Evolution of the sperm aster after microinjection of isolated human sperm centrosomes into meiotically mature human oocytes

This study examined the feasibility of isolating and transferringthe centrosome-containing region of spermatozoa to mature humanoocytes. The findings demonstrate that individual sperm centrosomescan be transferred and are capable of nucleating maternal tubulinto form a well-developed sperm aster in the recipient oocyte.The results are discussed with respect to centrosome functionin early human development and applications in clinical in-vitrofertilization in the treatment of certain forms of male factorinfertility.     

MISE AU POINT D'UNE TECHNIQUE D'ADMINISTRATION IN VIVO DES VECTEURS GENETIQUES DANS LEMUSCLE CARDIAQUE

Objective In order to improve the in vivo gene transfer into the heart muscle, we have designed a ECG-synchronized microinjection system that allows sequential gene delivery to the myocardium.Methods A cannula was introduced into the right carotid artery of the Wistar rat under general anesthesia.With the ECG-synchronized injection during diastole, the genetic vector (Ad CMV lacZ ) infusion was performed with various concentrations( l07 ～ l010pfu ) and different frequency ( the ratio of heart beats per injection from 1: 1 to 4: 1 ). The hearts of the rats were removed after 7 days for histological examination. Results Best results were obtained with a total vector amount of l09 pfu and a good ratio 3: 1 between heart frequency and injection frequency. The transfection efficiency was increased by use of vasodilators and by an increase of vascular permeability. No signs of myocardial ischemia or ventricular arrythmia were observed. Conclusion We have established a novel and safe method for in vivo gene transfer into the heart. Transgene expression suggests that this method may be useful technique to study cardiac function of treat cardiac diseases by means of gene theratpy.     

Computer image sperm selection as a novel approach to subzonal insemination in the human

Utilizing real-time computer image analysis, individual spermatozoawere selected using microaspiration. Selection criteria werebased on potential hyperactivation motility characteristics;the amplitude of lateral head displacement >7.5 µm,curvilinear velocity >70 µm/s and linearity of <30%.For this pilot study, 16 patients (eight in each group) wererecruited. Using subzonal insemination (SUZI), up to five (mean= 4.4 ± 0.3) spermatozoa selected using computer-imagesperm selection (CISS) were microinjected, or up to 15 (mean= 12.8 ± 1.3 SD) unselected spermatozoa. In the groupwhich utilized CISS, 28 out of 49 (57%) oocytes were fertilizedcompared with 13 out of 52 (25%) utilizing conventional SUZI(P < 0.04); polyspermy was 20% (n = 10) and 2% (n = 1) respectively.CISS with SUZI showed increased efficiency in achieving fertilizationand is a novel approach to studying individual sperm functionin a sperm egg bioassay where gamete ratios are close to unity.     

Gene Therapy: The Potential Applicability of Gene Transfer Technology to the Human Germline

The theoretical possibility of applying gene transfer methodologies to the human germline is explored. Transgenic methods for genetically manipulating embryos may in principle be applied to humans. In particular, microinjection of retroviral vector appears to hold the greatest promise, with transgenic primates already obtained from this approach. Sperm-mediated gene transfer offers potentially the easiest route to the human germline, however the requisite methodology is presently underdeveloped. Nuclear transfer (cloning) offers an alternative approach to germline genetic modification, however there are major health concerns associated with current nuclear transfer methods. It is concluded that human germline gene therapy remains for all practical purposes a future possibility that must await significant and important advances in gene transfer technology.     

GABA-receptor activation in the globus pallidus and entopeduncular nucleus: opposite effects on reaction time performance in the cat

The possible role of GABAergic mechanisms in the control of the basal ganglia output structures, the globus pallidus (GP) and the entopeduncular nucleus (EP), was studied in cats performing a conditioned flexion movement triggered by an auditory stimulus. The effects of discrete unilateral microinjections of low doses of the GABA_A receptor agonist (muscimol 5–100 ng/ 0.5 l) and antagonist (bicuculline methiodide 25–150 ng/0.5 l) in the GP and the EP were tested on the motor performance of eight animals trained to release a lever in a simple reaction time (RT) schedule after an auditory stimulus. Control injections in neighboring structures did not induce any effect except with five- to tenfold higher doses in the closest injection sites. The dose of 20 ng muscimol injected into the ventral and medial part of the GP produced an arrest of the performance after a few unsuccessful trials (over the RT reinforcement limit of 500 ms), while muscimol injected in sites located in the lateral GP resulted in a dose-dependent lengthening in RTs, with a concomitant increase in the force change latency. In most of the subjects, the force exerted on the lever was higher after muscimol than after vehicle injection. Force change velocity was then significantly increased. In contrast, muscimol injected in the ventral and rostral region of the EP produced a decrease in RTs or a complete cessation of responding after a high number of anticipatory responses (release of the lever before the trigger stimulus). No significant changes in the force change latency could be observed while there was a non-significant tendency for the force levels to be lowered. Bicuculline injections in the EP were found to increase RTs with a concomitant increase in force change latency and a slowness of velocity, while no significant effect was observed following injections in the GP. These results suggest that a balance between GABAergic activity in the two output nuclei of the basal ganglia, the GP and the EP, is crucial for the correct initiation and execution of the conditioned motor task.     

