Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations

Background Potassium channels are important for the structure and function of the spermatozoa. As a potassium transporter, the mSlo3 is essential for male fertility as Slo3 knockout male mice were infertile with the series of functional defects in sperm cells. However, no pathogenic variant has been detected in human SLO3 to date. Here we reported a human case with homozygous SLO3 mutation. The function of SLO3 in human sperm and the corresponding assisted reproductive strategy are also investigated. Methods We performed whole-exome sequencing analysis from a large cohort of 105 patients with asthenoteratozoospermia. The effects of the variant were investigated by quantitative RT-PCR, western blotting, and immunofluorescence assays using the patient spermatozoa. Sperm morphological and ultrastructural studies were conducted using haematoxylin and eosin staining, scanning and transmission electron microscopy. Results We identified a homozygous missense variant (c.1237A > T: p.Ile413Phe) in the sperm-specific SLO3 in one Chinese patient with male infertility. This SLO3 variant was rare in human control populations and predicted to be deleterious by multiple bioinformatic tools. Sperm from the individual harbouring the homozygous SLO3 variant exhibited severe morphological abnormalities, such as acrosome hypoplasia, disruption of the mitochondrial sheath, coiled tails, and motility defects. The levels of SLO3 mRNA and protein in spermatozoa from the affected individual were reduced. Furthermore, the acrosome reaction, mitochondrial membrane potential, and membrane potential during capacitation were also afflicted. The levels of acrosome marker glycoproteins and PLCζ1 as well as the mitochondrial sheath protein HSP60 and SLO3 auxiliary subunit LRRC52, were significantly reduced in the spermatozoa from the affected individual. The affected man was sterile due to acrosome and mitochondrial dysfunction; however, intra-cytoplasmic sperm injection successfully rescued this infertile condition. Conclusions SLO3 deficiency seriously impact acrosome formation, mitochondrial sheath assembly, and the function of K+ channels. Our findings provided clinical implications for the genetic and reproductive counselling of affected families.

Age features of morphological changes of the hematotesticular barrier in experimental diabetes mellitus.

Background. Diabetes mellitus is an acute medical and social problem in all parts of the world due to the constant increase in the incidence of the disease, severe complications, disability and high mortality. Hyperglycemia leads to oxidative stress and increased processes of formation of active oxygen species, which in turn make a significant contribution to the development of male infertility. Objective. Therefore, the aim of our study was to establish morphological changes in the vessels of the hemomicrocirculatory flow and testicular sustentocytes of adult rats with experimental streptozotocin diabetes mellitus (SDM). Methods. The material for the study were the testicles of 20 sexually mature 6-month-old rats (weighing 150-180 g). SDM in animals of the experimental group was simulated by a single intraperitoneal injection of streptozotocin (dissolved in 0.1 M citrate buffer solution with a pH of 4.5) at a dose of 6 mg per 100 g of mass. An equivalent dose of 0.1 M citrate buffer was injected intraperitoneally to animals of the control group. Histological, electron microscopic, biochemical, morphometric and statistical research methods were used. Results. It was found that in the early stages of SDM (28th day) on the background of hyperglycemia in the hemomicrocirculatory flow of the testes there is a spasm of the vessels of the afferent link, which is confirmed by a decrease in the lumen of arterioles and an increase in their Wagenworth index. On the 70th day of SDM on the background of elevated levels of glucose and glycosylated hemoglobin in the links of the hemomicrocirculatory flow of the testis there are signs of diabetic microangiopathy, manifested by: hemorheological disorders in micro-hemo-vessels, (erythrocyte sludge, adhesion of erythrocytes and platelets, microclasmatosis), decrease in the capacity of arterioles and capillaries (increase in the Wagenworth index, respectively by 1.8 and 1.9 times), microclasmatosis, thickening and proliferation of the basement membrane of capillaries. Against the background of diabetic microangiopathy, there is a decrease in the number of sustentocytes by 0.01 mm2 of testicular parenchyma by 1.8 times, compared with control indicators, the area of their profile increases by 2.2 times (in all cases p<0.05). The nucleolar areas probably do not change, which leads to an increase in the nuclear-cytoplasmic ratio by 2.9 times (p<0.05). Such morphometric changes of sustentocytes are caused by development of vacuolar hydropic dystrophies in them, and an apoptosis that is confirmed by data of histo-ultrastructural studies. Changes in sustentocytes against the background of the development of diabetic microangiopathy lead to a violation of the hematotesticular barrier and to dystrophic changes in the spermatogenic epithelium of the testis. Conclusion. Thus, on the 70th day of SDM in the hemomicrocirculatory flow of the testis, the development of diabetic microangiopathy is observed, which leads to a violation of the hematotesticular barrier, and as a consequence, to a violation of spermatogenesis.

