Novel properties of the depolarization-induced endogenous sodium conductance in the Xenopus laevis oocyte

 It has been shown by means of the two-microelectrode voltage-clamp technique that in membranes of Xenopus laevis oocytes a Na⁺-selective permeability can be activated by long-lasting or repetitive depolarization (R.T. Kado and C. Baud, Journal of Physiology, Paris, 77:1113–1117, 1981). In this study the permeability in inside-out giant membrane patches with diameters of 20–30 μm was analysed. Once induced, the Na⁺ permeability has a voltage-dependent open probability that increases with positive potentials and half-maximally activates at about 0 mV. Sudden changes of membrane potential elicit transient currents with strongly voltage-dependent time constants of from less than 1 ms at –150 mV to several hundreds of milliseconds at positive potentials. In contrast to the on-cell configuration, the permeability ceases completely within a few minutes in the cell-free inside-out configuration. This rundown can be prevented by including MgATP, but not Mg²⁺ or ATP alone, in the intracellular solution. Intracellular Mg²⁺ ions, in addition to being a co-factor for ATP in the activation process, decrease the permeability in a dose-dependent manner. Steady-state fluctuation analysis gave no evidence that an increased noise level is caused by open–close kinetics of an ion channel, suggesting that the single-channel conductance is below 1 pS if a channel-like structure is the origin of the endogenous Na⁺ permeability. Received: 14 July 1998 / Received after revision: 4 November 1998 / Accepted: 18 January 1999     

Molecular basis for pharmacological differences between brain and cardiac sodium channels

Sodium channels from brain and heart, whose primary structures are known, differ in their sensitivity to block by the guadinium toxins tetrodotoxin and saxitoxin and to block by external Zn²⁺ and Cd²⁺. Studies using site-directed mutagenesis have identified the SS2 and adjacent regions of all four repeats as critical determinants for toxin sensitivity. Within and in the immediate vicinities of the SS2 segments, there are only two amino-acid differences between rat brain sodium channel II and rat heart I sodium channel, both located in repeat I. Here we show that replacement of phenylalanine 385 of brain sodium channel by cysteine that is present at the equivalent position in heart channel (F385C) not only reduces sensitivity to the guadinium toxins but also increases sensitivity to Zn²⁺ and Cd²⁺, thus conferring properties of heart sodium channel on brain sodium channel. Replacement of asparagine at the second non-conserved position by arginine (N388R) only marginally affects sensitivity to the toxins, Zn²⁺ or Cd²⁺, but this mutation markedly reduces sensitivity to block by Ca²⁺ and Co²⁺. The double mutant channel (F385CN388R) shows combined properties of the two mutant channels. These results give a structural insight into the different properties of the two channel proteins.     

Oocyte cryopreservation beyond cancer: tools for ethical reflection

Purpose

This article offers physicians a tool for structured ethical reflection on challenging situations surrounding oocyte cryopreservation in young healthy women.

Methods

A systematic literature review offers a comprehensive overview of the ethical debate surrounding the practice. Ethical Counseling Methodology (ECM) offers a practical approach for addressing ethical uncertainties. ECM consists of seven steps: (i) case presentation; (ii) analysis of possible implications; (iii) presentation of ethical question(s); (iv) explanation of ethical terms; (v) presentation of the ethical arguments in favor of and against the procedure; (vi) examination of the individual patient’s beliefs and wishes; and (vii) conclusive summary.

Results

The most problematic aspects in the ethical debate include the distinction between medical and non-medical use of oocyte cryopreservation, safety and efficiency of the procedure, and marketing practices aimed at healthy women. Female empowerment and enhanced reproductive choices (granted oocyte cryopreservation is a safe and efficient technique) are presented as ethical arguments supporting the practice, while ethical reservations towards oocyte cryopreservation are based on concerns about maternal and fetal safety and wider societal implications.

Conclusions

Oocyte cryopreservation is gaining popularity among healthy reproductive age women. However, despite promised benefits it also involves risks that are not always properly communicated in commercialized settings. ECM offers clinicians a tool for structured ethical analysis taking into consideration a wide range of implications, various ethical standpoints, and patients’ perceptions and beliefs.     

