Individual Tubules Research Articles

Identification of spermatogenesis in individual seminiferous tubules and testicular tissue of adult normal and busulfan-treated mice employing Raman spectroscopy and principal component analysis

The present study aims to identify spermatogenesis in testicular seminiferous tubules (ST) and testicular tissue of adult normal and busulfan-treated mice utilizing PCA and Raman spectroscopy. Raman measurements were conducted on single tubules and testes samples from adult and immature mice, comparing them with those from busulfan-treated adult mice, with validation through histological examination. The analysis revealed a higher signal variability (30 %–40 % at the peaks), prompting scrutiny of individual Raman spectra as a means of spermatogenesis measurement. However, principal component analysis (PCA) demonstrated significant cluster separation between the ST of mature and immature mice. Similar investigations were performed to compare ST from normal mature mice and those from busulfan-treated (BS-treated) mature mice, revealing substantial separation along PC1 and PC2 for all comparison sets. Additionally, comparing testicular samples from mature and immature mice revealed distinct separation in PCA. The study concludes that the combined approach of PCA and Raman spectroscopy proves to be a noninvasive and potentially valuable method for identifying spermatogenesis in seminiferous tubules and testicular samples.

STAGES OF MORPHOLOGICAL CHANGES OF TARGET ORGANS IN EXPERIMENTALLY SIMULATED ACUTE PERITONITIS

One of the urgent problems in urgent abdominal surgery remains acute disseminated peritonitis (AP), the mortality rate of which remains high due to the development of multiple organ failure syndrome, despite the use of modern methods of diagnosis and treatment.The purpose of the study. To substantiate the possibility of using laparoscopic methods of treatment depending on the severity and stage of morphological changes of the target organs and the duration of peritonitis simulated in the experiment.Material and methods. The experiment used 150 sexually mature non-linear female rats, weighing 180.0-220.0 grams, which were kept on the standard ration of the vivarium of the Central Research Laboratory with maintenance of food and drinking regimes recommended by the norms of keeping laboratory animals. The animals were divided into 5 groups – a control group (30 rats) and 4 experimental groups (120 rats), which underwent HP simulation by intraperitoneal injection of 1 ml of 30 % autocal. The animals of the research group were removed from the experiment under sodium thiopental anesthesia by bloodletting on the 1st, 3rd, 5th, and 7th days. Results of the study. Morphohistochemical study of the target organs on the 1st day of the experiment: singleorgan changes were observed (examination of the peritoneum revealed its pronounced swelling). A slight vascular reaction of the basal membrane of the mucous membrane was established in the small intestine. On the 3rd day, the changes remained monooraneous with increases in infl ammatory phenomena. On the 5th day, the spread of the systemic infl ammatory response to the lungs and kidneys was confi rmed. Areas with foci of dystrophic- necrotic changes in the epithelium of the tubules were visualized in individual excretory tubules in the kidneys.In the lungs: the number of erythrocytes and fi brin increased in the cellular component. On the 7th day of the experiment, the heart and liver were involved in the process. In the heart, dystonia and paresis of microcirculatory vessels, erythrocyte stasis, and the formation of cell aggregates in the lumen of vessels were observed. In the liver, it is observed: congestion of the central veins, manifestations of venous stasis.Conclusion. Based on the stages of morphological changes of the studied organs at diff erent times, the simulation of acute peritonitis allowed us to theoretically justify a diff erentiated approach to the selection of the type of surgical interventions.

Safety Study of an Original Mesenchymal Stromal Cell Secretome-Based Medicinal Product for Spermatogenesis Restoration

