氢表雄酮建立PCOS小鼠模型),每组各10只。对照组小鼠于颈背部连续皮下注射20天芝麻油溶液(0.1 m L/100 g)。实验组应用脱氢表雄酮(6 mg/100 g)联合芝麻油溶液(0.1 m L/100 g)连续颈背部皮下注射20天。经苏木精-伊红染色观察两组小鼠卵巢组织病理学改变。应用实时荧光定量PCR法对小鼠卵巢颗粒细胞中Cd24a分子和前列腺素代谢相关酶m RNA表达量进行检测。结果:实验组体重显著高于对照组(P0.05);实验组小鼠卵巢呈多囊样改变,卵泡中颗粒细胞数量减少,闭锁卵泡增多,闭锁卵泡直径明显大于对照组;实验组Cd24a分子和前列腺素代谢相关酶m RNA表达量较对照组存在显著差异(P0.05)。结论:PCOS小鼠卵巢颗粒细胞中Cd24a分子和前列腺素代谢相关酶表达量异常,提示Cd24a分子可能与PCOS疾病发生相关。     

玉竹提取物对大鼠多囊卵巢综合症的疗效研究

目的 观察玉竹提取物（POD）对多囊卵巢综合症（PCOS）模型大鼠卵巢功能及炎症的影响。方法 本研究选择3周龄雌性SD大鼠,采用来曲唑进行灌胃21 d复制PCOS模型,造模成功后,采用不同浓度POD对PCOS模型大鼠进行灌胃处理。记录大鼠每日饮水及摄食量、体重变化情况及动情周期,检测血液相关指标、测定血清睾酮水平,检测大鼠糖耐量,采用苏木素-伊红（HE）染色观察卵巢组织形态学变化,通过蛋白质免疫印迹法（WB）、实时荧光定量反转录聚合酶链反应（qRT-PCR）及免疫组织化学法（IHC）检测卵巢组织内抗苗勒激素（AMH）、炎性细胞因子白介素-1β (IL-1β)、肿瘤坏死因子α (TNF-α)的表达。结果 与对照组相比,PCOS模型大鼠动情周期紊乱、卵巢呈多囊样改变,血清睾酮水平显著上升、卵巢AMH表达上调、糖耐量异常（P<0.01）,IL-1β和TNF-α表达增加（P<0.01）;不同浓度POD处理PCOS模型大鼠后,大鼠动情周期逐渐恢复正常、卵巢中黄体数量增加、囊性卵泡数量减少、血清睾酮水平下调、糖耐量异常缓解（P<0.01）,卵巢组织炎性细胞因子IL-1β、TNF-...     

高雄激素相关的慢性炎症与多囊卵巢综合征的研究进展

多囊卵巢综合征是育龄妇女常见的慢性炎症代谢性疾病,70%以上患者出现高雄激素血症。下丘脑-垂体-性腺相关激素是月经周期和卵巢活动的调节器,脑神经元分泌的Kisspeptin、褪黑素通过下丘脑-垂体-卵巢轴调节Gn RH神经元AMH、Gn RH的表达,卵巢促性腺激素、雄激素水平增高,有利于慢性炎症的形成,促进多囊卵巢综合征的发生发展。本文综述了近几年多囊卵巢综合征中雄激素相关的慢性炎症研究,并根据相关研究提出了一些见解,希望能为多囊卵巢综合征的研究提供一些新的思路。     

敲减miR-155对PCOS小鼠卵巢形态学及生殖内分泌的影响

[目的]研究miR-155敲减基因对多囊卵巢综合征(PCOS)小鼠卵巢形态及生殖内分泌的影响。[方法]取20只小鼠,采用CMC-来曲唑悬浮液灌胃建立PCOS模型,造模成功后将其随机分为miR-155基因敲减组与阴性对照组,各10只。检测两组小鼠血脂及血清性激素变化、卵巢形态、卵巢组织中GLUT4蛋白表达水平。[结果]miR-155基因敲减组中无优势卵泡,有较多小卵泡,无黄体,颗粒细胞减少,卵泡膜明显增厚,间质细胞增生显著;miR-155基因敲减组除FSH水平低于阴性对照组;其余血脂及血清性激素均高于阴性对照组,差异有统计学意义(P<0.05);miR-155基因敲减组中GLUT4蛋白低于阴性对照组,差异有统计学意义(P<0.05)。[结论]miR-155基因敲减可通过降低GLUT4蛋白分泌影响PCOS小鼠卵巢形态、内分泌及胰岛素抵抗功能。     

Physiological role of metastin/kisspeptin in regulating gonadotropin-releasing hormone (GnRH) secretion in female rats

