Introduction of a Geminin mScarlet Reporter into H2B-mTurq2 hiPSCs for Live-cell Imaging of Proliferation and Cell Cycling.

We previously generated a doxycycline-inducible H2B-mTurq2 reporter in hiPSCs to track cells and study cell division and apoptosis. To improve visualization of cycling cells, we introduced a ubiquitously transcribed mScarletI-Geminin (GMMN) (1-110) into the previously untargeted second AAVS1 allele. Fusion to the N-terminal part of GMNN provided tightly controlled mScarletI expression during the cell cycle. mScarletI fluorescence increased gradually from the S-phase through the M-phase of the cell cycle and was lost at the metaphase-anaphase transition. The resulting hiPSC reporter line generated, which we named ProLiving, is a valuable tool to study cell division and cell cycle characteristics in living hiPSC-derived cells.

Generation of LUMCi041-A-2: Equipping a PAX3 reporter iPSC line with doxycycline inducible H2B-mTurquoise2 for live cell imaging.

An induced pluripotent stem cell (iPSC) line, in which a H2B-fluorescent protein fusion is temporally expressed, is a valuable tool to track cells and study cell divisions and apoptosis. To this end we introduced a 3rd generation "all-in-one" doxycycline-inducible H2B-mTurquoise2 vector into the AAVS1 locus of PAX3-Venus iPSCs via CRISPR/Cas9. H2B-mTurquoise2 expression is absent but readily induced by doxycycline allowing quantification of cell divisions and imaging of living cells. Besides being a universal reporter in iPSC-based differentiation and toxicity assays, the generated pluripotent and genomically normal LUMCi041-A-2 line is particularly suited to study PAX3-positive stages of development.

Advanced in silico validation framework for three-dimensional traction force microscopy and application to an in vitro model of sprouting angiogenesis.

In the last decade, cellular forces in three-dimensional hydrogels that mimic the extracellular matrix have been calculated by means of Traction Force Microscopy (TFM). However, characterizing the accuracy limits of a traction recovery method is critical to avoid obscuring physiological information due to traction recovery errors. So far, 3D TFM algorithms have only been validated using simplified cell geometries, bypassing image processing steps or arbitrarily simulating focal adhesions. Moreover, it is still uncertain which of the two common traction recovery methods, i.e., forward and inverse, is more robust against the inherent challenges of 3D TFM. In this work, we established an advanced in silico validation framework that is applicable to any 3D TFM experimental setup and that can be used to correctly couple the experimental and computational aspects of 3D TFM. Advancements relate to the simultaneous incorporation of complex cell geometries, simulation of microscopy images of varying bead densities and different focal adhesion sizes and distributions. By measuring the traction recovery error with respect to ground truth solutions, we found that while highest traction recovery errors occur for cases with sparse and small focal adhesions, our implementation of the inverse method improves two-fold the accuracy with respect to the forward method (average error of 23% vs. 50%). This advantage was further supported by recovering cellular tractions around angiogenic sprouts in an in vitro model of angiogenesis. The inverse method recovered higher traction peaks and a clearer pulling pattern at the sprout protrusion tips than the forward method. STATEMENT OF SIGNIFICANCE: Biomaterial performance is often studied by quantifying cell-matrix mechanical interactions by means of Traction Force Microscopy (TFM). However, 3D TFM algorithms are often validated in simplified scenarios, which do not allow to fully assess errors that could obscure physiological information. Here, we established an advanced in silico validation framework that mimics real TFM experimental conditions and that characterizes the expected errors of a 3D TFM workflow. We apply this framework to demonstrate the enhanced accuracy of a novel inverse traction recovery method that is illustrated in the context of an in vitro model of sprouting angiogenesis. Together, our study shows the importance of a proper traction recovery method to minimise errors and the need for an advanced framework to assess those errors.

Intrauterine management of fetal supraventricular tachycardia (SVT) with cardiac failure.

