Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).     

High nucleotide sequence variation in a region of low recombination in Drosophila simulans is consistent with the background selection model

We surveyed nucleotide sequence variation at glucose dehydrogenase (Gld), in a region of low recombination on chromosome 3R, from a population sample of Drosophila simulans. The levels of nucleotide variation were surprisingly high. There was no departure from the expectation of a neutral model for the level of polymorphism, indicating no evidence of a selective sweep in this region. There was a significant deficiency of singleton polymorphisms according to the Fu and Li test, although Tajima and Hudson, Kreitman, and Aguade (HKA) tests do not provide evidence of a significant elevation of variation due to balancing selection. Genetic map data for the D. simulans third chromosome were used to calculate expected values of pi for Gld under a current model of background selection, varying the values for the parameter sh (selection coefficient against deleterious mutations). We show that the recombinational landscape of D. simulans is sufficiently different from that of D. melanogaster that we expect higher variation under the background selection model, even when effective population sizes are assumed to be equal. The data for Gld were tested against the predictions using computer simulations of the distribution of the number of segregating sites conditioned on pi. Background selection alone can explain our observations as long as sh is larger than 0.005 and species-level effective population size is assumed to be several- fold larger than in D. melanogaster. Alternatively, the deleterious mutation rate may be smaller in D. simulans, or balancing selection may be acting nearby, thereby reducing the effect of background selection.      

MAP2 IHC detection: a marker of antigenicity in CNS tissues

Immunohistochemistry (IHC) is used to detect antibody-specific antigens in tissues; the results depend on the ability of the primary antibodies to bind to their antigens. Therefore, results depend on the quality of preservation of the specimen. Many investigators have overcome the deleterious effects of over-fixation on the binding of primary antibodies to specimen antigens using IHC, but if the specimen is under-fixed or fixation is delayed, false negative results could be obtained despite certified laboratory practices. Microtubule-associated protein 2 (MAP2) is an abundant microtubule-associate protein that participates in the outgrowth of neuronal processes and synaptic plasticity; it is localized primarily in cell bodies and dendrites of neurons. MAP2 immunolabeling has been reported to be absent in areas of the entorhinal cortex and hippocampus of Alzheimer’s disease brains that were co-localized with the dense-core type of amyloid plaques. It was hypothesized that the lack of MAP2 immunolabeling in these structures was due to the degradation of the MAP2 antigen by the neuronal proteases that were released as the neurons lysed leading to the formation of these plaques. Because MAP2 is sensitive to proteolysis, we hypothesized that changes in MAP2 immunolabeling may be correlated with the degree of fixation of central nervous system (CNS) tissues. We detected normal MAP2 immunolabeling in fixed rat brain tissues, but MAP2 immunolabeling was decreased or lost in unfixed and delayed-fixed rat brain tissues. By contrast, two ubiquitous CNS-specific markers, myelin basic protein and glial fibrillary acidic protein, were unaffected by the degree of fixation in the same tissues. Our observations suggest that preservation of various CNS-specific antigens differs with the degree of fixation and that the lack of MAP2 immunolabeling in the rat brain may indicate inadequate tissue fixation. We recommend applying MAP2 IHC for all CNS tissues as a pre-screen to assess the quality of the tissue preservation and to avoid potentially false negative IHC results.     

鼻咽上皮细胞的调节性容积回缩能力及机制

用图像分析系统和通道阻断法研究了原代人胎儿鼻咽上皮细胞的调节性容积回缩(regulatoryvolumedecrease,RVD)能力及其机制。结果发现,低渗刺激可诱发鼻咽上皮细胞产生RVD,在160-240mOsmol/L范围内,RVD强弱与渗透压呈“S”形负相关(r=-0.99,P<0.05),与细胞肿胀程度呈“S”形正相关(=0.99,P<0.05)。Cl~-通道阻断剂tamoxifen(20μmol/L),ATP(10mmol/L)或NPPB(100μmol/L)对RVD阻抑率分别为100%(P<0.01),76.3%(P<0.01)和62.7%(P<0.01)。本研究表明,鼻咽上皮细胞受到低渗刺激时可产生RVD,Cl~-通道开放是其RVD的关键机制。     

Fibronectin Regulates Wnt7a Signaling and Satellite Cell Expansion

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Highlights? Fzd7 and Sdc4 form a coreceptor complex in satellite cells ? FN stimulates Wnt7a activity through the Fzd7/Sdc4 coreceptor complex ? Activating satellite cells remodel their niche with FN ? FN regulates the satellite cell pool during muscle regeneration     

Fitness variation in response to artificial selection for reduced cell area,cell number and wing area in natural populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background

Genetically based body size differences are naturally occurring in populations of Drosophila melanogaster, with bigger flies in the cold. Despite the cosmopolitan nature of body size clines in more than one Drosophila species, the actual selective mechanisms controlling the genetic basis of body size variation are not fully understood. In particular, it is not clear what the selective value of cell size and cell area variation exactly is. In the present work we determined variation in viability, developmental time and larval competitive ability in response to crowding at two temperatures after artificial selection for reduced cell area, cell number and wing area in four different natural populations of D. melanogaster.

Results

No correlated effect of selection on viability or developmental time was observed among all selected populations. An increase in competitive ability in one thermal environment (18°C) under high larval crowding was observed as a correlated response to artificial selection for cell size.

Conclusion

Viability and developmental time are not affected by selection for the cellular component of body size, suggesting that these traits only depend on the contingent genetic makeup of a population. The higher larval competitive ability shown by populations selected for reduced cell area seems to confirm the hypothesis that cell area mediated changes have a relationship with fitness, and might be the preferential way to change body size under specific circumstances.

     

拐芹根化学成分研究Ⅱ

从伞型科当归属植物拐芹(Angelica polymorpha Maxim)的根及根茎中又分得4个结晶性化合物。经物理常数测定、光谱分析,分别鉴定为欧前胡素Ⅰ,异氧化前胡内酯Ⅱ,Pabulenol Ⅲ,Phellopterin Ⅳ。     

The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster

In a recent study, we reported that the combined average mutation rate of 10 di-, 6 tri-, and 8 tetranucleotide repeats in Drosophila melanogaster was 6.3 x 10(-6) mutations per locus per generation, a rate substantially below that of microsatellite repeat units in mammals studied to date (range = 10(-2)-10(-5) per locus per generation). To obtain a more precise estimate of mutation rate for dinucleotide repeat motifs alone, we assayed 39 new dinucleotide repeat microsatellite loci in the mutation accumulation lines from our earlier study. Our estimate of mutation rate for a total of 49 dinucleotide repeats is 9.3 x 10(-6) per locus per generation, only slightly higher than the estimate from our earlier study. We also estimated the relative difference in microsatellite mutation rate among di-, tri-, and tetranucleotide repeats in the genome of D. melanogaster using a method based on population variation, and we found that tri- and tetranucleotide repeats mutate at rates 6.4 and 8.4 times slower than that of dinucleotide repeats, respectively. The slower mutation rates of tri- and tetranucleotide repeats appear to be associated with a relatively short repeat unit length of these repeat motifs in the genome of D. melanogaster. A positive correlation between repeat unit length and allelic variation suggests that mutation rate increases as the repeat unit lengths of microsatellites increase.