Search

The transport and distribution of charged molecules in polyelectrolyte solutions are of both fundamental and practical importance. A practical example, which is the specific subject addressed in the present paper, is the transport and distribution of charged species into cartilage. The charged species could be a contrast agent or a drug molecule involved in diagnosis or treatment of the widespread degenerative disease osteoarthritis, which leads to degradation of articular cartilage. Associated scientific issues include the rate of transport and the equilibrium concentrations of the charged species in the cartilage and the synovial fluid. To address these questions, we present results from magnetic resonance micro-imaging experiments on a model system of articular cartilage. The experiments yield temporally and spatially resolved data on the transport of a negatively charged contrast agent (charge = -2), used in medical examinations of cartilage, into a polyelectrolyte solution, which is designed to capture the electrostatic interactions in cartilage. Also presented is a theoretical analysis of the transport where the relevant differential equations are solved using finite element techniques as well as treated with approximate analytical expressions. In the analysis, non-ideal effects are included in the treatment of the mobile species in the system. This is made possible by using results from previous Monte Carlo simulations. The results demonstrate the importance of taking non-idealities into account when data from measurements of transport of charged solutes in a system with fixed charges from biological polyelectrolytes are analyzed.

Water transport across cell membranes can be measured non-invasively with diffusion NMR. We present a method to quantify the intracellular lifetime of water in cell suspensions with short transverse relaxation times, T2, and also circumvent the confounding effect of different T2 values in the intra- and extracellular compartments. Filter exchange spectroscopy (FEXSY) is specifically sensitive to exchange between compartments with different apparent diffusivities. Our investigation shows that FEXSY could yield significantly biased results if differences in T2 are not accounted for. To mitigate this problem, we propose combining FEXSY with diffusion-relaxation correlation experiment, which can quantify differences in T2 values in compartments with different diffusivities. Our analysis uses a joint constrained fitting of the two datasets and considers the effects of diffusion, relaxation and exchange in both experiments. The method is demonstrated on yeast cells with and without human aquaporins.

The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, in vitro germ cell differentiation, and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition, we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL, our results suggest a post-transcriptional regulation mechanism in hES cells. In addition, we found that DAZL suppressed the translation of OCT4, and affected the transcription of several genes associated with germ cells, cell cycle arrest, and cell migration. Furthermore, DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo.

Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

Nutrients and environmental chemicals, including endocrine disruptors, have been incriminated in the current increase in male reproductive dysfunction, but the underlying mechanisms remain unknown. The gastrointestinal tract represents the largest surface area exposed to our environment and thereby plays a key role in connection with exposure of internal organs to exogenous factors. In this context the gut microbiome (all bacteria and their metabolites) have been shown to be important contributors to body physiology including metabolism, cognitive functions and immunity. Pivotal to male reproduction is a proper development of the testis, including the formation of the blood-testis barrier (BTB) that encapsulates and protects germ cells from stress induced environmental cues, e.g. pathogenic organisms and xenobiotics. Here we used specific pathogen free (SPF) mice and germ-free (GF) mice to explore whether gut microbiota and/or their metabolites can influence testis development and regulation of BTB. Lumen formation in the seminiferous tubules, which coincides with the development of the BTB was delayed in the testes of GF mice at 16 days postpartum. In addition, perfusion experiments (Evans blue) demonstrated increased BTB permeability in these same mice. Reduced expressions of occludin, ZO-2 and E-cadherin in GF testis suggested that the microbiota modulated BTB permeability by regulation of cell-cell adhesion. Interestingly, exposure of GF mice to Clostridium Tyrobutyricum (CBUT), which secrete high levels of butyrate, restored the integrity of the BTB and normalized the levels of cell adhesion proteins. Moreover, the GF mice exhibited lower serum levels of gonadotropins (LH and FSH) than the SPF group. In addition, the intratesticular content of testosterone was lower in GF compared to SPF or CBUT animals. Thus, the gut microbiome can modulate the permeability of the BTB and might play a role in the regulation of endocrine functions of the testis.

It is well known that estrogens and estrogen-like endocrine disruptors can suppress steroidogenic gene expression, attenuate androgen production and decrease differentiation of adult Leydig cell lineage. However, there is no information about the possible link between the potency of Leydig cells to produce androgens and their sensitivity to estrogenic stimuli. Thus, the present study explored the relationship between androgen production potential of Leydig cells and their responsiveness to estrogenic compounds. To investigate this relationship we selected mouse genotypes contrasting in sex hormone levels and differing in testosterone/estradiol (T/E2) ratio. We found that two mouse genotypes, CBA/Lac and C57BL/6j have the highest and the lowest serum T/E2 ratio associated with increased serum LH level in C57BL/6j compared to CBA/Lac. Analysis of steroidogenic gene expression demonstrated significant upregulation of Cyp19 gene expression but coordinated suppression of LHR, StAR, 3βHSDI and Cyp17a1 in Leydig cells from C57BL/6j that was associated with attenuated androgen production in basal and hCG-stimulated conditions compared to CBA/Lac mice. These genotype-dependent differences in steroidogenesis were not linked to changes in the expression of estrogen receptors ERα and Gpr30, while ERβ expression was attenuated in Leydig cells from C57BL/6j compared to CBA/Lac. No effects of estrogenic agonists on steroidogenesis in Leydig cells from both genotypes were found. In contrast, xenoestrogen bisphenol A significantly potentiated hCG-activated androgen production by Leydig cells from C57BL/6j and CBA/Lac mice by suppressing conversion of testosterone into corresponding metabolite 5α-androstane-3α,17β-diol. All together our data indicate that developing mouse Leydig cells with different androgen production potential are resistant to estrogenic stimuli, while xenoestrogen BPA facilitates hCG-induced steroidogenesis in mouse Leydig cells via attenuation of testosterone metabolism. This cellular event can cause premature maturation of Leydig cells that may create abnormal intratesticular paracrine milieu and disturb proper development of germ cells.