Gross, Microanatomical and Ultrastructural Studies of Interdigital Gland in Madras Red Sheep (Ovis aries)

Background: Madras Red sheep is a well-recognized meat type breed reared only on free range system to yield tasty meat. The aim of this study was to determine the gross, microanatomical and ultrastructural details of the interdigital gland in Madras Red sheep. Methods: The interdigital glands were removed immediately after slaughter and subjected for gross morphological, histological, immunohistochemical studies and ultrastructural observations. Result: The results revealed that a well-developed tobacco-pipe shaped interdigital gland was present in all the four limbs which was composed of an orifice, excretory duct, body and bent or flexure. Histologically, wall of the gland was composed of epidermis, dermis and capsule from within outward. Epidermis was made of stratified squamous keratinized epithelium. Dermis was composed of dense irregular connective tissue with sweat, sebaceous glands, arrectores pili muscle and hair follicles were embedded within. The sweat gland appeared as group of tortuous tubules under scanning electron microscope. The oily secretion of the gland helps in maintaining the healthiness of foot during movement of the animal.

Dermatomyositis: Muscle Pathology According to Antibody Subtypes

Background and Objectives:Discoveries of dermatomyositis specific antibodies (DMSAs) in dermatomyositis patients raised awareness of various myopathological features among antibody subtypes. However, only perifascicular atrophy and perifascicular myxovirus resistant protein A (MxA) overexpression were officially included as the definitive pathological criteria for dermatomyositis classification. We aimed to demonstrate myopathological features in MxA-positive dermatomyositis to determine characteristic myopathological features in different DMSA subtypes.Method:We performed a retrospective pathology review of muscle biopsies of dermatomyositis patients diagnosed between January 2009 and December 2020 in a tertiary laboratory for muscle diseases. We included all muscle biopsies with sarcoplasmic expression for MxA and seropositivity for DMSAs. MxA-positive muscle biopsies which tested negative for all DMSAs were included as seronegative dermatomyositis. We evaluated histological features stratified according to four pathology domains (muscle fiber, inflammatory, vascular, and connective tissue) and histological features of interest by histochemistry, enzyme histochemistry, and immunohistochemical study commonly used in the diagnosis of inflammatory myopathy. We performed ultrastructural studies of 54 available specimens.Result:A total of 256 patients were included. Of these, 249 patients were positive for one of the five DMSAs (seropositive patients: 87 anti-TIF1-γ; 40 anti-Mi-2; 29 anti-MDA5; 83 anti-NXP-2; and 10 anti-SAE DM) and 7 patients were negative for all five DMSAs (seronegative patients). Characteristic myopathological features in each DMSA subtype were as follows: anti-TIF1-γ with vacuolated/punched out fibers (64.7%, P<.001) and perifascicular enhancement in HLA-ABC stain (75.9%, P<.001); anti-Mi-2 with prominent muscle fiber damage (score 4.8±2.1, P<.001), inflammatory cell infiltration (score 8.0±3.0, P=.002), perifascicular atrophy (67.5%, P=.02), perifascicular necrosis (52.5%, P<.001), increased perimysial alkaline phosphatase activity (70.0%, P<.001), central necrotic peripheral regenerating fibers (45.0%, P<.001), and sarcolemmal membrane attack complex deposition (67.5%, P<.001); anti-MDA5 with scattered/diffuse staining pattern of MxA (65.5%, P<.001) with less muscle pathology and inflammatory features; anti-NXP2 with microinfarction (26.5%, P<.001); and anti-SAE and seronegative DM with HLA-DR expression (50.0%, P=.02 and 57.1%, P=.02, respectively).Discussion:We described a comprehensive serological-pathological correlation of DM primarily using MxA expression as an inclusion criterion. In our study, DMSAs were associated with distinctive myopathological features suggesting different underlying pathobiological mechanisms in each subtype.

