Developmental expression patterns of chemokines CXCL11, CXCL12 and their receptor CXCR7 in testes of common marmoset and human.

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.

Raman microspectroscopic discrimination of TCam-2 cultures reveals the presence of two sub-populations of cells.

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.

Experimental endocrine manipulation by contraceptive regimen in the male marmoset (Callithrix jacchus).

Marmosets are used as preclinical model in reproductive research. In contrast to other primates, they display short gestation times rendering this species valid for exploration of effects on fertility. However, their peculiar endocrine regulation differs from a those of macaques and humans. We subjected male marmosets to previously clinically tested hormonal regimens that are known to effectively suppress spermatogenesis. Beside a control group, seven groups (each n=6) were investigated for different periods of up to 42 months: regimen I, (four groups) received testosterone undecanoate (TU) and norethisterone enanthate (NETE); regimen II, (two groups) received TU and NETE followed by NETE only; and regimen III, (one group) received NETE only. Testicular volume, cell ploidy and histology, endocrine changes and fertility were monitored weekly. TU and NETE and initial TU and NETE treatment followed by NETE failed to suppress spermatogenesis and fertility. Testicular volumes dropped, although spermatogenesis was only mildly affected; however, testicular cellular composition remained stable. Serum testosterone dropped when NETE was given alone but the animals remained fertile. Compared with controls, no significant changes were observed in sperm motility and fertility. Administration of TU and NETE affected testicular function only mildly, indicating that the regulatory role of chorionic gonadotrophin and testosterone on spermatogenesis is obviously limited and testicular function is maintained, although the endocrine axis is affected by the treatment. In conclusion, marmosets showed a different response to regimens of male contraception from macaques or men and have to be considered as a problematic model for preclinical trials of male hormonal contraception.

Routine cryopreservation of spermatozoa is safe--evidence from the DNA methylation pattern of nine spermatozoa genes.

PURPOSE: Assess short- and mid-term impact of cryopreservation on DNA methylation status of different genes in spermatozoa. METHODS: Semen samples from 10 healthy normozoospermic men were collected at the Department of Clinical Andrology of the Centre of Reproductive Medicine and Andrology (Muenster, Germany). Each was divided into four equal aliquots: 1) untreated, 2) diluted in cryoprotectant, 3) short term (2 days) cryopreserved and 4) mid term (4 weeks) cryopreserved. Samples were "swim-up" purified prior to analysis. DNA fragmentation was measured using comet assay and Flow cytometric evaluation with Acridine Orange (FCEAO). The degree of methylation of nine genes was determined by bisulfite pyrosequencing of genomic DNA. RESULT(S): Analysis of three maternally imprinted genes (LIT1, SNRPN, MEST), two paternally imprinted genes (MEG3, H19), two repetitive elements (ALU, LINE1), one spermatogenesis-specific gene (VASA) and one gene associated with male infertility (MTHFR) in semen samples demonstrated no alteration in methylation pattern regardless of duration of cryopreservation. CONCLUSION(S): The lack of any changes in the sub-fraction of the genome examined in our study, implies that sperm DNA methylation is unaffected by cryopreservation and suggests that this daily clinical routine is safe in terms of DNA methylation.

Effects of the FSH receptor gene polymorphism p.N680S on cAMP and steroid production in cultured primary human granulosa cells.

The study was designed to evaluate in vitro the cellular mechanisms of the single nucleotide polymorphism (SNP) p.N680S of the FSH receptor gene (FSHR) in human granulosa cells (GC) and included patients homozygous for the FSHR SNP (NN/SS) undergoing ovarian stimulation. GC were isolated during oocyte retrieval and cultured for 1­7 days. Basal oestradiol and progesterone concentrations were measured after short-term culture. The kinetics of cAMP, oestradiol and progesterone concentrations in response to various amounts of FSH were analysed in a 6­7 day culture. Basal oestradiol, but not progesterone, concentrations on day 1 of GC culture, were significantly higher in NN compared with SS (P = 0.045), but non-responsive to FSH stimulation. Immunofluorescence microscopy demonstrated the re-appearance of FSHR expression with increasing days in culture. Upon stimulation with FSH, GC cultured for 6­7 days displayed a dose-dependent increase of cAMP, oestradiol and progesterone but no difference in the EC50 values between both variants. Primary long-term GC cultures are a suitable system to study the effects of FSH in vitro. However, the experiments suggest that factors down-stream of progesterone production or external to GC might be involved in the clinically observed differences in an FSHR variant-mediated response to FSH.

