Molecular markers of human sperm functions

Fertilization is a stepwise process that allows two mature gametes to reach each other, fuse and eventually give rise to a new individual. Despite the tremendous importance of reproduction for species development and maintenance, fertility is decreasing worldwide, with peaks in western countries. It is estimated that about 7% of men experiences problems in conceiving a child because of sperm defects. In such a situation, understanding which are the essential sperm players in each of the steps of the fertilization process is essential for the development of new pharmacological strategies to treat the infertile men, for genetic screening of idiopathic male infertility as well as to produce effective male contraceptive agents. The present review will summarize recent evidence for the identification and characterization of molecular markers of sperm functions with emphasis on post-ejaculatory maturation events and the process of sperm–oocyte interaction.     

Human Neurospheres as Three-Dimensional Cellular Systems for Developmental Neurotoxicity Testing

Background

Developmental neurotoxicity (DNT) of environmental chemicals is a serious threat to human health. Current DNT testing guidelines propose investigations in rodents, which require large numbers of animals. With regard to the “3 Rs” (reduction, replacement, and refinement) of animal testing and the European regulation of chemicals [Registration, Evaluation, and Authorisation of Chemicals (REACH)], alternative testing strategies are needed in order to refine and reduce animal experiments and allow faster and less expensive screening.

Objectives

The goal of this study was to establish a three-dimensional test system for DNT screening based on human fetal brain cells.

Methods

We established assays suitable for detecting disturbances in basic processes of brain development by employing human neural progenitor cells (hNPCs), which grow as neurospheres. Furthermore, we assessed effects of mercury and oxidative stress on these cells.

Results

We found that human neurospheres imitate proliferation, differentiation, and migration in vitro. Exposure to the proapoptotic agent staurosporine further suggests that human neurospheres possess functioning apoptosis machinery. The developmental neurotoxicants methylmercury chloride and mercury chloride decreased migration distance and number of neuronal-like cells in differentiated hNPCs. Furthermore, hNPCs undergo caspase-independent apoptosis when exposed toward high amounts of oxidative stress.

Conclusions

Human neurospheres are likely to imitate basic processes of brain development, and these processes can be modulated by developmental neurotoxicants. Thus, this three-dimensional cell system is a promising approach for DNT testing.     

CD45-positive cells of haematopoietic origin enhance chondrogenic marker gene expression in rat marrow stromal cells

Adult mesenchymal stem cells (MSCs) can be readily isolated from bone marrow, expanded in culture and subsequently subjected to differentiation into various connective tissue lineages. In general, for animal studies separation of MSCs from other bone marrow-derived cells is achieved by sole adherence to plastic surface of tissue culture flasks; however, this procedure produces a heterogeneous cell population containing CD45-positive haematopoietic cells (HCs) and haematopoietic stem cells (HSCs). It is known, that mixed cell cultures consisting of cocultures of differentiated somatic cells with adult stem cells promote differentiation towards specific cell lineages. For determining the effect of the CD45-positive cell population on the differentiation potential of MSCs, we sorted out the bone marrow-derived adherent cells by immunomagnetic technique (MACS) to attain a subpopulation of CD45-depleted cells. Herein, we show that the presence of adherent CD45-positive HCs not only promote expression of the chondrogenic marker genes Col2a1, COMP and Sox9, but also of Col1a1, Col10a1 and to a certain degree Cbfa1 in MSCs when cultured in an appropriate three-dimensional environment. These observations constitute a step towards unravelling the influence of haematopoietic cells on chondrogenic differentiation of MSCs.     

