Germinal stage vitrification is superior to MII stage vitrification in prepubertal mouse oocytes

This study investigated if in vitro maturation (IVM) before or after vitrification would be more successful for prepubertal oocytes. To mimic prepubertal conditions in an experimental setup, oocytes were collected from healthy 14, 21 and 28day old Swiss albino mice. The germinal vesicle (GV) stage oocytes and in vitro matured MII oocytes were subjected to vitrification-warming. Both structural (meiotic spindle morphology, mitochondrial integrity, cortical granules) and functional (sperm zona binding, fertilization) characteristics were assessed in oocytes after warming. This study demonstrated that IVM was more detrimental to prepubertal oocytes than to young adults. Further, vitrification of the IVM oocytes resulted in an increase in the number of abnormal meiotic spindles, a change in the cortical distribution pattern, a reduction in sperm zona binding and the fertilization rate. Importantly, oocyte integrity was better when prepubertal oocytes were vitrified before, rather than after, IVM. The above observations support GV stage vitrification for prepubertal oocytes requiring fertility preservation. Understanding the mechanisms behind the differing outcomes for oocytes from immature females will help in refining current protocol, thereby retaining the oocytes' maximum structural and functional integrity Further investigation is necessary to determine whether human prepubertal oocytes also behave in a similar way. It is to be noted here, with great emphasis, that a major limitation of this study is that the oocytes’ abilities were tested only until fertilisation, as a consequence of which the study cannot reveal the developmental potentials of the embryos beyond fertilisation.     

Ultrastructure of bovine oocytes exposed to Taxol prior to OPS vitrification

Our objective was to document potential subcellular consequences of treatment with the microtubule stabilizer Taxol with or without subsequent vitrification of cow and calf oocytes by the open pulled straw (OPS) method. Oocytes were divided into four experimental groups for cows and four groups for calves: (1) a control group fixed immediately after maturation; (2) an OPS group cryopreserved by conventional OPS; (3) a Taxol/CPA group exposed to 1 microM Taxol and cryoprotective agents (CPAs); and (4) a Taxol/OPS group vitrified by OPS including 1 microM Taxol to the vitrification solution. All oocytes were processed for light and transmission electron microscopy. The main injuries were observed on the metaphase plate and the spindle. In control oocytes, the metaphase appeared as condensed chromosomes arranged in a well-organized metaphase plate and the spindle showed well organized microtubules in both cow and calf oocytes. However, in cow OPS oocytes, the metaphase plate was disorganized into scattered chromosomes or the chromosomes were condensed into a single block of chromatin. In addition, microtubules were not organized as typical spindles. In contrast, cow Taxol/OPS oocytes as well as both cow and calf Taxol/CPAs oocytes showed well-organized metaphase plates and normal spindle morphology. All calf OPS and calf Taxol/OPS oocytes displayed a single block of chromatin and no microtubules could be observed around the chromosomes. In conclusion, treatment with 1 microM Taxol before and during vitrification did not induce adverse changes in the oocyte cytoplasm or metaphase spindles in adult bovine oocytes, but stabilized the metaphase and spindle morphology.     

From the first protein structures to our current knowledge of protein folding: delights and scepticisms

Every breakthrough that opens new vistas also removes the ground from under the feet of other scientists. The scientific joy of those who have seen the new light is accompanied by the dismay of those whose way of life has been changed for ever. The publication of the first structures of proteins at atomic resolution 50 years ago astounded and inspired scientists in every field, but caused others to flee or scoff. That advance and every subsequent paradigm-shifting breakthrough in protein science have met with some resistance before universal acceptance. I relate these events and their impact on the field of protein folding.     

