Reinkeovi kristali u muškaraca s kriptorhizmom

Reinke's crystals were for the first time described in 1896th by the German anatomist Friedrich Reinke. They are found within the tissue of human testis, in the interstitial compartment adjacent to the seminiferous tubules. Leydig cells, which are unique features of the interstitium of testis, are responsible for the production of testosteron but also for the production of Reinke's crystals. Depending on the position in relation to the seminiferous tubules, Leydig cells can be divided into peritubular and perivascular group. The hypothesis of this research is that in patients with cryptorchidism number of Reinke's crystals is increased in unit volumen as well as in whole organ. Since Leydig cells with damaged endocrine function tend to offset with mechanisms such as hyperplasia and hypertrophy our hypothesis was that their number in unit volume will be increased, too. The aim of this thesis was to investigate morphological characteristics of Reinke's crystals on the level of light, confocal and electron microscopy and to determine their number in normal, healthy testes and cryptorchid testes. Cryptorchid group of patients is divided, depending on the position of testis, highscrotal and inguinal group. From testis biopsies of 24 patients with unilateral cryptorchidism, 15 were highscrotal and 9 inguinal. The results obtained were compared with 6 control samples. All biopsies were taken from patients age 18 to 30 years old. Paraffin sections of testes were stained with hemalaun-eosin and modified Masson's method. In both groups Reinke's crystals were found inside the nucleus and cytoplasm of Leydig cells. Crystals were often found lying freely inside the interstitium. Pictures taken with confocal microscope were used for analysis of correlation between crystals in the whole section and for the 3D reconstruction of crystal using softver for image analysis (Ellipse, VidiTo, slovakia). Transmission electron microscopy confirmed the hexagonal shape of the Reinke crystal. Besides regular shape of the crystals, many smaller, „irregular“ shapes with 5 or less edges were found with irregular planes for which is presumed that they are precursors of final, hexagonal shapes. The analysis of the crystal lattice is made by using fast Fourier transformation on the sections wuth thickness between 70 and 300 nm. The difference in morphology of Reinke's crystals between cryptorchid and control samples was not found. Average volume of the testis in the group of patients with cryptorchidism was 10,29 cm3, while the controls averaged double that volume, 21,06 cm3. Highscrotal group had an average testicular volume 11,81 cm3, whereas in inguinal group was significantly smaller (7,76cm3 in average). Concentrations of testosterone in serum were within referant values and did not differ significantly between the groups. Stereological part of the study showed multiple increase in number of Reinke's crystals in both subgroups of patients with cryptorchidism in comparison to controls. Increase in number of crystals is likely due to disturbed function of enzyme at higher temperatures than usual. In all groups, higher number of crystals was found perivasculary, as well as number of Leydig cells. This research provides a new insight into the number, morphology, and position of Reinke's crystals in the tissue of cryptorchid testes which may be of clinical importance in the diagnosis of pathological processes such as tumors of Leydig cells

A One-Step Real-Time Multiplex PCR for Screening Y-Chromosomal Microdeletions without Downstream Amplicon Size Analysis

BACKGROUND: Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. PRINCIPAL FINDINGS: The widely established "EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)" were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. SIGNIFICANCE: With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection

Sexual dimorphism of the extraorbital lacrimal glands in SF-1 knockout mice

Sexual dimorphism (SD) represents all the differences between males and females of the same species. SD of the murine lacrimal gland and the major effect of testosterone on its formation are well documented. Steroidogenic factor-1 (SF-1, NR5a1) is a nuclear receptor essential for the fetal development of steroid hormones producing organs and SF-1 knockout mice (Sf-1 KO) are therefore born without gonads and adrenal glands. The aim of this study was to investigate whether SD in lacrimal glands is present in the absence of exposure to sex hormones during development. Lacrimal glands from adult Sf-1 KO male and female mice without hormonal exposure, and from males that were treated with testosterone propionate (TP) prior to sacrifice, were examined. After sacrifice, glandular tissue was processed using standard histological procedures. Paraffin sections were analysed by stereology and immunostained against the androgen receptor (AR). Our results showed that there were no statistically significant differences in the mean volumes of acini, connective tissue or ductal system between males, females, and males on TP. The same pertains to the mean length of the ducts in all three groups. In the absence of sex hormones, sex chromosomes proved to be insufficient in inducing sexual dimorphism in LG. However, nuclei of the acinar cells in males on TP were positive for AR, whereas in males without TP no expression of AR was detected. Administration of TP induced the expression of AR in the nuclei of acinar cells of males but did not affect the morphology of LG. We conclude that SD in the lacrimal gland is not present in Sf-1 KO mice and this suggests that sex hormones have a major role in the development of SD in the lacrimal gland

