In the hypertrophic heart the myostructural afterload in the form of endoepicardial networks is predominant, which enhances myocardial hypertrophy. The intrinsic antagonism is derailed. Likewise, the connective tissue scaffold, i.e. the stromatogenic afterload, is enriched in the response to the derailment of antagonism in a hypertrophic heart up to regional captivation of the heart musculature. Due to the selective susceptibility of the auxotonic, contracting oblique transmural myocardial network for low dose negative inotropic medication, this promises to attenuate progress in myocardial hypertrophy. Volume reduction surgery is most effective in reducing wall stress as long as the myocardium is not critically fettered by fibrosis. The use of external mechanical circulatory support is then effective if the heart is supported in its resting mode, which means around a middle width and at minimal amplitude of motion. The takotsubo cardiomyopathy might possibly reflect an isolated, extreme stimulation of the intrinsic antagonism as a response to hormonally induced sensitization of the myocardium to catecholamine. A particular significant conclusion with respect to the diseased heart is that clinical diagnostics need new impulses with a focus on the analysis of local motion patterns and on myocardial stiffness reflecting disease-dependent antagonistic intensity. This would become a relevant diagnostic marker if corresponding (noninvasive) measurement techniques would become available.

Successful in vitro spermatogenesis was reported using immature mouse testicular tissues in a fragment culture approach, raising hopes that this method could also be applied for fertility preservation in humans. Although maintaining immature human testicular tissue fragments in culture is feasible for an extended period, it remains unknown whether germ cell survival and the somatic cell response depend on the differentiation status of tissue. Employing the marmoset monkey (Callithrix jacchus) we aimed to assess whether the maturation status of prepubertal and peri-/pubertal testicular tissues influence the outcome of testis fragment culture. Testicular tissue fragments from 4 and 8-month-old (n = 3, each) marmosets were cultured and evaluated after 0, 7, 14, 28 and 42 days. Immunohistochemistry was performed for identification and quantification of germ cells (melanoma-associated antigen 4) and Sertoli cell maturation status (anti-Müllerian hormone: AMH). During testis fragment culture, spermatogonial numbers were significantly reduced (p < 0.05) in the 4- but not 8-month-old monkeys, at day 0 versus day 42 of culture. Moreover, while Sertoli cells from 4-month-old monkeys maintained an immature phenotype (i.e. AMH expression) during culture, AMH expression was regained in two of the 8-month-old monkeys. Interestingly, progression of differentiation to later meiotic stage was solely observed in one 8-month-old marmoset, which was at an intermediate state regarding germ cell content, with gonocytes as well as spermatocytes present, as well as Sertoli cell maturation status. Although species-specific differences might influence the outcome of testis fragment experiments in vitro, our study demonstrated that the developmental status of the testicular tissues needs to be considered as it seems to be decisive for germ cell maintenance, somatic cell response and possibly the differentiation potential.

In der Tradition Harveys besteht nach Otto Frank die Herzmuskelwand allein aus tangential ausgerichteten Muskelfasern. Sie sollen aktiv nur die systolische Kammerentleerung betreiben. Tatsächlich ist der Herzmuskel ein räumlich vernetztes Flechtwerk mit 2/5 aller Myozytenketten, die in unterschiedlichen Winkeln von der tangentialen Anordnung abweichen. So entsteht neben einer konstriktiven auch eine im Vergleich zur konstriktiven wesentlich geringere dilative Kraft. Mit Nadelkraftaufnehmern haben wir entsprechend einen „Entlastungstyp“, der hauptsächlich konstriktiv wirkt und eine „auxotone“, z. T. dilativ wirkende Kontraktionskurve gemessen. Dieser Antagonismus unterstützt die Autoregulation der Herzmuskelfunktion nach Frank-Starling, wirkt formgebend, unterstützt die spätsystolische rasche Kammerdilation und begrenzt die systolische Konstriktion der Kammerwand. Die antagonistischen dilativen Kräfte reagieren besonders empfindlich auf Inotropika. Mit niedrig dosierten negativen Inotropika lassen sich die antagonistischen Kräfte isoliert abschwächen. Alle myokardialen Vernetzungsstrukturen verschiedener Richtungen arbeiten an 4 Nachlasten, jeweils an einer hämodynamischen, einer myostrukturellen, einer stromatogenen und an einer hydraulischen. Dieses Wechselspiel zwischen Myokardarbeit und Nachlast ändert sich funktions- und strukturabhängig; das erschwert die klinische Vermessung der Myokardfunktion. Die klinischen Auswirkungen sind weitreichend (siehe Teil II,  https://doi.org/10.1007/s00059-018-4735-x).