Various studies have attempted to unravel the physiological role of metastin/kisspeptin in the control of gonadotropin-releasing hormone (GnRH) release. A number of evidences suggested that the population of metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) is involved in generating a GnRH surge to induce ovulation in rodents, and thus the target of estrogen positive feedback. Females have an obvious metastin/kisspeptin neuronal population in the AVPV, but males have only a few cell bodies in the nucleus, suggesting that the absence of the surge-generating mechanism or positive feedback action in males is due to the limited AVPV metastin/kisspeptin neuronal population. On the other hand, the arcuate nucleus (ARC) metastin/kisspeptin neuronal population is considered to be involved in the regulation of tonic GnRH release. The ARC metastin/kisspeptin neurons show no sex difference in their expression, which is suppressed by gonadal steroids in both sexes. Thus, the ARC population of metastin/kisspeptin neurons is a target of estrogen negative feedback action on tonic GnRH release. The lactating rat model provided further evidence indicating that ARC metastin/kisspeptin neurons are involved in GnRH pulse generation, because pulsatile release of luteinizing hormone (LH) is profoundly suppressed by suckling stimulus and the LH pulse suppression is well associated with the suppression of ARC metastin/kisspeptin and KiSS-1 gene expression in lactating rats.     

Kisspeptin Signalling in the Hypothalamic Arcuate Nucleus Regulates GnRH Pulse Generator Frequency in the Rat

Background

Kisspeptin and its G protein-coupled receptor (GPR) 54 are essential for activation of the hypothalamo-pituitary-gonadal axis. In the rat, the kisspeptin neurons critical for gonadotropin secretion are located in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei. As the ARC is known to be the site of the gonadotropin-releasing hormone (GnRH) pulse generator we explored whether kisspeptin-GPR54 signalling in the ARC regulates GnRH pulses.

Methodology/Principal Findings

We examined the effects of kisspeptin-10 or a selective kisspeptin antagonist administration intra-ARC or intra-medial preoptic area (mPOA), (which includes the AVPV), on pulsatile luteinizing hormone (LH) secretion in the rat. Ovariectomized rats with subcutaneous 17β-estradiol capsules were chronically implanted with bilateral intra-ARC or intra-mPOA cannulae, or intra-cerebroventricular (icv) cannulae and intravenous catheters. Blood samples were collected every 5 min for 5–8 h for LH measurement. After 2 h of control blood sampling, kisspeptin-10 or kisspeptin antagonist was administered via pre-implanted cannulae. Intranuclear administration of kisspeptin-10 resulted in a dose-dependent increase in circulating levels of LH lasting approximately 1 h, before recovering to a normal pulsatile pattern of circulating LH. Both icv and intra-ARC administration of kisspeptin antagonist suppressed LH pulse frequency profoundly. However, intra-mPOA administration of kisspeptin antagonist did not affect pulsatile LH secretion.

Conclusions/Significance

These data are the first to identify the arcuate nucleus as a key site for kisspeptin modulation of LH pulse frequency, supporting the notion that kisspeptin-GPR54 signalling in this region of the mediobasal hypothalamus is a critical neural component of the hypothalamic GnRH pulse generator.     

Reproductive Hormone-Dependent and -Independent Contributions to Developmental Changes in Kisspeptin in GnRH-Deficient Hypogonadal Mice

Kisspeptin is a potent activator of GnRH-induced gonadotropin secretion and is a proposed central regulator of pubertal onset. In mice, there is a neuroanatomical separation of two discrete kisspeptin neuronal populations, which are sexually dimorphic and are believed to make distinct contributions to reproductive physiology. Within these kisspeptin neuron populations, Kiss1 expression is directly regulated by sex hormones, thereby confounding the roles of sex differences and early activational events that drive the establishment of kisspeptin neurons. In order to better understand sex steroid hormone-dependent and -independent effects on the maturation of kisspeptin neurons, hypogonadal (hpg) mice deficient in GnRH and its downstream effectors were used to determine changes in the developmental kisspeptin expression. In hpg mice, sex differences in Kiss1 mRNA levels and kisspeptin immunoreactivity, typically present at 30 days of age, were absent in the anteroventral periventricular nucleus (AVPV). Although immunoreactive kisspeptin increased from 10 to 30 days of age to levels intermediate between wild type (WT) females and males, corresponding increases in Kiss1 mRNA were not detected. In contrast, the hpg arcuate nucleus (ARC) demonstrated a 10-fold increase in Kiss1 mRNA between 10 and 30 days in both females and males, suggesting that the ARC is a significant center for sex steroid-independent pubertal kisspeptin expression. Interestingly, the normal positive feedback response of AVPV kisspeptin neurons to estrogen observed in WT mice was lost in hpg females, suggesting that exposure to reproductive hormones during development may contribute to the establishment of the ovulatory gonadotropin surge mechanism. Overall, these studies suggest that the onset of pubertal kisspeptin expression is not dependent on reproductive hormones, but that gonadal sex steroids critically shape the hypothalamic kisspeptin neuronal subpopulations to make distinct contributions to the activation and control of the reproductive hormone cascade at the time of puberty.     

