Parasperm: morphological and functional studies on nonfertile sperm

Sperm polymorphism, a phenomenon in which more than one type of sperm is produced within a species, occurs widely in animals from invertebrates to vertebrates. Sperm in this phenomenon can be categorized into fertile sperm and nonfertile sperm on the basis of fertilization ability. Nonfertile sperm can be further classified into parasperm and aberrant deformed sperm. Parasperm are sperm produced through a constant developmental process along with normal fertile eusperm, and they are readily distinguished from deformed sperm, which are irregularly crippled by unpredictable errors at certain stages during spermatogenesis. Sperm identified as parasperm occur widely in invertebrates but are presently quite limited in vertebrates. This may be the result of the deficiency both of studies on parasperm and of clear criteria to identify parasperm in vertebrates. Some vertebrates show unique spermatogenesis, such as symplastic spermatid and semicystic spermatogenesis. Thus, parasperm must be identified by comparing cells in each cyst and in semen, because irregularly shaped cells in the seminal duct could be either parasperm or normal spermatids. Although parasperm are identified by clear criteria in vertebrates, only in cottoid fishes to date, it is possible for parasperm to be discovered in other vertebrates. Recently, roles related to sperm competition have been reported in several species (e.g., the marine cottoid fish Hemilepidotus gilberti), and, with some of them, parasperm production is influenced by an intraspecific factor such as a sex ratio or the density of a population. Sperm competition is one of the important candidates for influencing evolution of parasperm functions, but not all parasperm seem to have a relation to sperm competition. Parasperm function may relate to the ecological conditions of each species that produces parasperm. Studies on parasperm function will be advanced by an ecological approach concerning male fertilization success as well as cytological investigation for parasperm. An erratum to this article is available at .     

Labeling of immune cells for in vivo imaging using magnetofluorescent nanoparticles

Observation of immune and stem cells in their native microenvironments requires the development of imaging agents to allow their in vivo tracking. We describe here the synthesis of magnetofluorescent nanoparticles for cell labeling in vitro and for multimodality imaging of administered cells in vivo. MION-47, a prototype monocrystalline iron oxide nanoparticle, was first converted to an intermediate bearing a fluorochrome and amine groups, then reacted with either HIV-Tat peptide or protamine to yield a nanoparticle with membrane-translocating properties. We describe how to assess optimal cell labeling with tests of cell phenotype and function. Synthesis of magnetofluorescent nanoparticles and cell-labeling optimization can be realized in 48 h, whereas nanoparticle uptakes and retention studies may generally take up to 120 h. Labeled cells can be detected by magnetic resonance imaging, fluorescence reflectance imaging, fluorescence-mediated tomography, confocal microscopy and flow cytometry, and can be purified based on their fluorescent or magnetic properties. The present protocol focuses on T-cell labeling but can be used for labeling a variety of circulating cells.     

兔红细胞的荧光标记及寿命检测法

目的 :建立一种简单有效的兔红细胞标记及寿命检测法 ,并对GMA保养液 4℃保存的红细胞质量进行评价。方法 :日本大耳白兔动脉取血 ,红细胞用异硫氰酸荧光素 (FITC)标记后自体回输 ,定期检测荧光标记的红细胞数所占百分比。应用SAS软件对所得数据进行回归分析 ,根据方程计算兔体内标记红细胞的 2 4h回收率和半寿期。结果 :正常兔红细胞的 2 4h回收率是 93 .76%± 5.40 % ,半寿期为 ( 2 2 .50± 4.3 7)d ,与有关文献相符。GMA液 4℃保存 2 1d的红细胞 2 4h回收率为 89.13 %± 7.10 % ,半寿期为 ( 11.41± 1.63 )d ,符合输注条件。结论 :与其他红细胞体内标记方法相比 ,FITC标记法简便实用 ,价格便宜 ,不具有放射性危害 ,可用于输注红细胞的质量评价和体内生物学特性分析     

