Modulation of GABAA Receptor Function by Alcohols: Effects of Subunit Composition and Differential Effects of Ethanol

We sought to test the hypotheses that closely related alcohols would have effects on GABA_A receptor function that were not predicted by differences in lipid solubility, and that the subunit structure of the GABA_A receptor would significantly affect the actions of different alcohols. Cloned subunits of human GABA_A receptors were expressed in Xenopus oocytes, and two-electrode voltage-clamp recording was used to quantify the membrane current response to GABA_A in the presence and absence of different alcohols. 1-Butanol and 2-butanol differentially potentiated the response to 20 μM GABA in oocytes expressing the α₁β₂γ_2L and α₂β₂γ_2L, receptor isoforms. In the α₁β₂γ_2L receptor construct, 1-butanol was more potent than 2-butanol to potentiate GABA, receptor function, but 2-butanol had a greater efficacy. In the α₂β₂ receptor construct, 1-butanol and 2-butanol were equipotent, but 2-butanol again had a greater efficacy. In the a2p2 receptor construct, both 1-butanol and 2-butanol produced large potentiations of the current response to 3 μM GABA. The efficacy for butanol potentiation of GABA responses in the absence of a γ_2L subunit was greater, but the potency was greatly reduced. Low concentrations (20 mM) of ethanol potentiated GABA responses in the α₁β₂γ_2L receptor construct. Ethanol potentiation of GABA_A receptor function was completely blocked by the benrodiazepine receptor partial inverse agonist R015-4513 at a concentration (0.5 μM) that did not alter the control GABA response. In contrast, R015-4513 did not block potentiation of GABA_A receptor activity induced by npropanol, 1-butanol, 2-butanol, 1-heptanol, or propofol (2,0-diisopropylphenol). These results suggest that alcohols have specific interactions with GABA_A receptors, and that ethanol may have unique effects not shared by other longer chain alcohols.     

Clinical outcomes from mature oocytes derived from preovulatory and antral follicles: reflections on follicle physiology and oocyte competence

Purpose

The aim of this retrospective study was to compare the competence of oocytes obtained from preovulatory and antral follicles.

Methods

Mature oocytes from preovulatory follicles were retrieved from women selected for standard IVF treatment (Group A). Mature oocytes from antral follicles were recovered from women undergoing hCG-primed in vitro maturation (IVM) treatment (Group B). Patients groups were matched for age, BMI, FSH, AMH and antral follicle count (AFC) values. In vivo matured oocytes from both groups were microinjected and resulting embryos were culture and selected on day 3 for embryo transfer.

Results

Oocyte pick-ups (OPU) were 315 and 204 in Groups A and B, respectively. Fertilization rates were comparable (72.8 and 75.9 %, respectively; P = 0.137). In Group A, in which the average number of embryos transferred was higher, clinical pregnancy rates per OPU (37.5 %) and embryo transfer (38.4 %) were superior in comparison to Group B (27.0 %, P = 0.013; 29.4 %, P = 0.041; respectively). On the other hand, implantation rates (Group A, 23.7 %; Group B, 20.8 %) and proportions of babies born per transferred embryo (Group A, 19.5 %; Group B, 16.9 %) were similar (P = 0.528 and 0.332, respectively).

Conclusions

Overall, this suggests that oocyte competence is already achieved at the antral stage of follicle development.     

Linking steroid hormone levels to sexual maturity index and energy reserves in Nereis diversicolor from clean and polluted estuaries

The objective of this work was to compare seasonal variations of reproduction physiology of the ragworm Nereis diversicolor --a key species in estuarine ecosystems--originating from a clean (Authie) and multi-polluted (Seine) estuaries. A particular attention was carried out in female worms, on relationships between sexual maturity stages, energy reserves (glycogen and lipids) and steroid hormone levels (progesterone, 17beta-estradiol, and testosterone). Sexual maturity index (SMI), energy reserves and steroid hormones are clearly influenced by season in worms from both sites. Depleted steroid hormone levels were depicted in specimens exhibiting high sexual maturity stage and energy reserves. Intersite analysis has revealed all over the sampling period:--a sexual precocity in worms from Seine,--glycogen concentrations generally higher in worms from Authie,--no clear tendency for lipids,--no differences in steroid hormone levels. Sexual precocity and lower glycogen levels in Seine could be explained by a specific strategy above all devoted to reproduction in these worms. Chemical stress could be a possible explanation of these observations.     