A Population of Kisspeptin/Neurokinin B Neurons in the Arcuate Nucleus May Be the Central Target of the Male Effect Phenomenon in Goats

Exposure of females to a male pheromone accelerates pulsatile gonadotropin-releasing hormone (GnRH) secretion in goats. Recent evidence has suggested that neurons in the arcuate nucleus (ARC) containing kisspeptin and neurokinin B (NKB) play a pivotal role in the control of GnRH secretion. Therefore, we hypothesized that these neurons may be the central target of the male pheromone. To test this hypothesis, we examined whether NKB signaling is involved in the pheromone action, and whether ARC kisspeptin/NKB neurons receive input from the medial nucleus of the amygdala (MeA)—the nucleus suggested to relay pheromone signals. Ovariectomized goats were implanted with a recording electrode aimed at a population of ARC kisspeptin/NKB neurons, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA) volleys, was measured. Pheromone exposure induced an MUA volley and luteinizing hormone (LH) pulse in control animals, whereas the MUA and LH responses to the pheromone were completely suppressed by the treatment with an NKB receptor antagonist. These results indicate that NKB signaling is a prerequisite for pheromone action. In ovariectomized goats, an anterograde tracer was injected into the MeA, and possible connections between the MeA and ARC kisspeptin/NKB neurons were examined. Histochemical observations demonstrated that a subset of ARC kisspeptin/NKB neurons receive efferent projections from the MeA. These results suggest that the male pheromone signal is conveyed via the MeA to ARC kisspeptin neurons, wherein the signal stimulates GnRH pulse generator activity through an NKB signaling-mediated mechanism in goats.     

Sex steroid control of hypothalamic Kiss1 expression in sheep and rodents: comparative aspects

In recent years, the Kiss1 gene has been cast into the reproductive spotlight. In the short period since the discovered link between kisspeptins, the encoded peptides of Kiss1, and fertility, these peptides are now known to be critical for the neuroendocrine control of reproduction. Kisspeptin producing cells in the hypothalamus are poised to become the 'missing link' in the sex steroid feedback control of GnRH secretion. These cells contain all the necessary components to relay information of the sex steroid environment to GnRH neurons, which possess the kisspeptin receptor, GPR54. Sex steroids regulate Kiss1 mRNA, and kisspeptin expression in the hypothalamus, in a manner consistent with both negative and positive feedback control of GnRH. The precise nature of sex steroid effects, in particular those of estrogen, on Kiss1 expression have been extensively studied in the female rodent and ewe. In the arcuate nucleus (ARC) of both species, kisspeptin cells appear to forward signals pertinent to negative feedback regulation of GnRH, although in the ewe it appears this population of Kiss1 cell is also responsible for positive feedback regulation of GnRH at the time of the preovulatory GnRH/LH surge. In rodents, these positive feedback signals appear to be mediated by kisspeptin cells exclusively within the anteroventral periventricular nucleus (AVPV). There are no Kiss1 cells in the ovine AVPV, but there is a population in the preoptic area. The role these preoptic area cells play in the sex steroid feedback regulation of GnRH secretion, if any, is yet to be revealed.     

An eGFP-expressing subpopulation of growth hormone secretagogue receptor cells are distinct from kisspeptin,tyrosine hydroxylase,and RFamide-related peptide neurons in mice