SCIENTIFIC RELEVANCE. Currently, there are no effective and safe medicinal products for idiopathic male infertility. Previous studies in two animal models of infertility (short-term cryptorchidism in rats and doxorubicin-induced testicular injury in mice) have shown the effectiveness of an originator medicinal product based on the mesenchymal stromal cell (MSC) secretome.AIM. The aim of the study was to evaluate the toxicity profile of the MSC secretome-based medicinal product in rats after local intratesticular or intramuscular administration.MATERIALS AND METHODS. The MSC secretome is a combination of factors secreted by MSCs in low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG) for MSC conditioning. In the single-dose toxicity study, the MSC secretome-based medicinal product was injected under the testicular tunica albuginea of male Wistar rats (15 per group) at doses of 15 and 25 relative units (RU) per animal, which are 1.5 and 2.5 times higher than the therapeutic dose (10 RU). In the repeat-dose toxicity study, male Wistar rats (10 per group) received intramuscular thigh injections of the medicinal product on days 1, 6, and 12 at doses of 15 and 25 RU per animal. The local tolerance study involved histopathological examination of the testes and thighs at the injection site. All studies included control groups of intact animals and animals similarly injected with blank DMEM-LG. The early follow-up period was 14 days, and the late follow-up period was 42 days.RESULTS. The rats showed no changes in the general condition after single and repeated doses of the MSC secretome-based medicinal product. Single subtunical doses induced moderate irritation; its signs included pathological changes in individual seminiferous tubules: epithelial atrophy (70% of the animals on day 14; 55% at late follow-up) and sperm stasis (70% of the animals). Similar changes were observed in the blank DMEM-LG group (up to 80% of the animals). There were no pathological changes in the tissues after repeated injections. A transient increase in alkaline phosphatase activity was detected in animals after their third intramuscular injection at a dose of 25 RU; the other biochemical parameters were normal in all study groups.CONCLUSIONS. The MSC secretome-based medicinal product has a favourable safety profile following both intratesticular and intramuscular administration, as it does not cause any permanent changes in the studied organs and tissues.

MORPHO-FUNCTIONAL CHARACTERISTICS OF THE TESTICLES OF MEN WITH DIFFERENT FORMS OF PATHOSPERMIA

The most difficult form of male infertility to treat is azoospermia. It is identified as a complete absence of sperm in the ejaculate. Depending on the nature and causes of impaired spermatogenesis, azoospermia is divided into obstructive (excretory, OA) and non-obstructive (secretory, NOA). Testicular biopsy is the most effective method of diagnosis and a component of possible treatment for azoospermia. It is the only objective method of differential diagnosis between non-obstructive and obstructive forms of azoospermia.
 The aim: histological analysis of testicular biopsies of men with various forms of azoospermia.
 Мaterials and Methods. 78 infertile men with azoospermia were examined. They were divided into two groups: the first group, men with NOA (n = 28); the second group, men with OA (n = 50). The biopsy was preceded by a mandatory ultrasound diagnosis of the portal system. The biopsy was performed using the method of open operative access. Biopsies were mostly taken from the more palpable testicle or from both testicles. Biopsies were fixed in buffered 10% formalin (pH 7.2). After 1 day, they were dehydrated in 70% ethanol and embedded in paraffin. For histological studies, sections with a thickness of 5 μm were stained with hematoxylin and eosin. Biopsies were evaluated in accordance with previously described methods.
 Results. Histological analysis of testicular biopsies from 28.7% of patients with a non-obstructive form of azoospermia showed swelling of the testicular stroma, destructive changes in testosterone-producing cells, disruption of the structure of the syncytial complexes of the spermatogenic epithelium, and the complete absence of the process of spermatogenesis in individual tortuous seminiferous tubules, the absence of contacts between sustentocytes, and in erythrocyte sludge in the lumen of vessels. 42.8% of patients had fibrosis of the testicular stroma, stroma swelling, thinning of the wall of convoluted seminiferous tubules, violation of the structure of the syncytial complexes of the spermatogenic epithelium, proliferation of the wall of the convoluted seminiferous tubules into their lumen, and infiltration of the testicular stroma with lymphocytes. In 54.0% of patients with preserved spermatogenesis and an obstructive form of azoospermia, it was possible to find a history of orchoepididymitis in the anamnesis; one patient (2.0%) underwent bilateral orchopexy at the age of 5 years due to cryptorchidism; 6.0% recalled the trauma calculi in the anamnesis; and 38.0% denied any factors affecting fecundity in the anamnesis.
 Conclusions. The non-obstructive form of azoospermia is characterized by the following parameters: mostly a violation of the structure of the spermatogenic epithelium, a complete absence of the process of spermatogenesis in individual convoluted seminiferous tubules, a violation of the structure of the hematotesticular barrier, and a violation of blood microcirculation. The histological picture of preserved spermatogenesis is of the same type in 88.0% of patients with an obstructive form of azoospermia. In most tubules, a fixed number of cell rows is preserved, and cells of various stages of spermatogenesis are determined in them: spermatogonies, spermatocytes, a moderate number of spermatids.
 In the lumen of the tubules, exfoliated cells and a moderate number of spermatozoa are found.

Renal protein reabsorption impairment related to a myxosporean infection in the grass frog (Rana temporaria L.).

