The Gut Microbiota and Developmental Programming of the Testis in Mice

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.     

Proinsulin C-peptide interferes with insulin fibril formation

Insulin aggregation can prevent rapid insulin uptake and cause localized amyloidosis in the treatment of type-1 diabetes. In this study, we investigated the effect of C-peptide, the 31-residue peptide cleaved from proinsulin, on insulin fibrillation at optimal conditions for fibrillation. This is at low pH and high concentration, when the fibrils formed are regular and extended. We report that C-peptide then modulates the insulin aggregation lag time and profoundly changes the fibril appearance, to rounded clumps of short fibrils, which, however, still are Thioflavine T-positive. Electrospray ionization mass spectrometry also indicates that C-peptide interacts with aggregating insulin and is incorporated into the aggregates. Hydrogen/deuterium exchange mass spectrometry further reveals reduced backbone accessibility in insulin aggregates formed in the presence of C-peptide. Combined, these effects are similar to those of C-peptide on islet amyloid polypeptide fibrillation and suggest that C-peptide has a general ability to interact with amyloidogenic proteins from pancreatic β-cell granules. Considering the concentrations, these peptide interactions should be relevant also during physiological secretion, and even so at special sites post-secretory or under insulin treatment conditions in vivo.     

Lost in translation: The search for an in vitro screen for spermatogenic toxicity

The last two decades have seen an increasing search for in vitro models that can replace the use of animals for safety testing. We adapted the methods from a recent nonquantitative report of spermatogenesis occurring in ex vivo mouse testis explants and tried to develop them into a screening assay. The model consisted of small pieces of neonatal mouse testis (testis “chunks”), explanted and placed on pillars of agarose or chamber inserts, and cultured at the air–liquid interface. A peripheral torus‐shaped zone in these explants would often contain tubules showing spermatogenesis, while the middle of each chunk was often necrotic, depending on the thickness of the tissue. The endpoint was histology: what proportion of tubules in the “permissive torus” actually contained healthy pachytene spermatocytes or spermatids? Extensive statistical modeling revealed that a useful predictive model required more than 60% of these tubules to show spermatogenesis. Separately, the logistics of running this as a predictive assay require that the controls consistently produce ≥ 60% tubules with pachytenes and round spermatids, and achieving this level of spermatogenesis reliably and consistently every week proved ultimately not possible. Extensive trials with various media additions and amendments proved incapable of maintaining the frequency of spermatogenic tubules at consistently ≥ 60%. Congruent with Schooler's “decline effect”; generally, the more often we ran these cultures, the worse the performance became. We hope that future efforts in this area may use our experience as a starting point on the way to a fully productive in vitro model of spermatogenesis.     

Modular Coils with Low Hydrogen Content Especially for MRI of Dry Solids

IntroductionRecent advances have enabled fast magnetic resonance imaging (MRI) of solid materials. This development has opened up new applications for MRI, but, at the same time, uncovered new challenges. Previously, MRI-invisible materials like the housing of MRI detection coils are now readily depicted and either cause artifacts or lead to a decreased image resolution. In this contribution, we present versatile, multi-nuclear single and dual-tune MRI coils that stand out by (1) a low hydrogen content for high-resolution MRI of dry solids without artifacts; (2) a modular approach with exchangeable inductors of variable volumes to optimally enclose the given object; (3) low cost and low manufacturing effort that is associated with the modular approach; (4) accurate sample placement in the coil outside of the bore, and (5) a wide, single- or dual-tune frequency range that covers several nuclei and enables multinuclear MRI without moving the sample.

Materials and Methods

The inductors of the coils were constructed from self-supporting copper sheets to avoid all plastic materials within or around the resonator. The components that were mounted at a distance from the inductor, including the circuit board, coaxial cable and holder were manufactured from polytetrafluoroethylene.

Results and Conclusion

Residual hydrogen signal was sufficiently well suppressed to allow ¹H-MRI of dry solids with a minimum field of view that was smaller than the sensitive volume of the coil. The SNR was found to be comparable but somewhat lower with respect to commercial, proton-rich quadrature coils, and higher with respect to a linearly-polarized commercial coil. The potential of the setup presented was exemplified by ¹H / ²³Na high-resolution zero echo time (ZTE) MRI of a model solution and a dried human molar at 9.4 T. A full 3D image dataset of the tooth was obtained, rich in contrast and similar to the resolution of standard cone-beam computed tomography.     

Reassembly of somatic cells and testicular organogenesis in vitro

Testicular organogenesis in vitro requires an environment allowing a reassembly of testicular cell types. Previous in vitro studies using male murine germ cells cultured in a defined three-dimensional environment demonstrated tubulogenesis and differentiation into spermatozoa. Combining scaffolds as artificial culture substrates with testicular cell culture, we analysed the colonization of collagen sponges by rat testicular cells focusing on cell survival and reassembly of tubule-like-structures in vitro. Isolated testicular cells obtained from juvenile Sprague Dawley and eGFP transgenic rats were cultured on collagen sponges (DMEM high glucose + Glutamax, 35 °C, 5% CO₂ with or without gonadotropins). Live cell imaging revealed the colonization of cells across the entire scaffold for up to 35 days. After two days, histology showed cell clusters attached to the collagen fibres and displaying signs of tubulogenesis. Clusters consisted mainly of Sertoli and peritubular cells which surrounded some undifferentiated spermatogonia. Flow cytometry confirmed lack of differentiation as no haploid cells were detected. Leydig cell activity was detected by a rise of testosterone after gonadotropin stimulation. Our approach provides a novel method which is in particular suitable to follow the somatic testicular cells in vitro an issue of growing importance for the analysis of germ line independent failure of spermatogenesis.     

A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells

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.     

Single-cell analysis of the developing human testis reveals somatic niche cell specification and fetal germline stem cell establishment

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