Ghrelin acts on the growth hormone secretagogue receptor (GHSR) in the brain to elicit changes in physiological functions. It is associated with the neural control of appetite and metabolism, however central ghrelin also affects fertility. Central ghrelin injection in rats suppresses luteinizing hormone (LH) concentrations and pulse frequency. Although ghrelin suppresses LH and regulates kisspeptin mRNA in the anteroventral periventricular/periventricular nucleus (AVPV/PeN), there is no neuroanatomical evidence linking GHSR neural circuits to kisspeptin neurons. In this study, we first determined coexpression of GHSR and GnRH neurons using a GHSR-eGFP reporter mouse line. Using dual-label immunohistochemistry, we saw no coexpression. GHSR-eGFP expressing cells were present in the AVPV/PeN and over 90% of these expressed estrogen receptor-α (ERα). Despite this, we observed no evidence of GHSR-eGFP/kisspeptin coexpressing neurons in the AVPV/PeN. To further examine the phenotype of GHSR-eGFP cells in the AVPV/PeN, we determined coexpression with tyrosine hydroxylase (TH) and showed virtually no coexpression in the AVPV/PeN (<2%). We also observed no coexpression of GHSR-eGFP and RFamide-related peptide-3 (RFRP3) neurons in the dorsomedial hypothalamic nucleus. Importantly, we observed that approximately half of the GHSR-eGFP cells in the AVPV coexpressed Ghsr mRNA (as determined by in situ hybridization) so these data should be interpreted accordingly. Although ghrelin influences the hypothalamic reproductive axis, our data using a GHSR-eGFP reporter suggests ghrelin regulates neurons expressing ERα but does not directly act on GnRH, kisspeptin, TH, or RFRP3 neurons, as little or no GHSR-eGFP coexpression was observed.     

Comparative analysis of kisspeptin-immunoreactivity reveals genuine differences in the hypothalamic Kiss1 systems between rats and mice

Kiss1 mRNA and its corresponding peptide products, kisspeptins, are expressed in two restricted brain areas of rodents, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC). The concentration of mature kisspeptins may not directly correlate with Kiss1 mRNA levels, because mRNA translation and/or posttranslational modification, degradation, transportation and release of kisspeptins could be regulated independently of gene expression, and there may thus be differences in kisspeptin expression even in species with similar Kiss1 mRNA profiles. We measured and compared kisspeptin-immunoreactivity in both nuclei and both sexes of rats and mice and quantified kisspeptin-immunoreactive nerve fibers. We also determined Kiss1 mRNA levels and measured kisspeptin-immunoreactivity in colchicine pretreated rats. Overall, we find higher levels of kisspeptin-immunoreactivity in the mouse compared to the rat, independently of brain region and gender. In the female mouse AVPV high numbers of kisspeptin-immunoreactive neurons were present, while in the rat, the female AVPV displays a similar number of kisspeptin-immunoreactive neurons compared to the level of Kiss1 mRNA expressing cells, only after axonal transport inhibition. Interestingly, the density of kisspeptin innervation in the anterior periventricular area was higher in female compared to male in both species. Species differences in the ARC were evident, with the mouse ARC containing dense fibers, while the rat ARC contains clearly discernable cells. In addition, we show a marked sex difference in the ARC, with higher kisspeptin levels in females. These findings show that the translation of Kiss1 mRNA and/or the degradation/transportation/release of kisspeptins are different in mice and rats.     

新疆维汉民族多囊卵巢综合征患者CYP17基因多态性及与发病机制的关系

目的:探讨细胞色素P450酶17(CYP17)基因多态性及其与多囊卵巢综合征(PCOS)发病机制的关系。方法:选择2015年1月~2017年2月我院收治的新疆维汉民族PCOS患者260例为PCOS组,另选取同期在我院门诊检查的健康育龄妇女237例为对照组,采用限制性片段长度多态性聚合酶链反应(PCR-RFLP)技术检测两组受试者的CYP17基因多态性,比较两组等位基因及基因频率的分布,结合其临床资料分析CYP17基因多态性与PCOS发病机制的关系。结果:PCOS组体质量指数(BMI)、卵泡刺激素(FSH)水平低于对照组,黄体生成激素(LH)、睾酮(TES)及黄体生成激素与卵泡刺激素的比值(LH/FSH)高于对照组,差异有统计学意义(P0.05)。PCOS组CYP17基因A1A1、A1A2、A2A2型频率分别为34.62%、41.92%、23.46%,与对照组的34.18%、43.88%、21.94%比较差异无统计学意义(P0.05);PCOS组等位基因A1、A2频率为55.58%、44.42%,与对照组的56.12%、43.88%比较差异无统计学意义(P0.05);PCOS组不同CYP17基因型者的FSH、LH及LH/FSH水平整体比较差异无统计学意义(P0.05);PCOS组BMI水平A2A2A1A2A1A1,TES水平A2A2A1A1A1A2,差异均有统计学意义(均P0.05);对照组不同CYP17基因型者的BMI、FSH、LH、TES及LH/FSH水平整体比较差异均无统计学意义(P0.05)。结论:CYP17基因-34bp处T/C单核苷酸多态性是人群中一种常见的碱基置换,与PCOS的发病机制无明显的相关性。     