Life-long sperm production leads to the assumption that male fecundity remains unchanged throughout life. However, recently it was shown that paternal age has profound consequences for male fertility and offspring health. Paternal age effects are caused by an accumulation of germ cell mutations over time, causing severe congenital diseases. Apart from these well-described cases, molecular patterns of ageing in germ cells and their impact on DNA integrity have not been studied in detail. In this study, we aimed to assess the effects of 'pure' ageing on male reproductive health and germ cell quality. We assembled a cohort of 198 healthy men (18-84 years) for which end points such as semen and hormone profiles, sexual health and well-being, and sperm DNA parameters were evaluated. Sperm production and hormonal profiles were maintained at physiological levels over a period of six decades. In contrast, we identified a germ cell-specific ageing pattern characterized by a steady increase of telomere length in sperm and a sharp increase in sperm DNA instability, particularly after the sixth decade. Importantly, we found sperm DNA methylation changes in 236 regions, mostly nearby genes associated with neuronal development. By in silico analysis, we found that 10 of these regions are located in loci which can potentially escape the first wave of genome-wide demethylation after fertilization. In conclusion, human male germ cells present a unique germline-specific ageing process, which likely results in diminished fecundity in elderly men and poorer health prognosis for their offspring.

Im hypertrophen Herzen dominiert die myostrukturelle Nachlast in Form der endoepikardialen Vernetzung. Ihre Verdichtung beschleunigt das Fortschreiten der Hypertrophie. Der Antagonismus ist entgleist. Auch die bindegewebige Stützstruktur, die stromatogene Nachlast also, reichert sich in Antwort auf die Entgleisung des Antagonismus im hypertrophen Herzen an bis hin zur regionalen Fesselung des Herzmuskels. Wegen der selektiven Empfindlichkeit der auxoton sich kontrahierenden, schräg transmuralen Vernetzungen des Herzmuskels für niedrig dosierte negative Inotropika versprechen diese, das Fortschreiten der Myokardhypertrophie zu dämpfen. Die Verkleinerung des Kammervolumens durch die Reduktionschirurgie erweist sich als hoch wirksam im Sinne einer Abnahme der Wandspannung, solange die Herzen nicht kritisch vernarbt sind. Eine Unterstützung der Herzfunktion durch eine externe Pumpe kommt dann zur vollen Wirkung, wenn das Herz um seinen Ruhemodus, nämlich um eine mittlere Weite bei minimaler Bewegungsamplitude, unterstützt wird. Die Tako-Tsubo-Kardiomyopathie beruht möglicherweise auf einer isolierten, extremen Stimulation des inneren Antagonismus nach hormonell induzierter Sensibilisierung des Myokards gegen Katecholamine. Die klinische Diagnostik bedarf einer Neuorientierung einerseits im Sinne einer Analyse des lokalen Bewegungsablaufs der Kammerwände, andererseits wird die diagnostisch potenziell interessante Zielgröße die Myokardsteife sein, welche die krankheitsbedingte Intensität der antagonistischen Wirkung reflektiert. Entsprechende (nicht-invasive) Messmethoden müssen dazu allerdings noch verfügbar gemacht werden.