采用新鲜和冻融精子进行卵母细胞胞质内单精子注射的临床效果比较

目的 比较采用新鲜和冻融的睾丸及附睾精子进行卵母细胞胞质内单精子注射(ICSI)的临床效果.方法 选择2006年9月-2007年5月因无精症于北京大学第三医院生殖医学中心行ICSI的患者208例,按患者意愿分为冻融组37例和新鲜组171例.冻融组在行ICSI前将冻存的睾丸或附睾精子解冻并复苏.观察冻融组睾丸和附睾精子的临床利用率;比较两组患者的临床结局(包括正常受精率、优质胚胎率、临床妊娠率及胚胎着床率等)和妊娠结局(包括流产率、分娩孕周及新生儿出生体重等).结果 (1)冻融组睾丸精子的临床利用率为92%(23/25),附睾精子为100%(12/12).(2)新鲜组患者的正常受精率、优质胚胎率、临床妊娠率及胚胎着床率分别为62.25%(973/1563)、78.9%(768/973)、44.4%(60/135)和29.3%(84/287),分别与冻融组[分别为64.53%(282/437)、79.1%(223/282)、46.9%(15/32)和33.3%(23/69)]比较,差异均无统计学意义(P＞0.05).(3)新鲜组患者的流产率、单胎妊娠分娩孕周、双胎妊娠分娩孕周、单胎妊娠平均新生儿出生体重及双胎妊娠平均新生儿出生体重分别为11%(6/55)、(39.0±1.4)周、(36.8±1.7)周、(3409±393)g和(2584±266)g,分别与冻融组[分别为7%(1/15)、(38.7±0.6)周、(36.3±1.2)周、(3350±383)g和(2635±171)g]比较,差异均无统计学意义(P＞0.05).结论 采用冻融的睾丸或附睾精子行ICSI安全、有效,值得在临床推广.     

孕酮对子宫内膜、卵细胞及胚胎发育的影响

孕酮是一种女性激素,在人类生殖中占有重要地位,可作用于卵巢及子宫,对卵细胞的发育成熟、排卵、胚胎发育、胚胎着床、维持妊娠等具有重要作用。研究认为孕酮分泌不足可能是妊娠早期流产的原因之一。孕酮通过调节子宫内膜的容受性,为胚胎的生长发育提供一个适宜的环境,同时通过调节黄体生成激素(LH)峰及其受体以及对优势卵泡发育的作用来影响卵细胞的成熟及质量。此外胚胎本身的发育也受到孕酮的作用,在体外培养胚胎时添加孕酮能提高囊胚的形成率及植入率。虽然目前对孕酮的药代学及药效学已研究得十分透彻,其人工合成制剂已经投入临床使用,但对其病理生理机制尚存在争议。综述孕酮对子宫内膜、卵细胞及胚胎发育的影响。     

卵母细胞体外成熟技术的临床应用进展

卵母细胞体外成熟(in vitro maturation,IVM)是将处于不同时期的未成熟卵母细胞体外培养成熟的技术,临床不同来源的未成熟卵母细胞在胚胎发育潜能、妊娠率和活产率等方面存在差异。IVM作为一种有效治疗手段可应用于治疗多囊卵巢综合征、卵巢高反应及低反应患者、癌症患者生育力保存等,该技术已在全世界应用并有数千名健康婴儿出生。临床IVM技术的改进主要包括IVM培养基的改良、培养环境及操作流程的优化。目前,随着体外受精(IVF)效率的提高和培养体系的改进,自然周期或温和刺激可能更适合于进行IVF治疗的女性。新的治疗方案提出将自然周期/温和刺激IVF与IVM结合,温和刺激IVF联合IVM治疗方案不仅有望成为目前标准治疗的可行替代方案,而且可能是潜在的一线治疗选择。     

外泌体与生殖细胞生长发育的研究进展

卵母细胞和精子生长发育都受局部微环境中细胞因子的调控。在卵泡局部存在血-卵屏障,睾丸局部存在血-睾屏障,生殖细胞所在微环境是如何与机体进行信号转导和物质交换,这一过程尚不清楚。外泌体作为一种体液中普遍存在的膜性囊泡,可以参与细胞间的信号转导和物质交换,介导免疫耐受和促进肿瘤的发生发展等一系列的生理和病理活动,在卵泡或睾丸局部发挥重要的作用。卵巢来源的外泌体在卵泡的发生发展过程中具有一定的作用。在体外储存期间,精液中的外泌体对于维持精子的功能具有重要的作用。关于外泌体的研究是生殖医学中的一个新的领域,将会为生殖医学带来一个新的契机。     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes: role of the α subunit in agonist sensitivity and desensitization

