In focus in HCB, Histochemistry and Cell Biology

Zw10 is a spindle assembly checkpoint protein that regulates meiotic maturation in mouse oocytes Abstract Faithful chromosome segregation during the cell cycle is ensured by the spindle assembly checkpoint (SAC). Although SAC activity is highly conserved and most organisms share common SAC components, additional proteins that regulate SAC activity to ensure high fidelity chromosome segregation are present in higher eukaryotes. Zw10 is one of these additional SAC components. Although Zw10 has been demonstrated to be involved in SAC activity during mitosis, little is known about its role during oocyte meiosis. Here, we report that Zw10 is localized at the kinetochore and is required for SAC activation during meiotic maturation. Knockdown of Zw10 led to precocious polar body extrusion by impairing Mad2 recruitment at kinetochores. Moreover, Zw10 knockdown impaired chromosome alignment and kinetochore–microtubule attachment, increasing the incidence of aneuploidy. Furthermore, Zw10 expression decreased with maternal age, suggesting that Zw10 is associated with the age-related increase in the incidence of aneuploidy. Together, our results demonstrate that Zw10 is localized at kinetochores and functions as an essential SAC component in mouse oocytes.

Suitability of RNA Later solution as a tissue-preserving reagent for immunohistochemical analysis Abstract Histological and immunohistochemical studies require high-quality paraffin blocks, where proper fixation of tissue samples with formalin is a key point. However, in some cases, the possibility to preserve biological samples prior to the formalin fixation or to use deposited tissues from biobanks is important. RNA-stabilizing reagent RNALater represents a potential option, but its suitability for pathological and immunohistochemical studies remains underinvestigated. Here, comparative study of formalin-fixed tissues and those had undergone preservation with RNALater was performed for different SCID mice tissues (brain, liver, kidney, and lung) using histological staining (hematoxylin–eosin and Weigert-van Gieson) or immunostaining for b-actin, glial fibrillary acidic protein, and glycosaminoglycan chondroitin sulfate. It was shown that RNALater preservation for 7–14 days was suitable for histological characterisation of mouse lung tissue, whereas all other tissues demonstrated some changes. Immunoreactivity of all the studied tissues was affected to a different extent, and the observed changes were detected at the 7th day already and continued to get worse by the 14th day. Overall, RNALater preservation affects immunogenicity of normal mouse tissues (brain, liver, kidney, and lung) making them unsuitable for immunohistochemistry. Some tissues retain their morphology (lung tissue) or demonstrate moderate changes (brain, liver, kidney), suggesting a restricted suitability of the RNALater-preserved tissues for histological analysis.

Depletion of the cellular cholesterol content reduces the dynamics of desmosomal cadherins and interferes with desmosomal strength Abstract Desmosomal cadherins, desmocollins, and desmogleins are cholesterol-dependent entities responsible for the stable adhesion of desmosomes in epithelial cells. Here, we investigated the influence of cellular cholesterol depletion on the dynamic properties of the desmosomal cadherin desmocollin, particularly the lateral mobility and distribution of desmocollin 2 (Dsc2-YFP) in the plasma membrane, and how these properties influence the adhesion strength of desmosomes. Depletion of cellular cholesterol decreased the lateral mobility of Dsc2-YFP and caused dispersion of Dsc2-YFP in the plasma membrane of epithelial MDCK cells. As a consequence of the altered Dsc2-YFP dynamics, the adhesive strength of desmosomes was weakened. Moreover, our study is the first to show and quantify the co-association of desmosomes with cholesterol/sphingomyelin-enriched membrane domains at the ultrastructural level. Taken together, our data emphasize a critical role for the cellular cholesterol content in regulating the lateral mobility and distribution of Dsc2 and show that cholesterol depletion reduces the strength of desmosomal adhesions.