In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising “ex vivo” method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe

Purpose

We present our center’s experience with 34 consecutive cases who underwent in vitro maturation (IVM) of oocytes obtained from ovariectomy specimens and compare our data with updated literature data.

Methods

Feasibility and efficiency of oocyte collection during ovarian tissue processing was assessed by the recovery rate, maturation rate, and embryological development after IVM.

Results

On average, 14 immature oocytes were retrieved per patient during ovarian tissue processing in 33/34 patients. The overall maturation rate after IVM was 36 %. The maturation rate correlated with the age of the patient and the duration of IVM. Predominately, oocyte vitrification was performed. Eight couples preferred embryo cryopreservation. Here, a 65 % fertilization rate was obtained and at least one good-quality day 3 embryo was cryopreserved in 7/8 couples. The retrieval of oocytes ex vivo resulted in mature oocytes or embryos available for vitrification in 79 % of patients. One patient with ovarian insufficiency following therapeutic embolization of the left uterine and the right ovarian artery because of an arteriovenous malformation had an embryo transfer of one good-quality warmed embryo generated after IVM ex vivo, which resulted in an ongoing clinical pregnancy.

Conclusions

IVM of oocytes obtained ex vivo during the processing of ovarian cortex prior to cryopreservation is a procedure with emerging promise for patients at risk for fertility loss, as illustrated by the reported pregnancy. However, more data are needed in order to estimate the overall success rate and safety of this novel approach.     

Downregulation of gene expression and activity of GRIM-19 affects mouse oocyte viability,maturation, embryo development and implantation

PurposeTo investigate the expression of GRIM-19 (Gene associated with retinoid-interferon-induced mortality 19) in mouse oocytes and preimplantation embryos, and to study the effect of GRIM-19 on the developmental competence of mouse oocytes and embryos.MethodsGRIM-19 was evaluated at both mRNA and protein levels. The expression of GRIM-19 gene was downregulated in mouse oocytes cultured in vitro by specific small interfering RNA (siRNA) injection, while the activity of GRIM-19 was decreased by microinjection of a GRIM-19 antibody into the cytoplasm of germinal vesicle (GV) oocytes. Oocytes matured in vitro were then fertilized by intracytoplasmic sperm injection (ICSI), followed by observation and evaluation of fertilization rate, cleavage rate, blastocyst formation rate and implantation rate.ResultsGRIM-19 is expressed throughout oocyte maturation and preimplantation embryo development stages. GRIM-19 was localized primarily in the cytoplasm of all cells examined. Downregulation of gene expression and activity of GRIM-19 resulted in decreased oocyte viability, potency of oocyte maturation, embryo development and implantation.ConclusionsGRIM-19 may play important roles in mouse oogenesis and early embryonic development and implantation.     

A novel strategy for targeted killing of tumor cells: Induction of multipolar acentrosomal mitotic spindles with a quinazolinone derivative mdivi‐1

Traditional antimitotic drugs for cancer chemotherapy often have undesired toxicities to healthy tissues, limiting their clinical application. Developing novel agents that specifically target tumor cell mitosis is needed to minimize the toxicity and improve the efficacy of this class of anticancer drugs. We discovered that mdivi‐1 (mitochondrial division inhibitor‐1), which was originally reported as an inhibitor of mitochondrial fission protein Drp1, specifically disrupts M phase cell cycle progression only in human tumor cells, but not in non‐transformed fibroblasts or epithelial cells. The antimitotic effect of mdivi‐1 is Drp1 independent, as mdivi‐1 induces M phase abnormalities in both Drp1 wild‐type and Drp1 knockout SV40‐immortalized/transformed MEF cells. We also identified that the tumor transformation process required for the antimitotic effect of mdivi‐1 is downstream of SV40 large T and small t antigens, but not hTERT‐mediated immortalization. Mdivi‐1 induces multipolar mitotic spindles in tumor cells regardless of their centrosome numbers. Acentrosomal spindle poles, which do not contain the bona‐fide centrosome components γ‐tubulin and centrin‐2, were found to contribute to the spindle multipolarity induced by mdivi‐1. Gene expression profiling revealed that the genes involved in oocyte meiosis and assembly of acentrosomal microtubules are highly expressed in tumor cells. We further identified that tumor cells have enhanced activity in the nucleation and assembly of acentrosomal kinetochore‐attaching microtubules. Mdivi‐1 inhibited the integration of acentrosomal microtubule‐organizing centers into centrosomal asters, resulting in the development of acentrosomal mitotic spindles preferentially in tumor cells. The formation of multipolar acentrosomal spindles leads to gross genome instability and Bax/Bak‐dependent apoptosis. Taken together, our studies indicate that inducing multipolar spindles composing of acentrosomal poles in mitosis could achieve tumor‐specific antimitotic effect, and mdivi‐1 thus represents a novel class of compounds as acentrosomal spindle inducers (ASI).     