A morphophysiological study of tubular reabsorption and mechanisms of protein endocytosis in the kidney of frogs (Rana temporaria L.) during parasitic infection was carried out. Pseudoplasmodia and spores of myxosporidia, beforehand assigned to the genus Sphaerospora, were detected in Bowman's capsules and in the lumen of individual renal tubules by light and electron microscopy. Remarkable morphological alteration and any signs of pathology in kidney tissue related to this myxosporean infection have not been noted. At the same time, significant changes in protein reabsorption and distribution of molecular markers of endocytosis in the proximal tubule (PT) cells in infected animals were detected by immunofluorescence confocal microscopy. In lysozyme injection experiments, the endocytosed protein and megalin expression in the infected PTs were not revealed. Tubular expression of cubilin and clathrin decreased, but endosomal recycling marker Rab11 increased or remained unchanged. Thus, myxosporean infection resulted in the alterations in lysozyme uptake and expression of the main molecular determinants of endocytosis. The inhibition of receptor-mediated clathrin-dependent protein endocytosis in amphibian kidneys due to myxosporidiosis was shown for the first time. Established impairment of the endocytic process is a clear marker of tubular cell dysfunction that can be used to assess the functioning of amphibian kidneys during adaptation to adverse environmental factors.

Diagnostic Value of Biochemical and Histopathological Characteristics of the Round Goby Neogobius melanostomus (Gobiidae) in Assessing of Aquatic Environment

Study of the transformation of biochemical reactions into histopathological alterations is of importance for their correct use as the biomarkers for early biodiagnostics of the aquatic environment quality. The purpose of the present investigation was to analyze the biochemical characteristics (activity of superoxide dismutase, catalase, aminotransferases, alkaline phosphatase, level of oxidized proteins and lipid peroxidation, albumin and urea concentrations) in tissues of round goby Neogobius melanostomus (Pallas, 1814) with different degrees of histopathological alterations. The results of histopathological studies in liver, gills and kidneys of this fish revealed twenty four pathological alterations referring to five types of disorders: circulatory disturbances, inflammatory reactions, progressive and regressive changes, and benign tumors. The relation between some pathological changes (necrosis of nephrocytes, necrosis and atrophy of individual renal tubules; necrosis of hepatocytes) and biochemical parameters (activity of alanine aminotransferase and alkaline phosphatase, level of lipid peroxidation) in round goby tissues was found. We demonstrate high diagnostic significance of the combined use of biochemical and histopathological parameters in round goby tissues for assessing the aquatic environment quality.

Accurate Quantitative Histomorphometric-Mathematical Image Analysis Methodology of Rodent Testicular Tissue and Its Possible Future Research Perspectives in Andrology and Reproductive Medicine.

Infertility is increasing worldwide; male factors can be identified in nearly half of all infertile couples. Histopathologic evaluation of testicular tissue can provide valuable information about infertility; however, several different evaluation methods and semi-quantitative score systems exist. Our goal was to describe a new, accurate and easy-to-use quantitative computer-based histomorphometric-mathematical image analysis methodology for the analysis of testicular tissue. On digitized, original hematoxylin-eosin (HE)-stained slides (scanned by slide-scanner), quantitatively describable characteristics such as area, perimeter and diameter of testis cross-sections and of individual tubules were measured with the help of continuous magnification. Immunohistochemically (IHC)-stained slides were digitized with a microscope-coupled camera, and IHC-staining intensity measurements on digitized images were also taken. Suggested methods are presented with mathematical equations, step-by-step detailed characterization and representative images are given. Our novel quantitative histomorphometric-mathematical image analysis method can improve the reproducibility, objectivity, quality and comparability of andrological-reproductive medicine research by recognizing even the mild impairments of the testicular structure expressed numerically, which might not be detected with the present semi-quantitative score systems. The technique is apt to be subjected to further automation with machine learning and artificial intelligence and can be named ‘Computer-Assisted or -Aided Testis Histology’ (CATHI).

Histological Tracking into the Third Dimension: Evolution of Early Tumorigenesis in VHL Kidney.