卵子激活剂对无精子症患者冻融睾丸精子ICSI后妊娠结局的影响

目的研究卵子激活剂CultActive在临床中能否提高无精子症患者冻融睾丸精子行卵胞浆内单精子显微注射(ICSI)后的妊娠结局。方法选择2015年1月~2019年12月我院收治的188例行冻融睾丸精子ICSI助孕治疗的无精子症患者作为研究对象,按照患者ICSI后是否进行应用卵子激活剂CultActive随机分为冻融睾丸精子组(n=107)和卵子激活组(n=81),对照组为新鲜睾丸精子组(n=129),分别比较了三组患者在年龄、不孕年限、促性腺激素用量、促性腺激素使用天数等临床资料的差异以及获卵数、受精率、2PN率、分裂率、优胚率、可利用胚胎率、临床妊娠率等胚胎发育情况的差异。结果三组患者在年龄、不孕年限、促性腺激素用量、促性腺激素使用天数、获卵数上均无统计学差异(P>0.05)。B组在2PN率、优胚率、可利用胚胎率上均高于A、C组,但无统计学差异(P>0.05)。三组患者在分裂率上也无统计学差异(P>0.05)。B组在受精率上明显高于C组(P<0.05),且显著高于A组(p<0.01)。B组在临床妊娠率上显著高于A、C组(p<0.01)。结论卵子激活剂CultActive可以提高冻融睾丸精子ICSI效率,值得在临床中推广。     

Enhanced cognitive flexibility in reversal learning induced by removal of the extracellular matrix in auditory cortex

During brain maturation, the occurrence of the extracellular matrix (ECM) terminates juvenile plasticity by mediating structural stability. Interestingly, enzymatic removal of the ECM restores juvenile forms of plasticity, as for instance demonstrated by topographical reconnectivity in sensory pathways. However, to which degree the mature ECM is a compromise between stability and flexibility in the adult brain impacting synaptic plasticity as a fundamental basis for learning, lifelong memory formation, and higher cognitive functions is largely unknown. In this study, we removed the ECM in the auditory cortex of adult Mongolian gerbils during specific phases of cortex-dependent auditory relearning, which was induced by the contingency reversal of a frequency-modulated tone discrimination, a task requiring high behavioral flexibility. We found that ECM removal promoted a significant increase in relearning performance, without erasing already established—that is, learned—capacities when continuing discrimination training. The cognitive flexibility required for reversal learning of previously acquired behavioral habits, commonly understood to mainly rely on frontostriatal circuits, was enhanced by promoting synaptic plasticity via ECM removal within the sensory cortex. Our findings further suggest experimental modulation of the cortical ECM as a tool to open short-term windows of enhanced activity-dependent reorganization allowing for guided neuroplasticity.Structural remodeling and stabilization of synaptic networks are key mechanisms underlying learning in the adult brain. During early life, high structural and functional plasticity is required for the experience-shaped development of basic neuronal circuits (1). With brain maturation, juvenile plasticity of so-called critical or sensitive periods is decreased and is accompanied by the appearance of the brain’s extracellular matrix (ECM) and its specialized compact form named “perineuronal net” (PNN) enwrappping cell bodies and synaptic contacts (2, 3). Enzymatic degradation of the ECM in adult animals has been demonstrated to restore such forms of developmental (juvenile) plasticity with respect to topographical map plasticity in the visual cortex (4), fear-response–mediating circuits in the amygdala (5), spinal cord injuries (6, 7), and song learning circuits of zebra finches (8). In addition, enzymatic ECM removal altered several forms of synaptic plasticity in vitro and in vivo (9–12). However, even though structural stability of networks acquired during developmental phases is essential for neuronal efficiency, mechanisms allowing synaptic remodeling are key events during learning and memory formation throughout life (13). We recently demonstrated that endogenous proteases moderately digesting specific components of the ECM are regulated in an activity-dependent manner (2, 14) and ECM removal modulates synaptic short-term plasticity by synaptic exchange of postsynaptic glutamate receptors (10, 15). Further, ECM modulation enhances synaptic short-term plasticity by affecting voltage-dependent calcium channels (9). These findings challenge the view of the purely stabilizing role of ECM in the brain and indicate a potential regulatory switch for plastic network adaptations within the adult brain at the level of individual synapses by modulating the extracellular space (16). However, it remains open to what extent ECM modulations influence learning-related plasticity in the adult brain and its specific effects on behavior during a cognitive task.In the present study, we aimed at evaluating the potential role of experimental ECM removal within the auditory cortex (ACx) of Mongolian gerbils, which has been found to be particularly rich in ECM (17), during a cognitively demanding auditory go/no go shuttle-box task. We selected discrimination and reversal learning of frequency-modulated (FM) tones as a cognitive task, which necessarily requires ACx plasticity (18, 19). Injections of the ECM-degrading enzyme hyaluronidase (HYase) into the ACx after the first acquisition phase significantly enhanced subsequent reversal learning compared with sham-treated animals (injection of 0.9% saline). Particularly, after ECM degradation, animals abandoned the inappropriate discrimination strategy from the initial acquisition phase faster and thus promoted successful discrimination performance of the new contingency during reversal learning. ECM removal did not further influence the initial acquisition learning or interfere with already established—that is, learned—capacities in later learning stages, suggesting an enhanced activity-dependent neuroplastic reorganization of established synaptic networks in ACx during reversal learning.Our findings suggest that experimental degradation of the ECM in sensory cortex, although not affecting general sensory learning, does, however, enhance the cognitive flexibility that can build on the learned behaviors. Thereby, ECM degradation could be used as a tool for guided neuroplasticity, which might also bear therapeutic potential.     