关注生命预防自杀

随着人类社会的文明和进步，人们清晰地认识到自杀是严重影响经济和社会的重大公共卫生问题，世界卫生组织确定每年的9月10日为“世界预防自杀日”，旨在唤醒全社会来重视自杀行为．及时建立自杀干预机制，预防和减少人类的自杀。进入高自杀率的中国，更应重视自杀原因的分析．干预机制的探索，广泛宣传，让全社会来关注生命，预防自杀，从而持续降低自杀行为的发生频率。     

From natural products discovery to commercialization: a success story

In order for a natural product to become a commercial reality, laboratory improvement of its production process is a necessity since titers produced by wild strains could never compete with the power of synthetic chemistry. Strain improvement by mutagenesis has been a major success. It has mainly been carried out by “brute force” screening or selection, but modern genetic technologies have entered the scene in recent years. For every new strain developed genetically, there is further opportunity to raise titers by medium modifications. Of major interest has been the nutritional control by induction, as well as inhibition and repression by sources of carbon, nitrogen, phosphate and end products. Both strain improvement and nutritional modification contribute to the new process, which is then scaled up by biochemical engineers into pilot scale and later into factory size fermentors.     

Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method

Vitrification is frequently referred to as a novel technology of cryopreservation in embryology, although some young embryologists were born after its first successful application. Unfortunately, in spite of the accumulated evidence regarding its enormous potential value, most domestic animal and human laboratories use exclusively the traditional slow-rate freezing with its compromised efficiency and inconsistency. The purpose of this paper is to clarify terms and conditions, to summarize arguments supporting or disapproving the use of vitrification, and to outline its role among assisted reproductive technologies. To provide evidence for the potential significance of vitrification, achievements with the Cryotop technology, an advanced version of the "minimal volume approaches" is analyzed. This technology alone has resulted in more healthy babies after cryopreservation of blastocysts than any other vitrification technique, and more successful human oocyte vitrification resulting in normal births than any other cryopreservation method. The value of this method is also demonstrated by achievements in the field of domestic animal embryology. A modification of the technique using a hermetically sealed container for storage may help to eliminate potential dangers of disease transmission and open the way for widespread application for cryopreservation at all phases of oocyte and preimplantation embryo development in mammals.     

Stability of the cytoskeleton of matured buffalo oocytes pretreated with cytochalasin B prior to vitrification

Stabilizing the cytoskeleton system during vitrification can improve the post-thaw survival and development of vitrified oocytes. The cytoskeleton stabilizer cytochalasin B (CB) has been used in cryopreservation to improve the developmental competence of vitrified oocytes. To assess the effect of pretreating matured buffalo oocytes with CB before vitrification, we applied 0, 4, 8, or 12 μg/mL CB for 30 min. The optimum concentration of CB treatment (8 μg/mL for 30 min) was then used to evaluate the distribution of microtubules and microfilaments, the expression of the cytoskeleton proteins actin and tubulin, and the developmental potential of matured oocytes that were vitrified-warmed by the Cryotop method. Western blotting demonstrated that vitrification significantly decreased tubulin expression, but that the decrease was attenuated for oocytes pretreated with 8 μg/mL CB before vitrification. After warming and intracytoplasmic sperm injection, oocytes that were pretreated with 8 μg/mL CB before vitrification yielded significantly higher 8-cell and blastocyst rates than those that were vitrified without CB pretreatment. The values for the vitrified groups in all experiments were significantly lower (P < 0.01) than those of the control groups. In conclusion, pretreatment with 8 μg/mL CB for 30 min significantly improves the cytoskeletal structure, expression of tubulin, and development capacity of vitrified matured buffalo oocytes.     

Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling

This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84 ± 5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol + 0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol + 15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV + MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium (p = 0.01) and vitrification (p < 0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification (p < 0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.     

Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling

This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84 ± 5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol + 0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol + 15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV + MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium (p = 0.01) and vitrification (p < 0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification (p < 0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.     