Fetal arrhythmias are not uncommon in pregnancy. The diagnosis can be established on routine ultrasound scan. Fetal supraventricular tachycardia (SVT) is the most common cause of fetal tachycardia. If left undiagnosed and untreated, these fetuses may develop cardiac failure, hydrops fetalis and eventually death. We report two fetuses diagnosed antenatally to have fetal SVT. Both fetuses were in cardiac failure and were successfully treated with maternal administration of antiarrhythmic medications. Digoxin, and in severe instances, a combination with flecanaide significantly improved fetal outcomes and prevented fetal mortality. The long term prognosis of such patients are good.

3D niche microarrays for systems-level analyses of cell fate.

The behaviour of mammalian cells in a tissue is governed by the three-dimensional (3D) microenvironment and involves a dynamic interplay between biochemical and mechanical signals provided by the extracellular matrix (ECM), cell-cell interactions and soluble factors. The complexity of the microenvironment and the context-dependent cell responses that arise from these interactions have posed a major challenge to understanding the underlying regulatory mechanisms. Here we develop an experimental paradigm to dissect the role of various interacting factors by simultaneously synthesizing more than 1,000 unique microenvironments with robotic nanolitre liquid-dispensing technology and by probing their effects on cell fate. Using this novel 3D microarray platform, we assess the combined effects of matrix elasticity, proteolytic degradability and three distinct classes of signalling proteins on mouse embryonic stem cells, unveiling a comprehensive map of interactions involved in regulating self-renewal. This approach is broadly applicable to gain a systems-level understanding of multifactorial 3D cell-matrix interactions.

Elucidating the role of matrix stiffness in 3D cell migration and remodeling.

Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena.

Large-displacement 3D structural analysis of an aortic valve model with nonlinear material properties.

Grafts used in aortic valve-sparing procedures should ideally not only reproduce the geometry of the natural aortic root but also its material properties. Indeed, a number of studies using the finite element method have shown the importance of the natural sinus shape of the root in the functioning of the normal aortic valve, and the relative increase in stresses due to the replacement of the valve by a stiffer synthetic graft. Because of the wide range in experimentally measured values of aortic wall and leaflet material properties, studies by different research groups have incorporated very different material properties in their models. The aim of the present study was to investigate the influence of material properties on aortic wall displacements, and to determine which material properties would most closely match reported experimental data. Two geometrically accurate 3D models corresponding to the closed and open valve configurations were created in Pro/Engineer CAD software. Loads corresponding to systolic and diastolic pressures were specified and large-displacement structural analyses were carried out using the ANSYS package. Results have indicated that the closest match to experiments using isotropic material properties occurred for a Young's modulus of about 2000 KPa. Nonlinear models based on experimental stress-strain curves have shown similar displacements, but altered strain distribution patterns and significantly lower stresses. These results suggest that an accurate comparison of potential new graft models would have to be made with natural aortic valve models incorporating nonlinear material behavior.

65Zn incorporation in the male reproductive organs following gossypol treatment.

Male hamsters and rats were administered gossypol 10 mg/kg/day for 45 and 56 days respectively. Twenty four hours before the last dose, animals were administered 65Zn (specific activity 0.258 uci/mg) subcutaneously. A marked decrease in 65Zn incorporation was observed in testis, epididymis, seminal vesicles and prostate following drug administration. A significant increase in 65Zn uptake was however observed in vas deferens in both rat and hamster following drug administration. Our results suggest that whatever the mechanism of gossypol action on testis-epididymis complex may be, the marked decrease in 65Zn uptake by testis--epididymis complex following gossypol treatment may be related to the antispermatogenic effect of gossypol.

Effect of gossypol on rats maintained on protein deficient and low potassium diets.