AbstractChildren of older fathers have higher risk for certain diseases. Nevertheless, how ageing specifically affects male germ cells is so far not completely understood. In a cohort of 197 healthy men (18-84 years), we found that semen and reproductive parameters remained normal over six decades. Along with an age-dependent increase in telomere length in sperm (r=0.41, p>0.001), we found accelerated DNA fragmentation, more prominent after the sixth decate of life, and with around 60% of men older than 66 showing abnormal levels of DNA breaks. At the epigenetic level, by whole genome bisulfite sequencing we identified 236 sperm-specific differentially methylated regions between the youngest and oldest group, affecting mostly regions associated with homeobox genes and nervous system development. Therefore, we propose that during ageing, male germ cells are affected by an intrinsic and specific ageing process, distinguishable from the soma. These age-dependent changes might have consequences for fertility and offspring of older men.

STUDY QUESTION:Is Raman micro-spectroscopy a valid approach to assess the biochemical hallmarks of sperm regions (head, midpiece and tail) in four different species?SUMMARY ANSWER:Non-invasive Raman micro-spectroscopy provides spectral patterns enabling the biochemical characterization of the three sperm regions in the four species, revealing however high similarities for each region among species.WHAT IS KNOWN ALREADY:Raman micro-spectroscopy has been described as an innovative method to assess sperm features having the potential to be used as a non-invasive selection tool. However, except for nuclear DNA, the identification and assignment of spectral bands in Raman-profiles to the different sperm regions is scarce and controversial.STUDY DESIGN SIZE, DURATION:Raman spectra from head, midpiece and tail of four different species were obtained. Sperm samples were collected and smeared on microscope slides. Air dried samples were subjected to Raman analysis using previously standardized procedures.PARTICIPANTS/MATERIALS, SETTING, METHODS:Sperm samples from (i) two donors attending the infertility clinic at the Centre of Reproductive Medicine and Andrology; (ii) two C57BL/6 -TgN (ACTbEGFP) 1Osb adult mice; (iii) two adult Cynomolgus monkeys (Macaca fascicularis) and (iv) two sea urchins (Arbacia punctulata) were used to characterize and compare their spectral profiles. Differences and similarities were confirmed by principal component analysis (PCA).MAIN RESULTS AND THE ROLE OF CHANCE:Several novel region-specific peaks were identified. The three regions could be differentiated by distinctive Raman patterns irrespective of the species. However, regardless of the specie, their main spectral pattern remains mostly unchanged. These results were corroborated by the PCA analysis and suggest that the basic constituents of spermatozoa are biochemically similar among species.LIMITATIONS REASONS FOR CAUTION:Further research should be performed in live sperm to validate the detected spectral bands and their use as markers of distinctive regions.WIDER IMPLICATIONS OF THE FINDINGS:Raman peaks that have never been described in the sperm cell were detected. Particularly important are those that are unique to the midpiece as they might be a reference to the identification of sperm mitochondria, whose function is highly correlated with that of sperm. In the future, Raman micro-spectroscopy has the potential to be applied in assessment of male fertility.LARGE SCALE DATA:N/A.STUDY FUNDING AND COMPETING INTEREST(S):This work was supported by BMBF project 'Sperm Ident' (FKZ:13N13024) and the DAAD-CRUP bilateral exchange program (AI A06/16-57213087). S.A. is a recipient of a fellowship from the Portuguese foundation for science and technology (FCT-SFRH/BPD/110160/2015) and R.DC. is a recipient of a DAAD PhD stipend (91590556). There is no competing interest.