Effects of pre-treating in vitro-matured bovine oocytes with the cytoskeleton stabilizing agent taxol prior to vitrification

The purpose of this study was to determine the efficacy of pre-treating mature bovine oocytes with Taxol before vitrification by the open pulled Straw method (OPS). We evaluated the effects of pre-treating the oocytes with 1 microM Taxol on chromosome organization, spindle morphology, cortical granule distribution and the ability of fertilized oocytes to develop to the blastocyst stage. After calf or cow oocyte vitrification without Taxol, significantly higher proportions of spindle abnormalities in the form of abnormal spindle structures or dispersed or decondensed chromosomes were observed compared to fresh control oocytes. In contrast, when we compared calf oocytes pre-treated with Taxol before vitrification with control calf oocytes, similar percentages of oocytes showing a normal spindle morphology were observed. The percentages of oocytes with a peripheral cortical granule (CG) distribution increased when the oocytes were pretreated with Taxol and vitrified, while oocytes vitrified without Taxol pre-treatment gave rise to higher cortical distribution percentages. Cleavage and blastocyst rates were significantly lower for vitrified versus untreated oocytes, both in cow and calf oocytes. Significantly higher cleavage rates were obtained when calf and cow oocytes were vitrified with Taxol. Pre-treatment with Taxol before cow oocyte vitrification yielded significantly higher blastocyst rates. Calf oocytes, however, were unable to develop to the blastocyst stage, irrespective of previous Taxol treatment. These results indicate that the pre-treatment of oocytes with Taxol before vitrification helps to reduce the damage induced by the cryopreservation process, and potentially improves the subsequent development of vitrified bovine oocytes. Summary sentence: Pre-treatment of oocytes with Taxol before vitrification helps to reduce the damage induced by vitrification and potentially improves the development of vitrified bovine oocytes.     

Transgenic RNAi in mouse oocytes: The first decade

RNA interference (RNAi), a sequence-specific mRNA degradation induced by double-stranded RNA (dsRNA), is a common approach employed to specifically silence genes. Experimental RNAi in plant and invertebrate models is frequently induced by long dsRNA. However, in mammals, short RNA molecules are used preferentially since long dsRNA can provoke sequence-independent type I interferon response. A notable exception are mammalian oocytes where the interferon response is suppressed and long dsRNA is a potent and specific trigger of RNAi. Transgenic RNAi is an adaptation of RNAi allowing for inducing sequence-specific silencing upon expression of dsRNA. A decade ago, we have developed a vector for oocyte-specific expression of dsRNA, which has been used to study gene function in mouse oocytes on numerous occasions. This review provides an overview and discusses benefits and drawbacks encountered by us and our colleagues while working with the oocytes-specific transgenic RNAi system.     

Nonthermal electromagnetic fields: From first messenger to therapeutic applications

Nonthermal pulsed electromagnetic fields, from low frequency to pulse-modulated radio frequency, have been successfully employed as adjunctive therapy for the treatment of delayed and non-union fractures, fresh fractures and chronic wounds. Recent increased understanding of the mechanism of action of electromagnetic fields (EMF) has permitted technologic advances allowing the development of EMF devices which are portable and disposable, can be incorporated into dressings, supports and casts, and can be used over clothing. This broadens the use of non-pharmacological, non-invasive EMF therapy to the treatment of postoperative pain and edema to enhance surgical recovery. EMF therapy is rapidly becoming a standard part of surgical care, and new, more significant, clinical applications for osteoarthritis, brain and cardiac ischemia and traumatic brain injury are in the pipeline. This study reviews recent evidence which suggests that calmodulin (CaM)-dependent nitric oxide signaling is involved in cell and tissue response to weak nonthermal EMF signals. There is abundant evidence that EMF signals can be configured a priori to increase the rate of CaM activation, which, in turn, can modulate the biochemical cascades living cells and tissues employ in response to external insult. Successful applications in pilot clinical trials, coupled with evidence at the cellular and animal levels, provide support that EMF is a first messenger that can modulate the response of challenged biological systems.