Molecular basis of reproductive senescence: insights from model organisms

PurposeReproductive decline due to parental age has become a major barrier to fertility as couples have delayed having offspring into their thirties and forties. Advanced parental age is also associated with increased incidence of neurological and cardiovascular disease in offspring. Thus, elucidating the etiology of reproductive decline is of clinical importance.MethodsDeciphering the underlying processes that drive reproductive decline is particularly challenging in women in whom a discrete oocyte pool is established during embryogenesis and may remain dormant for tens of years. Instead, our understanding of the processes that drive reproductive senescence has emerged from studies in model organisms, both vertebrate and invertebrate, that are the focus of this literature review.ConclusionsStudies of reproductive aging in model organisms not only have revealed the detrimental cellular changes that occur with age but also are helping identify major regulator proteins controlling them. Here, we discuss what we have learned from model organisms with respect to the molecular mechanisms that maintain both genome integrity and oocyte quality.     

辅助生殖技术取卵前医患认知差异调查分析及对策

目的了解辅助生殖技术取卵前医护人员与患者心理状况、健康知识需求的认知差异,调整健康教育方式和内容。方法利用自行设计的调查表,对2012年7～8月在我院生殖中心取卵的92例患者与生殖中心49例医护人员进行调查,内容包括一般情况、患者术前、术后、对辅助生殖技术的认知情况等七个方面共37个条目。结果患者与医护人员对取卵失败增加经济负担、取卵较多发生卵巢过度刺激综合征、取卵术后什么时候可以洗澡、胚胎质量、取卵后影响卵巢加速变老、取卵后影响性生活、取卵失败引起家庭的矛盾7项内容认知,差异有统计学意义（均P〈0．05）。结论患者在取卵前的心理状况与医护人员的认知存在差异,医护人员应在术前调整健康教育的内容,针对患者担心的问题进行宣教,消除其焦虑心理,保证手术顺利进行和康复。     

经阴道穿刺取卵术中不同镇痛方法的应用效果比较

目的探讨双氯芬酸钠栓与丙泊酚复合地佐辛在经阴道穿刺取卵术中的镇痛效果。方法 2013年2-4月行取卵术的387例患者根据用药情况分为双氯芬酸钠栓组226例（A组）和丙泊酚复合地佐辛组161例（B组）。观察并记录两组在取卵术中的镇痛效果及术后头晕、恶心、呕吐等不良反应情况。结果 B组和A组患者取卵术中镇痛有效率分别为100.00%、99.14%,两者比较差异无统计学意义（χ^2=0.228,P=0.633）。B组患者的头晕发生率、恶心发生率、呕吐发生率、注射部位疼痛发生率和呼吸抑制发生率均较高,与A组比较差异均有显著统计学意义（P〈0.01）。结论经阴道取卵术中使用双氯芬酸钠栓镇痛效果好,不良反应发生率低,对妊娠无影响,使用方便、安全,值得临床推广使用。     

世纪之交展望试管婴儿科技工作

简单回顾了我国大陆体外受精 胚胎移植（ＩＶＦ ＥＴ）技术的建立，发展和成就。自我国大陆首例试管婴儿于１９８８年３月１０日在北京医科大学诞生以来，为了病人需要，已有１０多个ＩＶＦ中心在我国各省、市大医院成立，特别在近３～４年来，工作进展迅速。２１世纪世界发展趋势提示今后ＩＶＦ研究将有广阔前景。