Raman Microspectroscopy represents an innovative tool for the assessment of sperm biochemical features otherwise undetectable by routine semen analysis. Previously, it was shown that induced DNA damage can be detected in smeared sperm by this technique. This novel readout may be of value for clinical settings especially if it can be transferred to living cells. Yet, starting with living sperms this study was carried-out using a variety of conditions to disclose the Raman features of sperm nuclei under different hydration conditions and UV exposure. Human sperm were immobilized and Raman spectra were obtained from individual sperm as repeated measurements. To create conditions with controlled DNA damage, sperm samples were exposed to ultraviolet light. Several media were used to evaluate their effect on Raman spectra in aqueous conditions. To substantiate differences between the experimental conditions, the spectra were analyzed by Principal Component Analysis. We observed that spectra of sperm nuclei obtained in different solutions showed a qualitatively unchanged spectral pattern showing the principal signals related to DNA. Evaluating the effect of ultraviolet light generated the finding that spectra representing DNA damage were only observed in dry conditions but not in aqueous medium. Thus, Raman microspectroscopy was successfully applied for sperm analysis in different conditions, among them in live spermatozoa in aqueous solution during the initial measurement, revealing the principle use of this technique. However, implementation of Raman spectroscopy as a technique for clinical sperm analysis and selection may be especially relevant when DNA evaluation can be established using live sperm.

STUDY QUESTION:Can enzymatically dispersed testicular cells from adult men reassemble into seminiferous cord-like structures in vitro?SUMMARY ANSWER:Adult human testicular somatic cells reassembled into testicular cord-like structures via dynamic interactions of Sertoli and peritubular cells.WHAT IS KNOWN ALREADY:In vitro approaches using dispersed single cell suspensions of human testes to generate seminiferous tubule structures and to initiate their functionality have as yet shown only limited success.STUDY DESIGN, SIZE, DURATION:Testes from 15 adult gender dysphoria patients (mean ± standard deviation age 35 ± 9.3 years) showing spermatogonial arrest became available for this study after sex-reassignment surgery. In vitro primary testicular somatic cell cultures were generated to explore the self-organizing ability of testicular somatic cells to form testis cords over a 2-week period. Morphological phenotype, protein marker expression and temporal dynamics of cell reassembly were analyzed.PARTICIPANTS/MATERIALS, SETTING, METHODS:Cell suspensions obtained by two-step enzymatic digestion were plated onto glass coverslips in 24-well plates. To obtain adherent somatic cells, the supernatant was discarded on Day 2. The culture of the attached cell population was continued. Reassembly into cord-like structures was analyzed daily by microscopic observations. Endpoints were qualitative changes in morphology. Cell types were characterized by phase-contrast microscopy and immunohistochemistry. Dynamics of cord formation were recorded by time-lapse microscopy.MAIN RESULTS AND THE ROLE OF CHANCE:Primary adult human testicular cells underwent sequential morphological changes including compaction and reaggregation resulting in round or elongated cord-like structures. Time-lapse video recordings within the first 4 days of culture revealed highly dynamic processes of migration and coalescence of reaggregated cells. The cellular movements were mediated by peritubular cells. Immunohistochemical analysis showed that both SRY-related high mobility box 9-positive Sertoli and α-smooth muscle actin-positive peritubular myoid cells interacted and contributed to cord-like structure formation.LARGE SCALE DATA:Not applicable.LIMITATIONS, REASONS FOR CAUTION:Owing to scarcity of normal human testicular tissue, testes from gender dysphoria patients were used in the study. The regressed status might influence the experimental responses of primary cells. We observed basic morphological features resembling in vivo testicular cords, however, the proof of functionality (e.g. support of germ cells) will need further studies.WIDER IMPLICATIONS OF THE FINDINGS:The proposed in vitro culture system may open opportunities for examination of testicular cell interactions during testicular tubulogenesis. Further refinement of our approach may enable initiation of ex vivo spermatogenesis.STUDY FUNDING/COMPETING INTERESTS:The work was supported by EU-FP7-PEOPLE-2013-ITN 603568: 'Growsperm'. No conflict of interests is declared.

