Human neurospheres: From stained sections to three-dimensional assembly

Human neurospheres are free-floating spherical clusters generated from a single neural stem cell and comprising cells at different stages of maturation in the neuronal and glial lineages. Although recent findings have disproved the original idea of clonally derived neurospheres according to the paradigm of one stem cell — one neurosphere, they still represent a valid model for growing neural stem cell cultures in vitro. While the immunocytochemical approach to the identification of stem cells, progenitor cells, and mature cells has been extensively used, scant data are available about the ultrastructural arrangement of different cell types within the neurosphere. This paper provides, by means of scanning electron microscopy, some new insights into the three-dimensional assembly of human neurospheres, trying to correlate some parameters such as cell density, shape and growing strategies with the immunolocalization of some antigens such as nestin, GFAP, α-internexin and βIII-tubulin. The major findings from this study are: a) regardless of the stage of in vitro maturation, the growth of the spheres is the result of mitotic divisions producing the aspect of an irregular budding mechanism in the outermost layer look like; b) analysis of the volumetric composition of the inner core has revealed the presence of two alternative shape pattern (pyramidal vs rounded cells) possibly related to both the ongoing maturation stages and GFAP and internexin expression.

Tracking sperm of a donor in a recipient: an immunocytochemical approach

In order to study the mechanisms of sperm competition and cryptic female choice we require an understanding of the patterns of sperm storage, sperm removal and sperm digestion. Current studies infer these patterns mainly from paternity data, which only reveal the ultimate outcomes of the interactions between male and female reproductive processes. However, only with a mechanistic understanding of the fate of received sperm, and the involved patterns of postcopulatory sexual selection, can we understand the evolution of male and female reproductive morphology and physiology. The currently available approaches for tracking donor sperm in a recipient are either very time consuming, spatially imprecise or limited to model organisms for which considerable genetic knowledge and molecular know-how is available. Using the free-living flatworm Macrostomum lignano we here present a novel sperm tracking approach that uses DNA-labelling with a halogenated pyrimidine and localisation of the label using immunocytochemistry. We first outline modifications to established protocols to allow visualisation of gametic cells, in addition to somatic cells, determine the duration and patterns of spermatogenesis, and then show that labelled sperm from labelled donors can be observed in unlabelled recipients. We further show that labelled worms have a mating behaviour that is comparable to that of unlabelled worms except in one parameter. We suggest ways in which this approach can be optimised, and that it should be readily transferable to other taxa. We conclude that this approach will be a valuable tool to study postcopulatory sexual selection.