Sexually mature male albino rats were divided into four groups of 5 animals each. Animals of group I served as control, whereas animals of group II received gossypol (20 mg/kg body weight/day) for 45 days. Animals of groups III and IV were maintained on protein deficient diet. Animals of group IV received 20 mg/kg gossypol in addition to the protein deficient diet. Animals of group III and IV received protein deficient diet for 45 days before initiating gossypol treatment. The total period of maintaining the animals on protein deficient diet was 90 days. In another experiment, the same experimental protocol was followed except that the animals were maintained on low potassium diet instead of protein deficient diet. A significant decrease in body weight of animals was observed following protein deficient and gossypol (group IV) treatment. Testis weight decreased significantly in the animals of group III (protein deficient) and group IV (protein deficient + gossypol). Similar observations were made in the animals maintained on low potassium diet. In both the experiments, sperm motility was reduced significantly. Histologically, in the testis of animals of group IV (protein deficient + gossypol) almost all the tubules were disorganised and vacuolated and total arrest of spermatogenesis could be observed in majority of the tubules.

Spermatogenic events in rat and mice following intratesticular administration of optical isomers of gossypol.

Male rats and mice were administered racemic and dextrorotatory gossypol intratesticularly at the dose of 200 micrograms/testis. In a separate experiment, 100 micrograms of dextrorotatory and levorotatory gossypol was administered to male mice. Animals were sacrificed 72 hours after drug treatment. In another experiment, mice were sacrificed 3, 10, 20, 30 and 60 days after racemic gossypol (200 micrograms/testis) administration. Marked degenerative changes in rat and mice tests were observed in the animals receiving racemic gossypol. Dextrorotatory gossypol had less pronounced effect, both in rat and mice. Stage 7 and 16 spermatids were most susceptible to the deleterious effects of racemic gossypol. In mice, 57.52% and 85.40% decrease in stage 7 and 16 spermatids were observed 72 hrs after drug administration. A progressive recovery was observed after drug treatment; the damage (stage 7 & 16) was reduced to 7.92% and 21.30% after 60 days. Histopathological changes in mice testis following 100 micrograms of levorotatory gossypol were distinctly different from those of the dextrorotatory (100 micrograms/testis) gossypol. On the basis of observations made by us, it can be suggested that mice equally respond to the antifertility effect of gossypol following intratesticular administration. The dextrorotatory gossypol, both in rat and mice, had less pronounced effect on the histoarchitecture of the testis in comparison to racemic and levorotatory gossypol. Our observations further suggest that this animal model provides meaningful information on the mechanism of action of gossypol, and recovery of spermatogenesis is possible after termination of drug treatment.

Response of mice testis to gossypol acetic acid.

Male mice were administered gossypol (2.5, 5.0, 10.0 and 20.0 mg/kg body weight) by oral intubation, subcutaneous and intraperitoneal routes for 30 days. The drug treatment did not have any effect on body weight and organ weights. The motility of cauda epididymis and vas deferens spermatozoa was not affected by gossypol treatment nor there was any effect of gossypol on the histoarchitecture of the testis irrespective of the dose and route of administration. Maximum mortality was observed in mice when animals were given gossypol by intraperitoneal route.

Recovery of normal testis function and sperm motility after cessation of gossypol treatment in hamster.

The present investigations evaluate the reversibility of the effect of gossypol on the histoarchitecture of the hamster testis and the motility of the vas deferens spermatozoa. Adult male hamsters of proven fertility were treated orally with gossypol (10 mg/kg/day) for 13 weeks (group III). Another group of animals in the Group IV were given gossypol (10 mg/kg/day) for 13 weeks and were sacrificed 13 weeks after termination of the drug treatment. Animals in groups I and II were given vehicle and served as controls for groups III and IV respectively. To all the animals, gossypol or vehicle was administered by oral intubation. A marked reduction in the spermatozoa motility was observed in the animals of group III. In contrast, the motility of the spermatozoa in the animals of group IV (recovery phase) was in the normal range. A large number of seminiferous tubules were affected by gossypol treatment (group III); these tubules showed degeneration of germ cells, vacuolization and loosening of the germinal epithelium. In the quantitative analysis of the spermatogenic events, the ratio of resting spermatocyte/'A' type spermatogonia was not affected but the ratios of pachytene spermatocytes/resting spermatocytes and stage 19 spermatids/stage 7 spermatids decreased by 21%, 47% and 52% respectively. The tubular diameter and germinal height were decreased by 14% and 33% respectively. In group IV, hamsters were allowed to recover from the gossypol treatment. The seminiferous tubules exhibited normal histoarchitecture and the tubular diameter and germinal height recovered markedly. Recovery in different stages of spermatogenic cycle was evidently clear in this group.(ABSTRACT TRUNCATED AT 250 WORDS)