Zusammenfassung In der Tradition Harveys besteht nach Otto Frank die Herzmuskelwand allein aus tangential ausgerichteten Muskelfasern. Sie sollen aktiv nur die systolische Kammerentleerung betreiben. Tatsächlich ist der Herzmuskel ein räumlich vernetztes Flechtwerk mit 2/5 aller Myozytenketten, die in unterschiedlichen Winkeln von der tangentialen Anordnung abweichen. So entsteht neben einer konstriktiven auch eine im Vergleich zur konstriktiven wesentlich geringere dilative Kraft. Mit Nadelkraftaufnehmern haben wir entsprechend einen „Entlastungstyp“, der hauptsächlich konstriktiv wirkt und eine „auxotone“, z. T. dilativ wirkende Kontraktionskurve gemessen. Dieser Antagonismus unterstützt die Autoregulation der Herzmuskelfunktion nach Frank-Starling, wirkt formgebend, unterstützt die spätsystolische rasche Kammerdilation und begrenzt die systolische Konstriktion der Kammerwand. Die antagonistischen dilativen Kräfte reagieren besonders empfindlich auf Inotropika. Mit niedrig dosierten negativen Inotropika lassen sich die antagonistischen Kräfte isoliert abschwächen. Alle myokardialen Vernetzungsstrukturen verschiedener Richtungen arbeiten an 4 Nachlasten, jeweils an einer hämodynamischen, einer myostrukturellen, einer stromatogenen und an einer hydraulischen. Dieses Wechselspiel zwischen Myokardarbeit und Nachlast ändert sich funktions- und strukturabhängig; das erschwert die klinische Vermessung der Myokardfunktion. Die klinischen Auswirkungen sind weitreichend (siehe Teil II, https://doi.org/10.1007/s00059-018-4735-x ).

Recent morphological studies provide evidence that the ventricular walls are arranged as a 3D meshwork of aggregated cardiomyocyte chains, exhibiting marked local structural variations. In contrary to previous findings, up to two-fifths of the chains are found to have a partially transmural alignment, thus deviating from the prevailing tangential orientation. Upon contraction, they produce, in addition to a tangential force, a radial force component that counteracts ventricular constriction and aids widening of the ventricular cavity. In experimental studies, we have provided evidence for the existence of such forces, which are auxotonic in nature. This is in contrast to the tangentially aligned myocytes that produce constrictive forces, which are unloading in nature. The ventricular myocardium is, therefore, able to function in an antagonistic fashion, with the prevailing constrictive forces acting simultaneously with a dilatory force component. The ratio of constrictive to dilating force varies locally according to the specific mural architecture. Such antagonism acts according to local demands to preserve the ventricular shape, store the elastic energy that drives the fast late systolic dilation and apportion mural motion to facilitate the spiralling nature of intracavitary flow. Intracavitary pressure and flow dynamics are thus governed concurrently by ventricular constrictive and dilative force components. Antagonistic activity, however, increases deleteriously in states of cardiac disease, such as hypertrophy and fibrosis. ß-blockade at low dosage acts selectively to temper the auxotonic forces.

Purpose: There are ongoing arguments as to how cardiomyocytes are aggregated together within the ventricular walls. We used pneumatic distension through the coronary arteries to exaggerate the gaps between the aggregated cardiomyocytes, analyzing the pattern revealed using computed tomography, and validating our findings by histology. Methods: We distended 10 porcine hearts, arresting 4 in diastole by infusion of cardioplegic solutions, and 4 in systole by injection of barium chloride. Mural architecture was revealed by computed tomography, measuring also the angulations of the long chains of cardiomyocytes. We prepared the remaining 2 hearts for histology by perfusion with formaldehyde. Results: Increasing pressures of pneumatic distension elongated the ventricular walls, but produced insignificant changes in mural thickness. The distension exaggerated the spaces between the aggregated cardiomyocytes, compartmenting the walls into epicardial, central, and endocardial regions, with a feathered arrangement of transitions between them. Marked variation was noted in the thicknesses of the parts in the different ventricular segments, with no visible anatomical boundaries between them. Measurements of angulations revealed intruding and extruding populations of cardiomyocytes that deviated from a surface-parallel alignment. Scrolling through the stacks of tomographic images revealed marked spiraling of the aggregated cardiomyocytes when traced from base to apex. Conclusion: Our findings call into question the current assumption that cardiomyocytes are uniformly aggregated together in a tangential fashion. There is marked heterogeneity in the architecture of the different ventricular segments, with the aggregated units never extending in a fully transmural fashion. Key Points: • Pneumographic computed tomography reveals an organized structure of the ventricular walls.• Aggregated cardiomyocytes form a structured continuum, with marked regional heterogeneity.• Global ventricular function results from antagonistic forces generated by aggregated cardiomyocytes. Citation Format: • Burg MC, Lunkenheimer P, Niederer P et al. Pneumatic Distension of Ventricular Mural Architecture Validated Histologically. Fortschr Röntgenstr 2016; 188: 1045 - 1053.