Human organ donor-derived vagus nerve biopsies allow for well-preserved ultrastructure and high-resolution mapping of myelinated and unmyelinated fibers

The vagus nerve provides motor, sensory, and autonomic innervation of multiple organs, and electrical vagus nerve stimulation (VNS) provides an adjunctive treatment option for e.g. medication-refractory epilepsy and treatment-resistant depression. The mechanisms of action for VNS are not known, and high-resolution anatomical mapping of the human vagus nerve is needed to better understand its functional organization. Electron microscopy (EM) is required for the detection of both myelinated and unmyelinated axons, but access to well-preserved human vagus nerves for ultrastructural studies is sparse. Intact human vagus nerve samples were procured intra-operatively from deceased organ donors, and tissues were immediately immersion fixed and processed for EM. Ultrastructural studies of cervical and sub-diaphragmatic vagus nerve segments showed excellent preservation of the lamellated wall of myelin sheaths, and the axolemma of myelinated and unmyelinated fibers were intact. Microtubules, neurofilaments, and mitochondria were readily identified in the axoplasm, and the ultrastructural integrity of Schwann cell nuclei, Remak bundles, and basal lamina was also well preserved. Digital segmentation of myelinated and unmyelinated axons allowed for determination of fiber size and myelination. We propose a novel source of human vagus nerve tissues for detailed ultrastructural studies and mapping to support efforts to refine neuromodulation strategies, including VNS.

Field-Emission Scanning Electron Microscope as a Tool for Large-Area and Large-Volume Ultrastructural Studies

The development of field-emission scanning electron microscopes for high-resolution imaging at very low acceleration voltages and equipped with highly sensitive detectors of backscattered electrons (BSE) has enabled transmission electron microscopy (TEM)-like imaging of the cut surfaces of tissue blocks, which are impermeable to the electron beam, or tissue sections mounted on the solid substrates. This has resulted in the development of methods that simplify and accelerate ultrastructural studies of large areas and volumes of biological samples. This article provides an overview of these methods, including their advantages and disadvantages. The imaging of large sample areas can be performed using two methods based on the detection of transmitted electrons or BSE. Effective imaging using BSE requires special fixation and en bloc contrasting of samples. BSE imaging has resulted in the development of volume imaging techniques, including array tomography (AT) and serial block-face imaging (SBF-SEM). In AT, serial ultrathin sections are collected manually on a solid substrate such as a glass and silicon wafer or automatically on a tape using a special ultramicrotome. The imaging of serial sections is used to obtain three-dimensional (3D) information. SBF-SEM is based on removing the top layer of a resin-embedded sample using an ultramicrotome inside the SEM specimen chamber and then imaging the exposed surface with a BSE detector. The steps of cutting and imaging the resin block are repeated hundreds or thousands of times to obtain a z-stack for 3D analyses.

The Relevance of Ultrastructural Studies of Metastatic Cells from Women with Breast Cancer History

The interaction between cancer cells and the surrounding microenvironment is determinant for metastasis success. In this study, the ultrastructural relevance of cells in the malignant pleural effusion (MPE) of women with breast cancer history was investigated. In MPE, it is possible to observe single cells and clusters. Women whose MPE presents carcinomas in aggregates have a better prognosis when compared to cases in which metastatic single cells are found. Samples were collected via fine-needle aspiration puncture (US-FNA). Subsequent to the material preparation and ultrathin cuts, they were observed using light and transmission electron microscopy (LM/TEM). LM and TEM images served as a basis for the creation of a digital sculpture using ZBrush® software. Clusters exhibited structural stability, en route vesicles allowing exocytosis of electron-dense fibrous elements, and cytoplasmic protrusions contributing to migratory and invasive skills. Single cells presented different necrotic phenotypes and many displayed leukocyte-like characteristics. Cluster cooperative relationships seem to be related to a long-term permanence in MPE. The absence of a collaborative network presumably triggers a more aggressive behavior of single cells. Its putative fusion with leukocytes can maximize the efficiency for transendothelial migration, increasing chances of metastatic success and, unfortunately, reducing survival of women with recidivism.