Simultaneous detection of sperm membrane integrity and DNA fragmentation by flow cytometry: A novel and rapid tool for sperm analysis.

BACKGROUND: Sperm DNA integrity has become one of the most discussed and promising biomarkers for the assessment of male fertility. However, an easy-to-apply method capable of estimating DNA fragmentation in the live fraction of spermatozoa has remained elusive, preventing this parameter from being fully applied in clinical settings. OBJECTIVES: To validate a novel co-staining for the analysis of DNA fragmentation in membrane-intact spermatozoa. MATERIALS AND METHODS: Normozoospermic semen samples were used to validate the co-staining consisting of acridine orange (AO) and LIVE/DEAD™ Fixable Blue Dead Cell Stain (LD), against established methods for the evaluation of cell viability, propidium iodide stain (PI), and DNA fragmentation, the sperm chromatin structure assay (SCSA), to rule out cross-interference. Furthermore, the accuracy of the method was tested by the evaluation of samples prepared with different amounts of membrane and DNA damage (20, 40, 60, 80, and 100%). RESULTS: No significant differences were observed between the co-staining and the established staining procedures (membrane integrity, p = 0.755; DNA fragmentation p = 0.976). Moreover, high R square values were obtained from the analysis of samples of known membrane (R2  = 0.9959) and DNA damage (R2  = 0.9843). The simultaneous assaying of sperm membrane integrity and nuclear DNA fragmentation allowed the analysis of four sperm categories and thereby to assess the proportion of membrane-intact spermatozoa with compromised DNA integrity. DISCUSSION AND CONCLUSION: This new protocol has the potential to provide clinically relevant information about the DNA fragmentation in membrane-intact spermatozoa. Thus, it has the potential of improving the diagnostic of male infertility and enabling a better understanding of sperm dysfunction.

A germ cell-specific ageing pattern in otherwise healthy men.

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.

Models versus established knowledge in describing the functional morphology of the ventricular myocardium.

The myocytes comprising the ventricular mass are arranged so as to function in antagonistic fashion, the walls having the capacity to generate both constrictive and dilatory forces. This dualistic activity is organized on the basis of a site-specific morphologic pattern, permitting marked regional specificity for mural motion and providing a target for regional therapy. Diseased regions can be removed surgically without danger of jeopardizing the remaining healthy mural segments. The sensitivity of the intruding population of myocytes to positive and negative inotropic medication is markedly more pronounced than that of the prevailing tangentially aligned myocytes. This asymmetrical action of inotropes in the setting of global ventricular imbalance promotes the potential to restore constrictive as opposed to dilatory actions.

Spectral features of nuclear DNA in human sperm assessed by Raman Microspectroscopy: Effects of UV-irradiation and hydration.

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.

What is the clinical significance of ventricular mural antagonism?

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.

An analysis of the spatial arrangement of the myocardial aggregates making up the wall of the left ventricle.

OBJECTIVE: We used the technique of peeling of myocardial aggregates, usually described as 'fibres', to determine the spatial arrangement of the myocytes in the left ventricular wall of a healthy autopsied human heart. METHODS: We digitised the left ventricular outer and inner boundaries, as well as the pathways in space, of almost 3000 aggregates harvested from the left ventricular myocardium. During the process of gradual peeling, we sought to identify the myocardial aggregates as uniformly as possible. Despite this, interpolation was necessary to complete the pattern so as to construct a unit vector field that represented the preferred direction of the myocardial aggregates throughout the entirety of the walls of the left ventricle of this individual human heart. RESULTS: Apart from the overall systematic arrangement of the aggregates necessary to achieve physiologic ventricular contraction, we documented substantial local heterogeneities in the orientation of the myocardial aggregates. In particular, a significant proportion of aggregates was found to intrude obliquely with respect to the ventricular boundaries, with markedly heterogeneous distribution. Moreover, the distribution of the helical angle of the aggregates relative to the ventricular base varied notably throughout the left ventricular free walls and the septum. Within the generally quite uniform and continuous structure of the ventricular mass, we were, however, unable to identify any organised tracts or functional subunits such as a 'helical ventricular band', nor did we find radial fibrous lamellas coursing across the ventricular wall. CONCLUSION: We suggest that the impact of local anatomical inhomogeneities, associated with gradients in regional contractile function on global ventricular dynamics, has been systematically underestimated in the past. Our analysis confirms furthermore the continuous nature of the myocardium associated with an overall gross organisation of the fibre direction field; however, there is no evidence of substructures compartmentalising the ventricles.