Reinke´s Crystals in Perivascular and Peritubular Leydig Cells

The human testis is composed of seminiferous tubules and interstitium. Within the interstitium, residing Leydig cells can occasionally bear Reinke´s crystals. The aim of the current study was to investigate Reinke´s crystals in perivascular and peritubular Leydig cells in control and infertile (cryptorchid) testes. For that purpose, bright field, confocal and transmission electron microscopy were applied. The crystal lattice was investigated by Fast Fourier Transformation and the number of crystals determined by stereology. Results of the study indicated a higher number of crystals in perivascular cells (in the both control and cryptorchid group). Moreover, when control and cryptorchid specimens were compared for the presence of the crystal, a higher number of Reinke´s crystals was recorded in cryptorchid testes. Thick sections of the crystal were extremely helpful in yielding crystallographic data which confirmed a trigonal crystal structure of the lattice. The exact molecular composition of crystal’s microfilaments still remains unknown. (doi: 10.5562/cca1814

Macrophages and Leydig Cells in Testicular Biopsies of Azoospermic Men

A number of studies have indicated that testicular macrophages play an important role in regulating steroidogenesis of Leydig cells and maintain homeostasis within the testis. The current paper deals with macrophages (CD68 positive cells) and Leydig cells in patients with nonobstructive azoospermia (NOA). Methods employed included histological analysis on semi- and ultrathin sections, immunohistochemistry, morphometry, and hormone analysis in the blood serum. Histological analysis pointed out certain structural changes of macrophages and Leydig cells in NOA group of patients when compared to controls. In the testis interstitium, an increased presence of CD68 positive cells has been noted. Leydig cells in NOA patients displayed a kind of a mosaic picture across the same bioptic sample: both normal and damaged Leydig cells with pronounced vacuolisation and various intensity of expression of testosterone have been observed. Stereological analysis indicated a significant increase in volume density of both CD68 positive and vacuolated Leydig cells and a positive correlation between the volume densities of these cell types. The continuous gonadotropin overstimulation of Leydig cells, together with a negative paracrine action of macrophages, could result in the damage of steroidogenesis and deficit of testosterone in situ

Flow Cytometry of Mouse and Human Adipocytes for the Analysis of Browning and Cellular Heterogeneity

Summary: Adipocytes, once considered simple lipid-storing cells, are rapidly emerging as complex cells with many biologically diverse functions. A powerful high-throughput method for analyzing single cells is flow cytometry. Several groups have attempted to analyze and sort freshly isolated adipocytes; however, using an adipocyte-specific reporter mouse, we demonstrate that these studies fail to detect the majority of white adipocytes. We define critical settings required for adipocyte flow cytometry and provide a rigid strategy for analyzing and sorting white and brown adipocyte populations. The applicability of our protocol is shown by sorting mouse adipocytes based on size or UCP1 expression and demonstrating that a subset of human adipocytes lacks the β2-adrenergic receptor, particularly in the insulin-resistant state. In conclusion, the present study confers key technological insights for analyzing and sorting mature adipocytes, opening up numerous downstream research applications. : Freshly isolated adipocytes are a notoriously difficult cell type to study. Hagberg et al. provide a detailed flow cytometry protocol for the analysis and sorting of mouse and human adipocytes by defining the critical factors and conditions required for studying this specialized cell type and pinpointing common pitfalls. Keywords: adipocyte, adipose tissue, flow cytometry, FACS, mouse, human, uncoupled protein 1, beta 2 adrenergic recepto