Identification of living spermatogenic cells of the mouse by transillumination-phase contrast microscopic technique for ‘in situ’ analyses of DNA polymerase activities

Summary The stages of spermatogenesis can be identified in freshly isolated, unstained adult mouse seminiferous tubules using a transillumination method. Late acrosome- and maturation phase spermatids, arranged in bundles at stages XII–VI give rise to a spotty transillumination pattern. Before spermiation, these cells form a continuous layer on the top of the seminiferous epithelium, recognized by a strong homogeneous central light absorption in the freshly isolated seminiferous tubules at stages VII and VIII. Other stages have a pale light absorption pattern. The accurate determination of the developmental stages of the germ cells was based on the morphology of the developing acrosomic system and of the nuclei of the spermatids, as revealed by phase contrast microscopy. Using this procedure, the activity levels of DNA polymerases and have been studied by autoradiography of squash preparations. Using endogenous templates, assay conditions that differentiate between the solubilized DNA polymerases and in vitro, were used to distinguish between these activities in situ in different stages of mouse spermatogenesis. Except in very late spermatids shortly before spermiation, DNA polymerases and were detectable in all cell types examined. Coinciding with the nuclear protein transitions, elongating spermatids at steps 10–12 and maturation phase spermatids at steps 13–14 showed high DNA polymerase activities. As no replication occurs in these cells, the observations support the view that both DNA polymerases and could be involved in repair DNA synthesis.     

Role of clozapine in the occurrence of chromosomal abnormalities in human bone-marrow cells in vivo and in cultured lymphocytes in vitro

Summary Bone-marrow chromosomes were examined from 38 mentally and physically retarded and two psychiatric patients who were being treated with a variety of neuropharmacologic drugs. Twenty of these patients used clozapine (Leponex^®). The clastogenic effects of clozapine in vitro were studied in the lymphocyte cultures of three patients-one free of hematologic disease and two who 6 months earlier had had agranulocytosis attributed to the use of clozapine. The mean frequency of cytogenetic abnormalities in the bonemarrow cells of patients who used clozapine was significantly increased (P> 0.05). The two patients who had had agranulocytosis had a greater frequency of cytogenetic abnormalities in their cultured lymphocytes in vivo and in vitro than the patient free of hematologic disease. A clone with a 13/14 chromosome translocation was detected in one of the patients. As all patients received a number of drugs during the in vivo and in vitro studies no definite conclusions could be drawn regarding the role played by clozapine in the occurrence of chromosomal abnormalities.     

Does polymyxin B nonapeptide increase outer membrane permeability in antibiotic supersensitive enterobacterial mutants?

Abstract The outer-membrane-disorganizing peptide (polymyxin B nonapeptide; PMBN) was able to sensitize even "antibiotic supersensitive" enterobacterial mutants to hydrophobic antibiotics. This resulted in an extreme sensitivity. The mutants included the "deep rough" lipopolysaccharide mutants, as well as the acrA mutant of Escherichia coli and the "class A, B, and C mutants of Salmonella typhimurium . Sensitization factors of approx. 30 or more were found for most antibiotics. Even minimum inhibitory concentrations as low as approx. 0.5 ng/ml (rifampicin), 1.5 ng/ml (erythromycin), 2 ng/ml (fusidic acid), 6 ng/ml (novobiocin), and 30 ng/ml (clindamycin) were achieved in the presence of 30 μg/ml of PMBN. The finding indicates that the mechanisms which mediate the increase in hydrophobic diffusion are different but synergistic in the mutants and in the PMBN-grown cells.     

Active movements of the chromatoid body. A possible transport mechanism for haploid gene products

Recent data indicate that the chromatoid body typical of rat spermatogenesis may contain RNA synthesized in early spermatids by the haploid genome. Analyses of living step-1 and step-3 spermatids by time-lapse cinephotomicrography have shown that the chromatoid body moves in relation to the nuclear envelope in two different ways. Predominantly in step 1, the chromatoid body moves along the nuclear envelope on a wide area surrounding the Golgi complex and has frequent transient contacts with the latter organelle. In step 3, the chromatoid body was shown to move perpendicular to the nuclear envelope. It was seen located very transiently at the top of prominent outpocketings of the nuclear envelope with apparent material continuities through nuclear pore complexes to intranuclear particles. The rapid movements of the chromatoid body are suggested to play a role in the transport of haploid gene products in the early spermatids, including probably nucleocytoplasmic RNA transport.     

Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis

The physiological apoptosis that occurs in immature testis appears to be necessary for the maturation of this tissue. Thus, inhibition of the early apoptotic wave associated with the first round of spermatogenesis is followed by accumulation of spermatogonia and infertility later in life. To identify the cell types undergoing apoptosis in immature rat testis and to characterize the relationship between this apoptosis and progression of the first wave of spermatogenesis, sequential viable segments of seminiferous tubules from 8-, 18-, and 26-day-old rats were examined under a phase-contrast microscope. One novel observation was the existence of pronounced stage-specificity during the peak of apoptosis at the very early postnatal ages of 18 and 26 days. Increased apoptosis of pachytene spermatocytes in stages VII-VIII was the major feature that distinguished immature spermatogenesis from the corresponding adult process. The frequency of apoptosis among type A spermatogonia in immature stages IX-I was also elevated in comparison to the corresponding mature stages. The age-related peak of apoptosis was mediated by caspase 3; furthermore, stage-dependent expression of Bax in midpachytene spermatocytes was observed in the 18- and 26-day-old testis. These observations suggest that this Bax-regulated, caspase 3-mediated, increased apoptosis of midpachytene spermatocytes during the first wave of immature spermatogenesis represents a major difference in comparison to apoptosis occurring in the mature testis, and it may play an important regulatory role in establishing spermatogenesis in the rat testis.     

Dispersal and the evolution of specialisation in a two-habitat type metapopulation

Metapopulation theory for the evolution of specialisation is virtually absent. In this article, therefore, we study a metapopulation model for consumers with a fitness trade-off between two habitats. We focus on effects of habitat abundance, dispersal rate and trade-off strength on the evolution of specialisation under two types of trade-off. Adaptation affects either the intrinsic growth rates r or the carrying capacities K. Depending on dispersal rate and trade-off strength, evolution can result in one generalist, one specialist or two specialist types. Higher dispersal rate and a weaker trade-off favour the evolution of a generalist, for both trade-off structures. However, we also find differences between the two trade-off structures. Our results are qualitatively similar to analyses of two-patch models, suggesting that insights from such simpler models can be extrapolated to metapopulation models. Additional effects, however, occur because in classical metapopulations patch lifetime depends on extinction rate. Counterintuitively, this favours the evolution of specialisation when the trade-off affects r.     

Fishing-induced changes in predation pressure by perch (<Emphasis Type="Italic">Perca fluviatilis</Emphasis>) regulate littoral benthic macroinvertebrate biomass,density, and community structure

We aimed to study whether the varying changes in predation pressure by perch (Perca fluviatilis) reflect the biomass, density, and community structure of the benthic macroinvertebrates. Prey preference is size-dependent, and overall predation pressure is density dependent, and thus the size structure of the P. fluviatilis population should affect the structure of the macroinvertebrate community, and the population density of P. fluviatilis should reflect the overall density of benthic macroinvertebrates. We sampled the littoral benthic community in a boreal lake that had been divided into two parts that were subjected to two different fishing procedures during 2007–2012 period and analyzed the macroinvertebrate diet of fish. The benthic macroinvertebrate community reflected the predation pressure. Total macroinvertebrate biomass increased during the study period in the lake division with a non-size-selective fishing procedure (NSF), i.e., all invertivorous perch size-classes targeted, but decreased in the section with negatively size-selective fishing procedure (SSF), i.e., large invertivorous individuals ≥ 16 cm were not targeted. This difference was a result of the increase in large-sized species, such as Odonata, for the NSF procedure and decrease in the SSF procedure. In contrast to total biomass, total macroinvertebrate density did not show a response to predator size structure but rather total macroinvertebrate density decreased with increasing fish density. The study demonstrates the effect of predation pressure of P. fluviatilis on benthic communities, thus highlighting the keystone predator role of the species in boreal lakes and gives more insight on the multiple effects of fish predation on littoral benthic communities.     

Stereospecific hydrolysis of 3-(4-methoxyphenyl)glycidic ester in supercritical carbon dioxide by immobilized lipase

In the hydrolysis of racemic 3-(4-methoxyphenyl)glycidic acid methyl ester by immobilized Mucor miehel lipase in supercritical CO₂ the initial hydrolysis rate of the (2S,3R)-form was faster than the rate of the (2R,3S) -form. The stereoisomeric excess of the (2R,3S)-form reached 87 % at 53 % total conversion level. The water content of the reaction mixture and the initial concentration of the 3-(4-methoxyphenyl) glycidic acid methyl ester had no effect on isomeric purity. The reaction rate in supercritical CO₂ was considerably faster than in toluene/water -mixture.     

Immunohistochemical localization of urokinase-type plasminogen activator in Sertoli cells and tissue-type plasminogen activator in spermatogenic cells in the rat seminiferous epithelium

The occurrence of plasminogen activators of the urokinase-type (u-PA) and tissue-type (t-PA) at various stages of the epithelial cycle was studied immunohistochemically in rat seminiferous tubule segments. u-PA immunoreactivity was detected exclusively at stages VII and VIII in Sertoli cells, displaying a distinct granular cytoplasmic staining. t-PA immunoreactivity was found during mid- and late pachytene and diakinesis (stages VII-XIII) in spermatogenic cells, displaying a granular cytoplasmic staining with maximal intensity in stages IX-XIII. The specificity of the stainings was supported by staining controls, including absorption of the antibodies with purified preparations of the activators. It was also supported by zymographic studies of the occurrence of u-PA and t-PA in extracts of tubular segments at different stages of the cycle, isolated by transillumination-assisted microdissection. The possible functions of the two types of plasminogen activators in the seminiferous epithelium are discussed.