Neuronal Modifications During Visuomotor Association Learning Assessed by Electric Brain Tomography

Summary In everyday life specific situations need specific reactions. Through repetitive practice, such stimulus-response associations can be learned and performed automatically. The aim of the present EEG study was the illustration of learning dependent modifications in neuronal pathways during short-term practice of visuomotor associations. Participants performed a visuomotor association task including four visual stimuli, which should be associated with four keys, learned by trial and error. We assumed that distinct cognitive processes might be dominant during early learning e.g., visual perception and decision making. Advanced learning, however, might be indicated by increased neuronal activation in integration- and memory-related regions. For assessment of learning progress, visual- and movement-related brain potentials were measured and compared between three learning stages (early, intermediate, and late). The results have revealed significant differences between the learning stages during distinct time intervals. Related to visual stimulus presentation, Low Resolution Electromagnetic Brain Tomography (LORETA) revealed strong neuronal activation in a parieto-prefrontal network in time intervals between 100–400 ms post event and during early learning. In relation to the motor response neuronal activation was significantly increased during intermediate compared to early learning. Prior to the motor response (120–360 ms pre event), neuronal activation was detected in the cingulate motor area and the right dorsal premotor cortex. Subsequent to the motor response (68–430 ms post event) there was an increase in neuronal activation in visuomotor- and memory-related areas including parietal cortex, SMA, premotor, dorsolateral prefrontal, and parahippocampal cortex. The present study has shown specific time elements of a visuomotor-memory-related network, which might support learning progress during visuomotor association learning.     

Glucuronidation of piceatannol by human liver microsomes: major role of UGT1A1, UGT1A8 and UGT1A10

Objectives Piceatannol, a dietary polyphenol present in grapes and wine, is known for its promising anticancer and anti‐inflammatory activity. The aim of this study was to analyse the concentration‐dependent glucuronidation of piceatannol in vitro. Methods To determine the glucuronidation of piceatannol, experiments were conducted with human liver microsomes as well as using a panel of 12 recombinant UDP‐glucuronosyltransferase isoforms. Furthermore, the chemical structures of novel glucuronides were identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Key findings Along with piceatannol it was possible to identify three metabolites whose structures were identified by LC‐MS/MS as piceatannol monoglucuronides (M1–M3). Formation of M1 and M3 exhibited a pattern of substrate inhibition, with apparent K_i and V_max/K_m values of 103 ± 26.6 µm and 3.8 ± 1.3 µl/mg protein per min, respectively, for M1 and 233 ± 61.4 µm and 19.8 ± 9.5 µl/mg protein per min, respectively, for M3. In contrast, formation of metabolite M2 followed classical Michaelis–Menten kinetics, with a K_m of 18.9 ± 8.1 µm and a V_max of 0.21 ± 0.02 nmol/mg protein per min. Incubation in the presence of human recombinant UDP‐glucuronosyltransferases (UGTs) demonstrated that M1 was formed nearly equally by UGT1A1 and UGT1A8. M2 was preferentially catalysed by UGT1A10 and to a lesser extent by UGT1A1 and UGT1A8. The formation of M3, however, was mainly catalysed by UGT1A1 and UGT1A8. Conclusions Our results elucidate the importance of piceatannol glucuronidation in the human liver, which must be taken into account in humans after dietary intake of piceatannol.     

Functionalization of fatty acid mimetics for solid-phase coupling and subsequent target identification

Fatty acid mimetics such as pirinixic acid (PA) derivatives and 2-(phenylthio)alkanoic acid derivatives are drug-like small molecules with an interesting pharmacological profile. Previously, we have characterized PA derivatives (e.g., 1) as dual agonists of peroxisome proliferator-activated receptors (PPARs) α and γ and as inhibitors of microsomal prostaglandin E(2)-synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO). 2-(Phenylthio)alkanoic acids (e.g., 2) were shown to act as highly active and selective PPARα agonists. Encouraged by these results, we would like to identify other target proteins and, thereby, further explore the pharmacological profile of these molecules. An elegant method to screen for potential interaction partners is the so-called "protein-fishing" approach. Requirement is coupling of a functionalized small molecule to a solid phase which is used for biological experiments. Ideally, the pharmacophore of the small molecule remains intact as far as possible. Here, we describe the successful design and synthesis of functionalized fatty acid mimetics, thus providing an eligible starting point for solid-phase coupling and subsequent "protein-fishing" experiments.