Further studies on the functional properties of spinal axons in vivo

Mammalian spinal tracts in situ demonstrate a phase of marked hyperexcitability during hypoxia or on the application of an excess of potassium or citrate ion. This is in keeping with the fact that they also show post-spike supernormality as well as hyperexcitability under cathodal polarization (17). Behavior of this kind indicates that central axons carry a well developed L fraction of membrane properties. The rhythmic state in central axons in situ, unlike peripheral nerve or spinal root, is not induced by the action of excess potassium ion. This appears to be related to the absence of a positive after-potential in dorsal columns (17). However, sodium citrate can elicit autonomous firing in central axons. When synchronized by an applied stimulus the resulting periodic oscillations have a fundamental frequency (340 to 400 C.P.S.) which is significantly greater than that of peripheral nerve.     

Electrophysiological properties of isthmic neurons in frogs revealed by in vitro and in vivo studies

The frog nucleus isthmi (parabigeminal nucleus in mammals) is a visually responsive, cholinergic and anatomically well-defined group of neurons in the midbrain. It shares reciprocal topographic projections with the ipsilateral optic tectum (superior colliculus in mammals) and strongly influences visual processing. Anatomical and biochemical information indicates the existence of distinct neural populations within the frog nucleus isthmi, which raises the question: are there electrophysiological distinctions between neurons that are putatively classified by their anatomical and biochemical properties? To address this question, we measured frog nucleus isthmi neuron cellular properties in vitro and visual response properties in vivo. No evidence for distinct electrophysiological classes of neurons was found. We thus conclude that, despite the anatomical and biochemical differences, the cells of the frog nucleus isthmi respond homogeneously to both current injections and simple visual stimuli.     

Intraocular transplants of a human gastrinoma in immuno-suppressed rats: morphological, chromatographic and functional studies

Tissue pieces of a metastatic human gastrinoma (ultrastructural Type II) were successfully transplanted to the anterior eye-chamber of rats immunosuppressed with Cyclosporin A. Immunocytochemical investigation of the transplants showed evidence for preserved endocrine activity of tumour cells with immunoreactivity towards the C-terminal of the gastrin/cholecystokinin molecule. Studies of gastric acid secretion in tumour-bearing rats and sham-operated controls with chronic gastric fistulas showed that the basal acid output did not differ between the groups during 3 weeks of study. However, the stimulated gastric acid secretion decreased after 5 days in both groups to remain significantly depressed throughout the study, an effect probably due to Cyclosporin A treatment of the groups. The concentration of immunoreactive gastrin in plasma from rats with tumours in oculo was 5 times higher than in sham-operated rats. Gastrin-34 was the major immunoreactive component in both patient serum and rat plasma. An immunoreactive fraction corresponding to component I was found in the patient serum, but not in the rat plasma, although present in the chamber fluid. Components corresponding to gastrin-17 were found both in the patient serum and in the rat plasma. The chromatographic pattern of the tumour was similar to that in rat chamber fluid. The dominating component corresponded to gastrin-17, while gastrin-34 represented the quantitatively smaller component. Gastrin-34 was, however, relatively more abundant in the tumour extract than in the chamber fluid. The study also indicates that a gastrin-producing tumour transplanted in oculo in immunosuppressed rats may increase the rat plasma concentration of the same molecular forms of gastrin as seen in the clinical situation.     

Bidirectional activity-dependent morphological plasticity in hippocampal neurons

Dendritic spines on pyramidal neurons receive the vast majority of excitatory input and are considered electrobiochemical processing units, integrating and compartmentalizing synaptic input. Following synaptic plasticity, spines can undergo morphological plasticity, which possibly forms the structural basis for long-term changes in neuronal circuitry. Here, we demonstrate that spines on CA1 pyramidal neurons from organotypic slice cultures show bidirectional activity-dependent morphological plasticity. Using two-photon time-lapse microscopy, we observed that low-frequency stimulation induced NMDA receptor-dependent spine retractions, whereas theta burst stimulation led to the formation of new spines. Moreover, without stimulation the number of spine retractions was on the same order of magnitude as the stimulus-induced spine gain or loss. Finally, we found that the ability of neurons to eliminate spines in an activity-dependent manner decreased with developmental age. Taken together, our data show that hippocampal neurons can undergo bidirectional morphological plasticity; spines are formed and eliminated in an activity-dependent way.     