Beta-blockade at low doses restoring the physiological balance in myocytic antagonism.

OBJECTIVE: The ventricular mass is organized in the form of meshwork, with populations of myocytes aggregated in a supporting matrix of fibrous tissue, with some myocytes aligned obliquely across the wall so as to work in an antagonistic fashion compared to the majority of myocytes, which are aggregated together in tangential alignment. Prompted by results from animal experiments, which showed a disparate response of the two populations of aggregated myocytes to negative inotropic medication, we sought to establish whether those myocytes that aggregated so as to extend obliquely across the thickness of the ventricular walls are more sensitive to beta-blockade than the prevailing population in which the myocytes are aggregated together with tangential alignment. If the two populations respond in similar differing fashion in the clinical situation, we hypothesize that this might help to explain why drugs blocking the beta-receptors improve function of the ventricular pump in the setting of congestive cardiac failure. METHODS: We implanted needle probes in 13 patients studied during open heart surgery, measuring the forces generated in the ventricular wall and seeking to couple the probes either to myocytes aggregated together with tangential alignment or to those aggregated in oblique fashion across the ventricular walls. In a first series of patients, we injected probatory doses intravenously, amounting to a total bolus of 40-100mg Esmolol, while in a second series, we gave fixed yet rising doses of 5, 10, and 20mg Esmolol in three separate boluses. RESULTS: Forces recorded in the aggregated myocytes with tangential alignment decreased insignificantly upon administration of low doses (57.1+/-12.4 mN-->56.6+/-7.6 mN), while forces recorded in the myocytes aggregated obliquely across the ventricular wall showed a significant decrease in the mean (59.3+/-11.6 mN-->47.4+/-6.4 mN). CONCLUSIONS: The markedly disparate action of drugs blocking beta-receptors at low dosage seems to be related to the heterogeneous extent, and time course, of systolic loading of the myocytes. This, in turn, depends on whether the myocytes themselves are aggregated together with tangential or oblique alignments relative to the thickness of the ventricular walls.

How are the myocytes aggregated so as to make up the ventricular mass?

Of late, it has become fashionable in the surgical literature to describe the ventricular mass as though arranged in the form of a continuous myocardial band, which starts at the aorta and ends at the pulmonary trunk. On the basis of this concept, its supporters have produced revisionist accounts of cardiac development and ventricular function, as well as using it as the basis for proposed surgical maneuvers. They seem unaware, however, that the original concept itself has never been supported by independent anatomic studies, while, to the best of our knowledge, they have not themselves performed anatomic investigations to prove its substance. Furthermore, the current proponents of the "unique myocardial band" ignore a large body of previous anatomic study which showed that the ventricular mass is arranged in the form of a modified blood vessel, with each myocyte anchored to its neighbor within a 3-dimensional myocardial mesh, rather than being arranged in a fashion analogous to skeletal muscles, with discrete origins and insertions of myocardial bands or tracts. In this review, we summarize the evidence showing that there are no anatomic structures within the ventricular myocardium that permit it to be unraveled in systematic fashion so as to produce the purported myocardial band. We also re-visit our own previous investigations, which supported the conventional approach, namely that the myocytes are aggregated together within a supporting fibrous matrix in the form of a 3-dimensional meshwork.

Heuristic problems in defining the three-dimensional arrangement of the ventricular myocytes.