Early events in rat testis after gossypol administration.

Eighty male rats were grouped into 8 groups of 10 animals each. Animals in groups I-IV were given gossypol (40 mg/kg/day) for 7, 14, 21 and 28 days respectively. Animals of groups V-VIII served as respective controls for groups I-IV. Marked changes in the activities of ATPase and SDH were observed following drug treatment. Decrease in the activity of testis LDH was evident even after 7 days of drug treatment. Activities of B-galactosidase, Glucose-6-phosphatase and Fructose-1-6-diphosphatase were not affected by gossypol treatment. Glycogen contents in testis were not different from those of the controls. A significant decrease in the tubular diameter and germinal height of the seminiferous tubules was observed after 21 days of drug treatment. Quantitative analysis of spermatogenic elements revealed marked decrease in the ratios of resting spermatocyte. A type spermatogonia, pachytene spermatocyte/resting spermatocyte, and stage 19 spermatids/stage 7 spermatids after 7 days of drug treatment. A progressive decrease in the ratios of these cell types was observed as the duration of the drug treatment was extended. Liver enzymes (except SDH and LDH after 28 days of drug treatment) were not affected by gossypol treatment. Our data strongly suggest that degenerative changes in the testis start after one week of drug administration. The histological changes visible at light microscopy level start appearing after 14 days of drug treatment.

Response of hamster to the antifertility effect of gossypol.

One hundred and five sexually mature male hamsters were divided in different groups. In the first experiment hamsters were administered gossypol, 10 mg/kg and 20 mg/kg/body weight/day, for twenty and thirty days. In the second experiment hamsters were administered gossypol, 5, 10 and 20 mg/kg/body weight/day, for sixty days. In the third experiment, hamsters were administered gossypol 5 mg, 10 mg, 20 mg and 40 mg/kg body weight/day for 45 days. Animals in all the groups were given gossypol by oral intubation every day. No significant effect on the body weight of hamsters following gossypol treatment was observed. At low doses the weights of testis and accessory sex organs were not statistically different from those of the controls. A significant decrease in testis and epididymis weight was however observed following high doses of gossypol. Low doses of gossypol treatment did not affect the motility of the vas deferens spermatozoa. The vas deferens spermatozoa were however immotile after 40 mg/kg/day gossypol treatment. Gossypol treatment induced a series of histological changes in the seminiferous epithelium of the hamster testis. The earliest sign of drug effect was seen in spermatids and with the increase in doses the effects became more pronounced and extended to the spermatocytes. At 40 mg/kg dose an almost complete arrest of spermatogenesis was observed. Quantitatively, the ratio of pachytene spermatocytes: resting spermatocytes and step 7 spermatids: pachytene spermatocytes decreased significantly. The step 7 spermatids did not mature to step 19 spermatids at all. Histochemically activities of ATPase, SDH and LDH decreased with the increasing doses of gossypol, the activity of 3B hydroxysteroid dehydrogenase was not affected by gossypol treatment. In testis the glucose-6-phosphatase activity was not affected significantly but the activities of fructose 1, 6-diphosphatase and glucose-6-phosphate isomerase decreased significantly with the increasing doses of gossypol. Amylase activity rose significantly at higher doses. Marked changes in LDH and LDH-X were however observed with the increase in gossypol dose. In liver the activity of glucose-6-phosphatase increased significantly while the activities of fructose 1, 6-diphosphatase, glucose-6-phosphate isomerase and amylase were not affected following gossypol treatment. The glycogen contents however increased significantly following high doses of gossypol. No changes in testosterone production and plasma levels of testosterone were observed following gossypol treatment.