Establishment and maintenance of the correct epigenetic code is essential for a plethora of physiological pathways and disturbed epigenetic patterns can provoke severe consequences, e.g. tumour formation. In recent years, epigenetic drugs altering the epigenome of tumours actively have been developed for anti-cancer therapies. However, such drugs could potentially also affect other physiological pathways and systems in which intact epigenetic patterns are essential. Amongst those, male fertility is one of the most prominent. Consequently, we addressed possible direct effects of two epigenetic drugs, decitabine and vorinostat, on both, the male germ line and fertility. In addition, we checked for putative transgenerational epigenetic effects on the germ line of subsequent generations (F1–F3). Parental adult male C57Bl/6 mice were treated with either decitabine or vorinostat and analysed as well as three subsequent untreated generations derived from these males. Treatment directly affected several reproductive parameters as testis (decitabine & vorinostat) and epididymis weight, size of accessory sex glands (vorinostat), the height of the seminiferous epithelium and sperm concentration and morphology (decitabine). Furthermore, after decitabine administration, DNA methylation of a number of loci was altered in sperm. However, when analysing fertility of treated mice (fertilisation, litter size and sex ratio), no major effect of the selected epigenetic drugs on male fertility was detected. In subsequent generations (F1–F3 generations) only subtle changes on reproductive organs, sperm parameters and DNA methylation but no overall effect on fertility was observed. Consequently, in mice, decitabine and vorinostat neither affected male fertility per se nor caused marked transgenerational effects. We therefore suggest that both drugs do not induce major adverse effects—in terms of male fertility and transgenerational epigenetic inheritance—when used in anti-cancer-therapies.

The chemokine receptor CXCR7 interacts with the chemokines CXCL11 and CXCL12. During development, this ligand receptor system (C-X-C) provokes cell-type-specific responses in terms of migration, adhesion or ligand sequestration. It is active in zebrafish and rodents but no data are available for its presence or function in primate testes. Real-time quantitative polymerase chain reaction was performed in monkeys to detect CXCL11 , CXCL12 and CXCR7 . At the protein level, CXCL12 and CXCR7 were localized in the testes of the marmoset ( Callitrix jacchus ) whereas CXCR7 patterns were determined for various stages in human testes. Morphometry and flow cytometry were applied to quantify CXCR7-positive cells in monkeys. Transcript levels and protein expression of CXCR7 were detectable throughout testicular development. In both species, CXCR7 protein expression was restricted to premeiotic germ cells. In immature marmoset testes, 69.9 % ± 9 % of the total germ cell population were labelled for CXCR7, whereas in the adult, 4.7 % ± 2.7 % were positive for CXCR7. CXCL12 mRNA was detectable in all developmental stages in marmosets. The CXCL12 protein was exclusively localized to Sertoli cells. This pattern of CXCL12/CXCR7 indicates their involvement in regulatory processes that possibly orchestrate the interaction between undifferentiated germ cells and Sertoli cells.