Using a novel three-dimensional (3D) approach, we tracked histological changes to elucidate the earliest stages of renal clear cell neoplasia in normal kidney tissue of patients with von Hippel-Lindau (VHL) disease. Tissue blocks of interest were procured, serially sectioned, and 3D reconstruction of the entirety of pathologic events was performed. The results reveal an abundance of foci with aberrant clear cell proliferation that initially develop along the tubular lining, but have the potential to aggregate within individual tubules. This stage is followed by the extension of clear cell aggregates beyond the tubular basement membrane, which allows for the recruitment of angiogenesis derived from interstitial vasculature. The results suggest that the most frequent pathologic event in VHL kidneys is the presence of isolated or aggregated clear cells within the tubular epithelium, potentially developing further into a protracted process of neoplasia. The abundance of independent pathologic events in VHL kidneys confirms developmental mechanisms to precede tumor initiation. To our knowledge, this is the first report demonstrating that tracking of histologic changes in the 3rd dimension enables the confirmation of the sequence of events from the earliest pathologic change in the VHL kidney to the neoplastic stage. This approach is not only useful for visualization and quantification of pathologic changes but also for targeted sampling allowing selective analysis of the earliest stages of clear cell carcinogenesis.

Three-Dimensional Analysis of Busulfan-Induced Spermatogenesis Disorder in Mice.

We examined if the distribution of impaired or normal spermatogenesis differs along the length of seminiferous tubules in disorders of spermatogenesis. For this purpose, three-dimensional (3D) reconstruction of seminiferous tubules was performed in mice with experimental spermatogenesis disorder induced by intraperitoneal injection of busulfan, and the areas of impaired and normal spermatogenesis were analyzed microscopically. The volume of the testis and length of seminiferous tubules decreased, and the proportion of tubule areas with impaired spermatogenesis increased depending on the dose of busulfan. With the highest dose of busulfan, although the proportion of impaired spermatogenesis was similar among individual seminiferous tubules, it was slightly but significantly higher in shorter tubules and in tubule areas near branching points. The tubule areas with impaired and normal spermatogenesis consisted of many segments of varying lengths. With increasing doses of busulfan, the markedly impaired segments increased in length without changing in number, whereas normal segments, although reduced in number and length, remained even with the highest dose of busulfan. Individual remaining normal segments consisted of several different stages, among which stage I and XII were found at higher frequencies, and stage VI at a lower frequency than expected in normal seminiferous tubules. We also examined if the distribution of impaired or normal spermatogenesis differs among different 3D positions in the testis without considering the course of seminiferous tubules. Although the proportions of impaired spermatogenesis with the minimum dose of busulfan and normal spermatogenesis with the highest dose of busulfan greatly varied by location within a single testis, there were no 3D positions with these specific proportions common to different testes, suggesting that the factors influencing the severity of busulfan-induced spermatogenesis disorder are not fixed in location among individual mice.

Effects of Oil Type on Sterol-Based Organogels and Emulsions

The present study investigates the effect of oil type on the formation, morphology and mechanical properties of phytosterol-based organogels. The formation of organogels can be satisfactorily predicted with a criterion based on Hansen Solubility Parameters (HSPs), provided that the sterol and sterol ester in these systems assemble as tubules. When structures other than tubules are formed, the predictability of the HSP-based criterion becomes void. In cases where organogelling occurred, the morphology and mechanical properties of the tubular network of the gels and water-in-oil emulsions were investigated. The findings revealed that the structure of the tubular network formed in oils with different compositions, could be grouped based on the dielectric constants of the oils. Curly and bundled tubules which formed networks, were observed in gels prepared with low dielectric constant oils (i.e. decane and limonene). For oils with a moderate dielectric constant (i.e. castor oil and sunflower oil), the tubules became less curly and straighter. Upon increasing the dielectric constant of the oil (eugenol), individual tubules were observed next to the bundled tubules. The results showed that straighter, bundled tubules are associated with firmer gels, whereas less straight (i.e. curly) tubules rendered weaker gels. The tubular network of the water-in-oil emulsions obtained for oils with a low dielectric constant appeared more open with straighter tubules. For oils with relatively high dielectric constant, the water-in-oil emulsions lost most of their tubular structure and only a few tubules could be observed. In the presence of emulsion droplets fewer tubules are formed, resulting in weaker networks.