Microinjection of a growth factor cocktail affects activated microglia in the neocortex of adult rats

Microglia, as the resident immune cells in the central nervous system, play important roles in regulating neuronal processes, such as neural excitability, synaptic activity, and apoptotic cell clearance. Growth factors can activate multiple signaling pathways in central nervous system microglia and can regulate their immune effects, but whether growth factors can affect the morphological characteristics and ultrastructure of microglia has not been reported. After microinjecting 300 nL of a growth factor cocktail, including 10 μg/mL epidermal growth factor, 10 μg/mL basic fibroblast growth factor, 10 μg/mL hepatocyte growth factor and 10 μg/mL insulin-like growth factor into adult rat cortex, we found that the number of IBA1-positive microglia around the injection area increased significantly, indicating local activation of microglia. All CD68-positive labeling co-localized with IBA1 in microglia. Cell bodies and protrusions of CD68-positive cells were strongly attached to or were engulfing neurons. Characteristic huge phagosomes were observed in activated phagocytes by electron microscopy. The phagosomes generally included non-degraded neuronal protrusions and mitochondria, yet they contained no myelin membrane or remnants, which might indicate selective phagocytosis by the phagocytes. The remnant myelin sheath after phagocytosis still had regenerative ability and formed "myelin-like" structures around phagocytes. These results show that microinjection of a growth factor cocktail into the cerebral cortex of rodents can locally activate microglia and induce selective phagocytosis of neural structures by phagocytes. The study was approved by the Institute of Laboratory Animal Science, Beijing Institute of Basic Medical Sciences(approval No. IACUC-AMMS-2014-501) on June 30, 2014.     

Demonstration of maturation-promoting and- inhibiting activities in mouse oocytes

The microinjection of cytoplasm from mature mouse oocytes into immature mouse oocytes induced the resumption of meiosis in the presence of 100 g/ml dibutyryl cyclic AMP or 100 M 3-isobutyl-1-methylxanthine. The volume of cytoplasm injected was critical to bringing about this maturation-promoting activity in mouse oocytes, and 20 pl of cytoplasm seems to be required to overcome the inhibitory effect of cyclic AMP on oocyte maturation. Furthermore, it was demonstrated that the microinjection of cytoplasm from immature mouse oocytes into immature mouse oocytes augmented the inhibitory effect of cyclic AMP on the resumption of meiosis. These results suggest that the appearance and disappearance of maturation-promoting and- inhibiting activities appear to be dependent on the meiotic stages in the mouse oocytes.     

小型微量注射泵的试制

通过对小型微量注射泵的试制,实现了微泵的技术功能,同时又能从价格上有新的突破,可以实现质量可靠、物美价廉的优点,适合大规模推广应用。