There is lack of consensus concerning the three-dimensional arrangement of the myocytes within the ventricular muscle masses. Bioengineers are seeking to model the structure of the heart. Although the success of such models depends on the accuracy of the anatomic evidence, most of them have been based on concepts that are far from anatomical reality, which ignore many significant previous accounts of anatomy presented over the past 400 years. During the 19th century, Pettigrew emphasized that the heart was built on the basis of a modified blood vessel rather than in the form of skeletal muscles. This fact was reemphasized by Lev and Simkins as well as Grant in the 20th century, but the caveats listed by these authors have been ignored by proponents of two current concepts, which state either that the myocardium is arranged in the form of a "unique myocardial band," or that the walls of the ventricles are sequestrated in uniform fashion by laminar sheets of fibrous tissue extending from epicardium to endocardium. These two concepts are themselves incompatible and are further at variance with the majority of anatomic studies, which have emphasized the regional heterogeneity to be found in the three-dimensional packing of the myocytes within a supporting matrix of fibrous tissue. We reemphasize the significance of this three-dimensional muscular mesh, showing how the presence of intruding aggregates of myocytes extending in oblique transmural fashion also contends against the notion that all myocytes are orientated with their long axes parallel to the epicardial and enodcardial surfaces.

Three-dimensional architecture of the left ventricular myocardium.

Concepts for ventricular function tend to assume that the majority of the myocardial cells are aligned with their long axes parallel to the epicardial ventricular surface. We aimed to validate the existence of aggregates of myocardial cells orientated with their long axis intruding obliquely between the ventricular epicardial and endocardial surfaces and to quantitate their amount and angulation. To compensate for the changing angle of the long axis of the myocytes relative to the equatorial plane of the ventricles with varying depths within the ventricular walls, the so-called helical angle, we used pairs of cylindrical knives of different diameters to punch semicircular slices from the left ventricular wall of pigs, the slices extending from the epicardium to the endocardium. The slices were pinned flat, fixed in formaldehyde, embedded in paraffin, sectioned, stained with azan or hematoxilin and eosin, and analyzed by a new semiautomatic procedure. We made use of new techniques in informatics to determine the number and angulation of the aggregates of myocardial cells cut in their long axis. The alignment of the myocytes cut longitudinally varied markedly between the epicardium and the endocardium. Populations of myocytes, arranged in strands, diverge by varying angles from the epicardial surface. When paired knives of decreasing diameter were used to cut the slices, the inclination of the diagonal created by the arrays increases, while the lengths of the array of cells cut axially decreases. The visualization of the size, shape, and alignment of the myocytic arrays at any side of the ventricular wall is determined by the radius of the knives used, the range of helical angles subtended by the alignment of the myocytes throughout the thickness of the wall, and their angulation relative to the epicardial surface. Far from the majority of the ventricular myocytes being aligned at angles more or less tangential to the epicardial lining, we found that three-fifths of the myocardial cells had their long axes diverging at angles between 7.5 and 37.5 degrees from an alignment parallel to the epicardium. This arrangement, with the individual myocytes supported by connective tissue, might control the cyclic rearrangement of the myocardial fibers. This could serve as an important control of both ventricular mural thickening and intracavitary shape.

The myocardium and its fibrous matrix working in concert as a spatially netted mesh: a critical review of the purported tertiary structure of the ventricular mass.

With the increasing interest now paid to volume reduction surgery, in which the cardiac surgeon is required to resect the ventricular myocardium to an extent unenvisaged in the previous century, it is imperative that we develop as precise knowledge as is possible of the basic structure of the ventricular myocardial mass and its functional correlates. This is the most important in the light of the adoption by some cardiac surgeons of an unvalidated model which hypothesises that the entire myocardial mass can be unravelled to produce one continuous band. It is our opinion that this model, and the phylogenetic and functional correlates derived from it, is incompatible with current concepts of cardiac structure and cardiodynamics. Furthermore, the proponents of the continuous myocardial band have made no effort to demonstrate perceived deficiencies with current concepts, nor have they performed any histological studies to validate their model. Clinical results using modifications of radius reduction surgery based on the concept of the continuous myocardial band show that the procedure essentially becomes ineffective. As we show in this review, if we understand the situation correctly, it was the erstwhile intention of the promoters of the continuous band to elucidate the basic mechanism of diastolic ventricular dilation. Their attempts, however, are doomed to failure, as is any attempt to conceptualise the myocardial mass on the basis of a tertiary structure, because of the underlying three-dimensional netting of the myocardial aggregates and the supporting fibrous tissue to form the myocardial syncytium. Thus, the ventricular myocardium is arranged in the form of a modified blood vessel rather than a skeletal muscle. If an analogy is required with skeletal muscle, then the ventricular myocardium possesses the freedom of motion, and the ability for shaping and conformational self-controlling that is better seen in the tongue. It is part of this ability that contributes to the rapid end-systolic ventricular dilation. Histologic investigations reveal that the fibrous content of the three-dimensional mesh is relatively inhomogeneous through the ventricular walls, particularly when the myocardium is diseased. The regional capacity to control systolic mural thickening, therefore, varies throughout the walls of the ventricular components. The existence of the spatially netted structure of the ventricular mass, therefore, must invalidate any attempt to conceptualise the ventricular myocardium as a tertiary arrangement of individual myocardial bands or tracts.