Comparative histological changes in the structure of the spleen and kidneys of experimental chickens exposed to the action of “Guidamis” as an adjuvant for vaccination against infectious bronchitis

The article provides a histological description of the internal organs – the spleen and kidneys of chickens exposed to the action of “Guidamis” as an adjuvant. A lysate was tested as part of a sample of the vaccine PB-07 against infectious bronchitis developed by researchers of the Department of Virology and Tumor Diseases of Birds of the Russian Agricultural Academy (Lomonosov). The experiments were carried out on Lohmann Brown chickens in the conditions of the Laishev branch of “Ak Bars” Poultry Company, the Republic of Tatarstan. The prolonged action of the vaccine against infectious bronchitis and the use of “Guidamis” as an adjuvant was manifested as an increase in the proliferation and differentiation of lymphoid tissues in the numerous lymph nodes. The use of the vaccine and the adjuvant had a weak reactogenic effect. In the spleen of control chickens, hyperplasia of lymphoid tissue cells is weakly expressed. In the periarterial zone of nodules of the thymus-dependent lymphocytes, the cell density is low. In the kidneys, for fourteen days after the vaccination against infectious bronchitis using Guidamis, the moderate granular dystrophy of the epithelium, mainly of individual distal tubules, and local moderate manifestations of edema of the capsule of the renal corpuscles were observed. In the kidneys, signs of pronounced edema of the capsule cavity, significant disturbances in the patency of the tubular network caused by swelling and partial desquamation of the epithelium, as well as the presence of epithelium and protein mass in the lumen of the tubules, were observed.

Organization of the endoplasmic reticulum in cells of effective and ineffective pea nodules (Pisum sativum L.)

Background. The endoplasmic reticulum (ER) is the largest membrane-bound organelle, which plays an important role in the functioning of a plant cell and participates in its differentiation.
 Materials and methods. Using the methods of transmission electron microscopy, the morphological features and dynamics of structural changes in the ER in symbiotic nodules of pea (Pisum sativum L.) wild-type and mutants blocked at different stages of nodule development were studied.
 Results. ER developed from a network of individual tubules in meristematic cells, to a developed network of cisterns around the nucleus and plasmalemma, and a network of granular and smooth tubules accompanying infection structures in colonized and infected cells and symbiosomes in infected cells.
 Conclusions. A correlation was found between the level of development of the ER network and the degree of bacteroid differentiation.

Identifying differential mRNA & miRNA expression patterns in microsurgically isolated individual seminiferous tubules reveals unique “niche” for spermatogenesis in men with severe forms of infertility

Non-obstructive azoospermia (NOA) is a cause of male infertility secondary to genetically driven defects in spermatogenesis. Testicular sperm extraction (TESE) is successful in identifying small number of sperm in 50% of men with NOA. During TESE, predominantly collapsed seminiferous tubules (ST) are identified with rare areas of dilated STs that are more likely to harbor viable sperm. Hence we hypothesize that miRNA regulated control of mRNA expression along STs leads to optimal environments for spermatogenesis within the human testis.

Abstract 2393: Kidneys of VHL patients reveal the origin of renal clear cell carcinoma

Abstract VHL disease is a classic tumor suppressor gene syndrome characterized by development of specific types of tumors in selective organs with nervous system and kidney being most consistently affected. Detailed studies on human spinal cord and cerebellum have previously revealed earliest stages of CNS tumorigenesis and the morphologic sequence resulting in development of frank tumors. To elucidate earliest stages of renal clear cell carcinoma, we performed a similar approach in kidney tissues of four VHL patients and three sporadic control cases. From all cases, blocks of interest were procured, followed by serial sectioning and 3dimensional reconstruction of potential precursor lesions. The results reveal an abundance of foci with aberrant mesonephric clear cell proliferations that initially develop along the tubular lining, but have the potential to aggregate within individual tubules. This stage is followed by microscopic invasive clear cell aggregations which represent tumor precursor structures. This study presents evidence for a consistent morphologic sequence for renal clear cell carcinogenesis. Molecular analysis of early steps within this sequence will allow for identification of earliest genetic and proteomic changes in the future. Therapeutic targeting of earliest changes may allow to develop preventive strategies for renal cancer development for VHL patients, potentially also for the non-VHL population. Citation Format: Mayyan Mubarak, Samuel Sommaruga, David Voigt, Xiacao Xu, Steve Kim, Ailin Song, Alexander O. Vortmeyer. Kidneys of VHL patients reveal the origin of renal clear cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2393.

Sperm retrieval in adolescent males with Klinefelter syndrome: medical and ethical issues.

Many men with conditions linked to azoospermia were formerly considered infertile. However, over the last two decades advances in assisted reproductive technology (ART) have been devised and have permitted these men to become biological fathers. Foremost among these have been testicular sperm extraction (TESE) and a “microdissection” advance in which individual spermatic tubules are punctured and sperm extracted (microTESE). In the former multiple biopsies of the subject’s testes are taken and sperm retrieved as available (1). The micro technique originally noted by Schlegel and colleagues considers individual tubules for micropuncture using optical magnification (2,3). Sperm retrieval rates (SRR) are somewhat higher when performing the latter technique on men with non-obstructive azoospermia or men with Klinefelter syndrome (KS) or one of its variants (2,4,5).

Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes

De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

Gene loss in keratinization programs accompanies adaptation of cetacean skin to aquatic lifestyle.

Gene loss in keratinization programs accompanies adaptation of cetacean skin to aquatic lifestyle.

Postnatal development of the testis in the rat: morphologic study and correlation of morphology to neuroendocrine parameters.

Histopathologic examination of the testis from juvenile rats is often necessary to characterize the safety of new drugs for pediatric use and is a required end point in male pubertal development and thyroid function assays. To aid in evaluation and interpretation of the immature testis, the characteristic histologic features of the developing rat testis throughout postnatal development are described and correlated with published neuroendocrine parameter changes. During the neonatal period (postnatal day [PND] 3-7), seminiferous tubules contained gonocytes and mitotically active immature Sertoli cells. Profound proliferation of spermatogonia and continued Sertoli cell proliferation occurred in the early infantile period (PND 8-14). The spermatogonia reached maximum density forming double-layered rosettes with Sertoli cells in the late infantile period (PND 15-20). Leptotene/zygotene spermatocytes appeared centrally as tubular lumina developed, and individual tubules segregated into stages. The juvenile period (PND 21-32) featured a dramatic increase in number and size of pachytene spermatocytes with the formation of round spermatids and loss of "infantile" rosette architecture. In the peri-pubertal period (PND 32-55), stage VII tubules containing step 19 spermatids were visible by PND 46. The presented baseline morphologic and endocrinologic information will help pathologists distinguish delayed development from xenobiotic effects, determine pathogenesis when confronted with nonspecific findings, and identify sensitive time points for targeted study design.

Diabetic nephropathy induces alterations in the glomerular and tubule lipid profiles

Diabetic nephropathy (DN) is a major life-threatening complication of diabetes. Renal lesions affect glomeruli and tubules, but the pathogenesis is not completely understood. Phospholipids and glycolipids are molecules that carry out multiple cell functions in health and disease, and their role in DN pathogenesis is unknown. We employed high spatial resolution MALDI imaging MS to determine lipid changes in kidneys of eNOS(-/-) db/db mice, a robust model of DN. Phospholipid and glycolipid structures, localization patterns, and relative tissue levels were determined in individual renal glomeruli and tubules without disturbing tissue morphology. A significant increase in the levels of specific glomerular and tubular lipid species from four different classes, i.e., gangliosides, sulfoglycosphingolipids, lysophospholipids, and phosphatidylethanolamines, was detected in diabetic kidneys compared with nondiabetic controls. Inhibition of nonenzymatic oxidative and glycoxidative pathways attenuated the increase in lipid levels and ameliorated renal pathology, even though blood glucose levels remained unchanged. Our data demonstrate that the levels of specific phospho- and glycolipids in glomeruli and/or tubules are associated with diabetic renal pathology. We suggest that hyperglycemia-induced DN pathogenic mechanisms require intermediate oxidative steps that involve specific phospholipid and glycolipid species.

New approaches to the Klinefelter syndrome

The Klinefelter syndrome (KS), with an incidence of 1 to 2 per 1000 male neonates, is one of the most frequent congenital chromosome disorders. The 47,XXY karyotype causes infertility, testosterone deficiency and a spectrum of further symptoms and comorbidities. In recent years, significant progress has been made in the elucidation of the pathophysiology and the treatment of the KS. It became clear that, to a large extent, the clinical picture is determined by gene dosage effects of the supernumerary X-chromosome. The origin of the extra X-chromosome from either the father or the mother influences behavioural features of patients with KS. The CAGn polymorphism of the androgen receptor, located on the X-chromosome, has a distinct impact on the KS phenotype. KS predisposes to the metabolic syndrome and its cardiovascular sequelae, contributing to the increased mortality of patients with KS. Neuroimaging studies have correlated anomalies in brain structures with psychosocial problems. The unexpected possibility to produce pregnancies and live birth with either ejaculated sperm – about 8% of KS men have a few sperm in semen – or with sperm extracted from individual tubules obtained by testicular biopsy can be considered a breakthrough. Testosterone substitution requires further optimisation in terms of when to initiate therapy and which preparations and dosages to use. Recently developed animal models help to further elucidation the genetic and pathopysiological basis and may lead to new therapeutic approaches to KS.