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 .     

Trachea and lung dimensions in nonsmoking twins: morphological and functional studies

To compare genetic and environmental factors that determine lung function and dimensions, chest radiographs and pulmonary function were measured in 17 pairs of nonsmoking twin adolescent boys (12 monozygotic pairs and 5 dizygotic pairs). Genetic factors dominated in tracheal width and lung dimensions (height, width, and apicofissural and fissurodiaphragmatic distances) at residual volume. Genetic factors also affected forced vital capacity, functional residual capacity, forced expiratory volume in 1 s, maximum expiratory flow at 25% vital capacity, and maximum flow at 50% vital capacity-to-forced vital capacity ratio. Peak expiratory flow correlated with tracheal width at residual volume. Age correlated with lung dimensions (width and depth) but not with tracheal width. These results indicate that genetic factors determine the dimensions and function of central airways, peripheral airways, and lung parenchyma in adolescent males. The effects of genetic factors on some functional measurements (airway resistance, closing volume-to-vital capacity ratio, and phase III in single-breath N2 washout) may be masked because of poor reproducibility of the tests.     

Isotocinergic neurons in the goldfish hypothalamus: Physiological and morphological studies on chemically identified cells

Summary Isotocinergic (IT) neurons show physiological and morphological characteristics that are similar to those of other preoptic neuroendocrine cells in the goldfish. Preoptic IT cells show resting membrane potentials of 20–55 mV, action potentials of up to 100mV, and physiological evidence of axonal branching. Dye-marked IT cells measure 14–56 m, their dendrites projecting to the ependyma and into the hypothalamic neuropil, their multiple beaded axons projecting to the pituitary. Indirect immunofluorescence identifies these dyemarked cells as IT. By combining electrophysiological, dye-marking and immunocytochemical techniques we can now, for the first time, study single, antidromically-identified peptidergic neurons of a specific type in vertebrate and invertebrate species.Supported by Grants from the USPHS (NS-13411 and NS-05696)The authors wish to thank Ms. S. Curtis for editorial assistance, Ms. D. Cronce for skillful technical assistance, Dr. W.W. Stewart for helpful suggestions and for his generous gift of Lucifer Yellow-CH, Dr. M. Manning for his generous gift of high quality peptides, and Dr. R.R. Dries and J.D. Fernstrom for kindly supplying antisera     

The use of neurotoxic dihydroxytryptamines as tools for morphological studies and localized lesioning of central indolamine neurons

Summary The usefulness of three neurotoxic dihydroxytryptamines — 5,6-dihydroxytryptamine, 5,7-dihydroxytryptamine and 4,5-dihydroxytryptamine — for fluorescence microscopical tracing and localized lesioning of central indolamine-containing axon bundles has been studied in the rat brain. The lesions produced by intraventricularly or intracerebrally administered dihydroxytryptamines were found to be much superior to mechanical or electrolytic lesions in producing extensive accumulations of fluorescence in the indolamine axon pathways. This greatly improves the possibilities for tracing of the normally non-fluorescent or weakly fluorescent indolamine axons from their cells of origin for long distances through the main fibre bundles and their branches. Much new information concerning the anatomy of the indolamine neuron systems is obtained with this technique, and some preliminary observations are presented.The efficiency of local, intracerebral injections of small amounts of dihydroxytryptamines for regional denervations in the CNS was also tested. It was found that local injections of 4 g of either of the three compounds into the ventromedial tegmentum and into the grey matter of the spinal cord produced extensive and probably rather selective damage to the ascending and descending indolamine fibre tracts and — although to a lesser and variable extent — the noradrenaline and dopamine systems. The denervating effects of the tegmental and the spinal cord injections were with respect to the serotonin-containing neurons comparable to those obtained by others after large lesions that destroy almost the entire midbrain raphe region, and after total transections of the spinal cord, respectively. The characteristics and the specificity of the dihydroxytryptamine-induced lesions are discussed.     

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.     

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.     

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     

Directed dimerization: an in vivo expression system for functional studies of type II phytochromes

Type II phytochromes (phy) in Arabidopsis form homodimers and heterodimers, resulting in a diverse collection of light‐stable red/far‐red (R/FR) sensing photoreceptors. We describe an in vivo protein engineering system and its use in characterizing the activities of these molecules. Using a phyB null mutant background, singly and doubly transgenic plants were generated that express fusion proteins containing the phyB–phyE N–terminal photosensory regions (NB–NE PSRs), a nuclear localization sequence, and small yeast protein domains that mediate either homodimerization or heterodimerization. Activity of NB/NB homodimers but not monomeric NB subunits in control of seedling and adult plant responses to R light is demonstrated. Heterodimers of the NB sequence with the chromophoreless NB^C357S sequence, which mimic phyB Pfr/Pr photo‐heterodimers, mediate R sensitivity in leaves and petioles but not hypocotyls. Homodimerization of the NC, ND and NE sequences and directed heterodimerization of these photosensory regions with the NB region reveal form‐specific R‐induced activities for different type II phy dimers. The experimental approach developed here of directed assembly of defined protein dimer combinations in vivo may be applicable to other systems.     

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.     

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.     

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.     

Diversity of functional morphological explanation

An introduction to the 19th Lochmühle conference on Architecture in living structure, organized by P. Dullemeijer, W.F. Gutmann and G.A. Zweers, held from March 15–17, 1984 in the Aussenstelle des Forschungsinstituts Senckenberg der Senckenbergischen Naturforschenden Gesellschaft, Frankfurt am Main.