Effect of gossypol on the fertility of male rats.

Thirty male rats were grouped into 5 groups of 6 animals each. Animals in groups II-V were given gossypol at a dose of 5 mg/kg, 10 mg/kg, 20 mg/kg and 40 mg/kg body weight per day for 45 days respectively. Animals of group I served as control. A significant decrease in body weight after administration of 40 mg/kg body weight of gossypol was observed; low doses of gossypol, however did not affect the body weight. Testis, epididymis, prostate and seminal vesicles weights decreased gradually with the increasing doses of gossypol. With the increasing doses of gossypol, a marked decrease in the vas deferens sperm motility was observed. At 40 mg/kg dose there was a total inhibition of sperm motility. Histological studies after 5 mg/kg revealed no apparent sign of degeneration, while after 10 mg/kg dose the changes in the individual cell types were accompanied by overall disorganisation of the germinal epithelium involving displacement of the spermatocytes. The rats treated with 20-40 mg/kg gossypol showed a pronounced deleterious effect on the histological structure of the testis. The drug effect was dose dependent developing sequentially; from the uppermost layer of elongated spermatids affecting round spermatids and finally spermatocytes. Quantitatively the ratios of pachytene spermatocytes: resting spermatocytes, stage 7 spermatids: pachytene spermatocytes, and stage 19 spermatids: stage 7 spermatids and tubular diameter and germinal height decreased significantly. The activities of glucose-6-phosphatase, fructose 1, 6-diphosphatase, glucose-6-phosphate isomerase in testis decreased significantly at high dose (40 mg/kg), while the activity of amylase and glycogen content increased significantly with the increasing doses of gossypol.(ABSTRACT TRUNCATED AT 250 WORDS)

Gossypol inhibition of Ca++ uptake and Ca++-ATPase in human ejaculated spermatozoal plasma membrane vesicles.

Gossypol, a plant-derived polyphenolic compound known to exert contraceptive actions in men, inhibits Ca++-transport and Ca++-activated ATPase in isolated plasma membranes of ejaculated human sperm cells. It also inhibits the membrane bound Mg++- and Na+ + K+-dependent ATPases, 5'-nucleotidase and alkaline phosphatase systems. Ca++-ATPase inhibition by gossypol is non-competitive. It abolishes the discontinuity in Arrhenius expression of temperature dependence of Ca++-ATPase and increases the energy of activation. Phosphatidyl choline and Na+-deoxycholate inhibit Ca++-transport activity in the membrane vesicles. The apparent similarity of Ca++-transport inhibition by gossypol and phosphatidyl choline may indicate the possible capability of this compound to induce changes in the lipid microenvironment of the membranes, wherein the integral proteins operate. Inhibitory effect of gossypol on the plasma membrane Ca++-pump suggests that gossypol may affect sperm motility by a mechanism which is related to the structure and functions of the plasma membrane.

Involvement of prostaglandin in the antifertility effects of gossypol.

This study was undertaken to determine the effects of gossypol alone and gossypol in combination with prostaglandin and aspirin. Rats were administered gossypol (40 mg/kg/day), gossypol and prostaglandin (PGF2 alpha-2 mg/kg/day), gossypol and aspirin (300 mg/kg) for 4 weeks. A marked effect of the gossypol-prostaglandin combination was observed on sperm motility and spermatogenesis. The effect of the gossypol-aspirin combination was less pronounced. The ratio of body weight to testicular and epididymal weights between the different groups showed no marked difference. No effect of drug treatment on plasma testosterone, LH and FSH was observed. The data presented in this paper suggest that prostaglandin plays an important role in the antifertility effects of gossypol.