Normal or induced secretory patterns of luteinising hormone and follicle-stimulating hormone in anoestrous gonadotrophin-releasing hormone-immunised and cyclic control heifers

The objective was to determine the effect of gonadotrophin-releasing hormone (GnRH), GnRH analogue (GnRH-A) or oestradiol administration on luteinising hormone (LH) and follicle-stimulating hormone (FSH) release in GnRH-immunised anoestrous and control cyclic heifers. Thirty-two heifers (477 ± 7.1 kg) were immunised against either human serum albumin (HSA; controls; n = 8), or a HSAGnRH conjugate. On day 70 after primary immunisation, control heifers (n = 4 per treatment; day 3 of cycle) received either (a) 2.5 μg GnRH or (b) 2.5 μg of GnRH-A (Buserelin®) and GnRH-immunised heifers (blocked by GnRH antibody titre; n = 6 per treatment) received either (c) saline, (d) 2.5 μg GnRH, (e) 25 μg GnRH or (f) 2.5 μg GnRH-A, intravenously. On day 105, 1 mg oestradiol was injected (intramuscularly) into control (n = 6) and GnRH-immunised anoestrous heifers with either low (13.4 ± 1.9% binding at 1:640; n = 6) or high GnRH antibody titres (33.4 ± 4.8% binding; n = 6). Data were analysed by ANOVA. Mean plasma LH and FSH concentrations on day 69 were higher (P < 0.05) in control than in GnRH-immunised heifers (3.1 ± 0.16 vs. 2.5 ± 0.12 ng LH ml⁻¹ and 22.5 ± 0.73 vs. 17.1 ± 0.64 ng FSH ml⁻¹, respectively). The number of LH pulses was higher (P < 0.05) in control than in GnRH-immunised heifers on day 69 (3.4 ± 0.45 and 1.0 ± 0.26 pulses per 6 h, respectively). On day 70, 2.5 μg GnRH increased (P < 0.05) LH concentrations in control but not in GnRH-immunised heifers, while both 25 μg GnRH and 2.5 μg GnRH-A increased (P < 0.05) LH concentrations in GnRH-immunised heifers, and 2.5 μg GnRH-A increased LH in controls. FSH was increased (P < 0.05) in GnRH-immunised heifers following 25 μg GnRH and 2.5 μg GnRH-A. Oestradiol challenge increased (P < 0.05) LH concentrations during the 13–24 h period after challenge with a greater (P < 0.05) increase in control than in GnRH-immunised heifers. FSH concentrations were decreased (P < 0.05) for at least 30 h after oestradiol challenge. In conclusion, GnRH immunisation decreased LH pulsatility and mean LH and FSH concentrations. GnRH antibodies neutralised low doses of GnRH (2.5 μg), but not high doses of GnRH (25 μg) and GnRH-A (2.5 μg). GnRH immunisation decreased the rise in LH concentrations following oestradiol challenge.     

Quantification of Rat Kisspeptin Using a Novel Radioimmunoassay

Kisspeptin is a hypothalamic peptide hormone that plays a pivotal role in pubertal onset and reproductive function. Previous studies have examined hypothalamic kisspeptin mRNA expression, either through in situ hybridisation or real-time RT-PCR, as a means quantifying kisspeptin gene expression. However, mRNA expression levels are not always reflected in levels of the translated protein. Kisspeptin-immunoreactivity (IR) has been extensively examined using immunohistochemistry, enabling detection and localisation of kisspeptin perikaya in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). However, quantification of kisspeptin-IR remains challenging. We developed a specific rodent radioimmunoassay assay (RIA) capable of detecting and quantifying kisspeptin-IR in rodent tissues. The RIA uses kisspeptin-10 as a standard and radioactive tracer, combined with a commercially available antibody raised to the kisspeptin-10 fragment. Adult female wistar rat brain samples were sectioned at 300 µm and the ARC and AVPV punch micro-dissected. Brain punches were homogenised in extraction buffer and assayed with rodent kisspeptin-RIA. In accord with the pattern of kisspeptin mRNA expression, kisspeptin-IR was detected in both the ARC (47.1±6.2 fmol/punch, mean±SEM n = 15) and AVPV (7.6±1.3 fmol/punch, mean±SEM n = 15). Kisspeptin-IR was also detectable in rat placenta (1.26±0.15 fmol/mg). Reverse phase high pressure liquid chromatography analysis showed that hypothalamic kisspeptin-IR had the same elution profile as a synthetic rodent kisspeptin standard. A specific rodent kisspeptin-RIA will allow accurate quantification of kisspeptin peptide levels within specific tissues in rodent experimental models.