The architecture of the ventricular mass and its functional implications for organ-preserving surgery.

It has generally been accepted that the myocardial fibres within the ventricular mass are arranged in syncytial fashion, precluding the identification of discrete and isolated muscular pathways. Recently, however, an entire hypothesis for surgical treatment has been proposed on the basis of the existence of a 'ventricular myocardial band', suggesting that this arrangement in itself points to detrimental results following partial ventriculectomy. In this review, we re-state the evidence supporting the accepted concept of the ventricular mass being made up of an undefined number of wedge-shaped functional units, each of them exerting its individually programmed contribution to the global activity of the ventricular walls. The wedge-shaped units consist of bundles of individual fibres which are arranged tangentially. An important subset of fibres intrudes into the ventricular wall, thus creating oblique pathways. Their angle of intrusion varies, and can be measured at up to 30 degrees . The steeper the angle of their intrusion, the more efficiently do the fibres counteract the systolic mural thickening. The network of supporting connective tissue, nonetheless, provides the necessary steep angulation towards the endocardium. This fibrous matrix serves as continuous chain for the transmission of forces, including that in the direction from the epicardium towards the endocardium, resulting in a dilating force. We have shown, using needle force probes, that in the hypertrophic heart the dynamic equilibrium of dilating and constricting forces acts at elevated diastolic and systolic levels, because the obliquity of the fibres increases due to the thickening of the wall, and there is a concomitant increase in connective tissue, causing an increase in the forces opposing systolic mural thickening. Then, in a vicious cycle, both populations of myocardial fibres stimulate each other to hypertrophy. Eventually, coronary perfusion becomes critically impaired, with still further deposition of connective tissue. Ultimately, the vector of the dilating force comes to dominate the constricting force, and the ventricle dilates. In this setting, partial left ventriculectomy remains a functionally sound intervention, since it is capable of improving global ventricular function by improving the geometrical state of the remaining anatomic myocardial units.

The anatomical arrangement of the myocardial cells making up the ventricular mass.

The architectural arrangement of the myocytes within the ventricular mass remains a highly contentious topic. It has recently been suggested by several distinguished surgeons that the overall myocardial structure is disposed in the form of a 'ventricular myocardial band'. There are, however, major anatomic deficiencies in this hypothesis, because the heart is formed on the basis of a modified blood vessel, rather than a collection of discrete muscular entities resembling the skeletal musculature. There is ample alternative evidence, nonetheless, already existing to provide a suitable explanation for the 'forceful reciprocal twisting' of the ventricular mass that is seen by cardiac surgeons during operative procedures. We provide here, therefore, a review of the anatomical studies we have performed separately and conjointly over a period of nearly 30 years. As before, we show that there is no anatomic evidence to support the concept of the 'ventricular myocardial band'. The overall arrangement is for the myocytes to be supported as the muscular components of a continuous and complex mass, the supporting collagenous fibrous matrix possessing epimysial, perimysial, and endomysial components. It had already been discussed at length during the previous century why there was no anatomic evidence to support the existence of separate 'muscles' within the ventricular continuum. There are no fibrous sheaths within the ventricular walls that permit the myofibres to be dissected on the basis of muscle bundles having a discrete origin and insertion, as is the case with the arrangement of the skeletal muscles. We have never sought ourselves, however, to deny the central helical nature of the overall architecture of the ventricular walls. The anatomic evidence supporting an overall helical nature for the ventricular myocardium has existed for over 150 years. All the available evidence, nonetheless, shows that these helical patterns are to be found throughout the walls, and in no way constitute a unique myocardial band.

Comparison of enzymatic digestion and mechanical dissociation of human testicular tissues.

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.

Direct but no transgenerational effects of decitabine and vorinostat on male fertility.

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.