Neuronal nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus laevis oocytes after nuclear injection of complementary deoxyribonucleic acid (cDNA) expression vectors. The two receptor subtypes 4/n1 and 3/n1 were readily distinguishable from one another by ACh sensitivity and desensitization. 3/n1 receptors showed lower ACh sensitivity and stronger desensitization than 4/n1 receptors. Furthermore, although the current/voltage relationship was very similar in both receptor subtypes, the voltage dependence of desensitization was found to be strikingly different. As the n1 subunit was unchanged, the subunits must be responsible for these functional differences. Symmetric hybrid cDNAs, 43 and 34, were constructed and functional receptors were obtained by co-injection with n1. These hybrid receptors displayed an ACh sensitivity that was mainly defined by the extracellular sequence of the subunit. In contrast, no part of the subunit was found fully to determine desensitization.     

小鼠卵母细胞的OPS法玻璃化冷冻保存技术

目的以小鼠卵母细胞为模型,利用开放式拉长细管(open pulled straw,OPS)法研究在不同条件下冷冻、解冻对其发育能力的影响,为家畜、濒危动物乃至人类探索一种简易、高效的卵母细胞冷冻保存提供理论与技术参考。方法在室温(25℃±0.5℃)条件下,以乙二醇(EG)、DMSO为主体抗冻保护剂配制成的玻璃化溶液(EFS、EDFS),采用OPS法对卵母细胞进行玻璃化冷冻保存。实验一:操作台温度分别为25℃±0.5℃和37℃±0.5℃时,卵母细胞在10%EG 10%DMSO或10%EG溶液中预处理30 s,移入EDFS或EFS中平衡15 s~45 s进行冷冻保存;实验二:操作台为37℃±0.5℃条件下,采用三种方法(卵母细胞在A:0.5 mol/L和0.3 mol/L的蔗糖中分别处理2 min和5 min;B:0.3 mol/L和0.15 mol/L的蔗糖中分别处理5 min和2 min;C:0.5 mol/L的蔗糖处理5 min)对EDFS30和EDFS40冷冻保存的卵母细胞进行解冻;实验三:解冻后的卵母细胞在SrCl2溶液中进行孤雌激活。结果与结论实验一中卵母细胞解冻后的形态正常率分别高达92.1%(25℃±0.5℃)和92.5%(37℃±0.5℃),均与对照组(98.9%)无显著性差异(P>0.05);实验二中卵母细胞解冻以C法效果最佳(形态正常率分别为92.5%和67.5%);实验三中解冻后在mCZB中培养0.5 h的卵母细胞激活效果优于未培养组(卵裂率和囊胚发育率分别为80.0%vs.57.7%;18.2%vs.0),均有显著或极显著差异(P<0.05或P<0.01);而培养组与新鲜对照组卵母细胞激活后卵裂率和桑椹胚发育率与对照组(80.0%vs.77.8%;52.8%vs.61.9%)均无显著性差异(P>0.05)。     

人窦前卵泡的体外发育及其卵母细胞成熟过程的观察

目的探讨在体外培养条件下,人窦前卵泡发育及其卵母细胞成熟的可能性。方法建立适宜的体外培养环境,应用正交设计将不同剂量的人绝经期促性腺激素（ｈＭＧ）、卵泡液、表皮生长因子（ＥＧＦ）组合,作用于来自体外受精－胚胎移植（ＩＶＦ－ＥＴ）抽吸的卵泡液及冲洗液中获得的窦前卵泡。结果窦前卵泡于培养第６～１２天形成窦卵泡,培养２１～２８天时直径≥３００μｍ的卵泡数显著增多（Ｐ＜０．００５）,当以ｈＭＧ１５０ＩＵ／Ｌ、卵泡液４０％、ＥＧＦ６μｇ／Ｌ作用于卵泡时,卵母细胞排出率与再经培养后成熟率均达最高,分别为８３％和３８％（Ｐ＜０．００５）。结论在适宜的培养条件下,来自ＩＶＦ－ＥＴ病人的抽吸卵泡液的窦前卵泡在体外能够发育,且其卵母细胞可达到成熟,是一新的人卵来源。窦前卵泡的体外发育和体内不同