Objective: To compare mechanical dissociation, employing the Medimachine system, and enzymatic digestion of human testicular tissues with respect to the proportion of spermatogonia and somatic cells, with the long-term objective of establishing human spermatogonial cultures.Design: Experimental basic science study.Setting: Reproductive biology laboratory.Patient(s): Testicular tissues were obtained from patients with gender dysphoria on the day of sex reassignment surgery. On the basis of the histological evaluation, tissue samples with complete spermatogenesis (fresh, n = 6; cryopreserved, n = 7) and with meiotic arrest (cryopreserved, n = 4) were selected.Intervention(s): None.Main Outcome Measure(s): The composition of testicular cell suspensions was assessed performing quantitative real-time polymerase chain reaction (qPCR) analyses for germ cell-specific (FGFR3, SALL4, UTF1, MAGE-A4) and somatic marker genes (ACTA2 and VIM). Additionally, flow-cytometric analyses were used to evaluate the percentage of SALL4- and vimentin-positive cells.Result(s): While Medimachine dissociation yielded higher cell numbers in all patient groups, viability of cells was highly variable and correlated with the histological status of the tissue. Interestingly, qPCR analysis revealed a significantly decreased expression of the somatic marker genes ACTA2 and VIM and an increased expression of the spermatogonial marker genes FGFR3 and SALL4 after Medimachine dissociation. These findings were corroborated by flow-cytometric analyses that demonstrated that the proportion of SALL4-positive cells was up to 4 times higher after mechanical dissociation.Conclusion(s): Medimachine dissociation of human testicular tissues is comparably fast and leads to an enrichment of SALL4-positive spermatogonia. The use of this method may therefore constitute an advantage for the establishment of human spermatogonial cell cultures. (C) 2015 by American Society for Reproductive Medicine.

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.

One of the consequences of oxygen metabolism is the production of reactive oxygen species (ROS) which in a situation of imbalance with antioxidants can damage several biomolecules, compromise cell function and even lead to cellular death. The particularities of the sperm cell make it particularly vulnerable to ROS attack compromising its functionality, mirrored in terms of fertility outcome and making the study of the origin of sperm ROS, as well as the alterations they cause very important. In the present work, we used UVB irradiation, an easy experimental approach known as a potent inducer of ROS formation, to better understand the origin of ROS damage without any confounding effects that usually exist in disease models in which ROS are reported to play a role. To address these issues we evaluated sperm mitochondrial ROS production using the Mitosox Red Probe, mitochondrial membrane potential using the JC-1 probe, lipid peroxidation through BODIPY probe and vitality using PI. We observed that UVB irradiation leads to an increase in sperm mitochondrial ROS production and lipid peroxidation that occur previously to an observable mitochondrial dysfunction. We concluded that sperm UVB irradiation appears to be a good and easily manipulated in vitro model system to study mitochondria-induced oxidative stress in spermatozoa and its consequences, which may be relevant in terms of dissecting the action pathways of many other pathologies, drugs and contaminants, including endocrine disruptors.

TCam-2 cells are the main in vitro model for investigations into seminomatous tumors. However, despite their widespread use, questions remain regarding the cells' homogeneity and consequently how representative they are of seminomas. We assess the TCam-2 cell line using routine and novel authentication methods to determine its homogeneity, identify any cellular sub-populations and resolve whether any changes could be due to generational differentiation. TCam-2, embryonal carcinoma cells (2102EP) and breast cancer cell (MCF7) lines were assessed using qRT-PCR, immunocytochemistry, flow cytometry and short tandem repeat analyses. Raman maps of individual cells (minimum of 10) and single scan spectra from 200 cells per culture were obtained. TCam-2s displayed the characteristic marker gene expression pattern for seminoma, were uniform in size and granularity and short tandem repeat analysis showed no contamination. However, based only on physical parameters, flowcytometry was unable to differentiate between TCam-2 and 2102EPs. Raman maps of TCam-2s comprised three equally distributed, distinct spectral patterns displaying large intercellular single spectral variation. All other cells showed little variation. Principal component, cluster and local spectral angle analyses indicated that the TCam-2s contained two different types of cells, one of which comprised two subgroups and was similar to some 2102EP cells. Protein expression corroborated the presence of different cells and generational differences. The detailed characterization provided by the Raman spectra, augmented by the routine methods, provide substantiation to the long-held suspicion that TCam-2 are not homogeneous but comprise differing cell populations, one of which may be embryonal carcinoma in origin.