In vivo functional studies of tumor-specific retrogene NanogP8 in transgenic animals

The current study was undertaken to investigate potential oncogenic functions of NanogP8, a tumor-specific retrogene homolog of Nanog (expressed in pluripotent cells), in transgenic animal models. To this end, human primary prostate tumor-derived NanogP8 was targeted to the cytokeratin 14 (K14) cellular compartment, and two lines of K14-NanogP8 mice were derived. The line 1 animals, expressing high levels of NanogP8, experienced perinatal lethality and developmental abnormalities in multiple organs, including the skin, tongue, eye, and thymus in surviving animals. On postnatal day 5 transgenic skin, for example, there was increased c-Myc expression and Ki-67⁺ cells accompanied by profound abnormalities in skin development such as thickened interfollicular epidermis and dermis and lack of hypodermis and sebaceous glands. The line 3 mice, expressing low levels of NanogP8, were grossly normal except cataract development by 4–6 mo of age. Surprisingly, both lines of mice do not develop spontaneous tumors related to transgene expression. Even more unexpectedly, high levels of NanogP8 expression in L1 mice actually inhibited tumor development in a two-stage chemical carcinogenesis model. Mechanistic studies revealed that constitutive NanogP8 overexpression in adult L1 mice reduced CD34⁺α6⁺ and Lrig-1⁺ bulge stem cells, impaired keratinocyte migration, and repressed the expression of many stem cell-associated genes, including Bmp5, Fgfr2, Jmjd1a, and Jun. Our study, for the first time, indicates that transgenically expressed human NanogP8 is biologically functional, but suggests that high levels of NanogP8 may disrupt normal developmental programs and inhibit tumor development by depleting stem cells.     

Rapid immunomagnetic negative enrichment of neutrophil granulocytes from murine bone marrow for functional studies in vitro and in vivo

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*10⁶ PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.     

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.Download video file.^{(54M, mov)}     

Simultaneous PET-MRI: a new approach for functional and morphological imaging

Noninvasive imaging at the molecular level is an emerging field in biomedical research. This paper introduces a new technology synergizing two leading imaging methodologies: positron emission tomography (PET) and magnetic resonance imaging (MRI). Although the value of PET lies in its high-sensitivity tracking of biomarkers in vivo, it lacks resolving morphology. MRI has lower sensitivity, but produces high soft-tissue contrast and provides spectroscopic information and functional MRI (fMRI). We have developed a three-dimensional animal PET scanner that is built into a 7-T MRI. Our evaluations show that both modalities preserve their functionality, even when operated isochronously. With this combined imaging system, we simultaneously acquired functional and morphological PET-MRI data from living mice. PET-MRI provides a powerful tool for studying biology and pathology in preclinical research and has great potential for clinical applications. Combining fMRI and spectroscopy with PET paves the way for a new perspective in molecular imaging.     

Comparative studies of some functional and morphological parameters in the livers of germfree, conventional and ex-germfree mice.

Some parameters of hepatic function and morphology were studied to compare germfree (GF) and conventional (CV) BALB/c mice. The levels of lipid peroxide (LPO) and aniline-hydroxylase (AH) activity in the livers and the serum total cholesterol (TC), triglyceride (TG) and phospholipid (PL) were significantly lower in GF than in CV 8-week-old mice. There were no significant differences in the histology and lectin-histochemistry of the livers in the GF and CV mice. On the other hand, in ex-GF mice which were induced by housing 4-week-old GF mice together with age-matched CV mice, the levels of LPO and AH activity in the liver and the serum TC, TG and PL contents increased rapidly within the first week and then approached values almost identical to those in CV mice 4 weeks later (i.e. at 8 weeks of age). The histologic picture of the liver was similar among the GF, CV and ex-GF mice.