Reorganization of the nuclear compartments involved in transcription and RNA processing in myonuclei of type I spinal muscular atrophy Abstract Type I spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by the loss or mutation of the survival motor neuron 1 (SMN1) gene. The reduction in SMN protein levels in SMA leads to the degeneration of motor neurons and muscular atrophy. In this study, we analyzed the nuclear reorganization in human skeletal myofibers from a type I SMA patient carrying a deletion of exons 7 and 8 in the SMN1 gene and two SMN2 gene copies and showing reduced SMN protein levels in the muscle compared with those in control samples. The morphometric analysis of myofiber size revealed the coexistence of atrophic and hypertrophic myofibers in SMA samples. Compared with controls, both nuclear size and the nuclear shape factor were significantly reduced in SMA myonuclei. Nuclear reorganization in SMA myonuclei was characterized by extensive heterochromatinization, the aggregation of splicing factors in large interchromatin granule clusters, and nucleolar alterations with the accumulation of the granular component and a loss of fibrillar center/dense fibrillar component units. These nuclear alterations reflect a severe perturbation of global pre-mRNA transcription and splicing, as well as nucleolar dysfunction, in SMA myofibers. Moreover, the finding of similar nuclear reorganization in both atrophic and hypetrophic myofibers provides additional support that the SMN deficiency in SMA patients may primarily affect the skeletal myofibers.

GSK3β and MCL-1 mediate cardiomyocyte apoptosis in response to high glucose Abstract Gestational diabetes mellitus is a risk factor for congenital heart defects. Our previous results indicated that a decrease in myocardial cells and an increase in apoptotic cells leads to heart defects under hyperglycemia, but much work remains to elucidate this important mechanism of myocardial cell apoptosis induced by high glucose (HG). In this study, we found that a decrease in GSK3β phosphorylation on Ser9 occurred concomitantly with HG-induced cardiomyocyte apoptosis and in the heart tissues of the offspring of diabetic rats in vitro and in vivo. Decreases in GSK3β (Ser9) phosphorylation in response to HG were remarkably restored after treatment with SC79, an activator of the Akt signaling pathway. SB216763, an effective inhibitor of the GSK3β signaling pathway, suppressed HG-induced apoptosis in cardiomyocytes. Further studies showed a decrease in the expression of the anti-apoptotic protein MCL-1 was associated with GSK3β-mediated apoptosis. MCL-1 overexpression partly inhibits HG-induced apoptosis in cardiomyocytes. Herein, this study revealed the roles of GSK3β and MCL-1 in modulating HG-induced cardiomyocyte apoptosis and maternal diabetes-induced abnormalities.

Immune response profile of primary tumour, sentinel and non-sentinel axillary lymph nodes related to metastasis in breast cancer: an immunohistochemical point of view Abstract Approximately 1.67 million new cases of breast cancer (BC) are diagnosed annually, and patient survival significantly decreases when the disease metastasizes. The axillary lymph nodes (ALNs) are the main doorway for BC tumoral cell escape, through which cells can disseminate to distant organs. The immune response, which principally develops in the lymph nodes, is linked to cancer progression, and its efficacy at controlling tumoral growth is compromised during the disease. Immunohistochemistry (IHC) is one of the most widely used research techniques for studying the immune response. It allows the measurement of the expression of particular markers related to the immune populations. This review focuses on the role of the immune populations in the primary tumour in the locoregional metastasis of the ALN, and the relationship of the immune response in these regions to distant metastasis. We considered only studies of immune cells using IHC techniques. In particular, lymphocytes, macrophages and dendritic cells all play important roles in BC and have been extensively studied. Although further research is needed, there is much evidence of their role in the invasion of the ALN and distant organs. Their association with tumoral growth or protection has not yet been demonstrated decisively and is very likely to be determined by a combination of factors. Moreover, even though IHC is a widely used technique in cancer diagnosis and research, there is still room for improvement, since its quantification needs to be properly standardized.

3D analysis of capillary network in skeletal muscle of obese insulin-resistant mice Abstract In obesity, the skeletal muscle capillary network regresses and the insulin-mediated capillary recruitment is impaired. However, it has been shown that in the early stage of advanced obesity, an increased functional vascular response can partially compensate for other mechanisms of insulin resistance. The present study aimed to investigate the changes in the capillary network around individual muscle fibres during the early stage of obesity and insulin resistance in mice using 3D analysis. Capillaries and muscle fibres of the gluteus maximus muscles of seven high-fat-diet-induced obese and insulin-resistant mice and seven age-matched lean healthy mice were immunofluorescently labelled in thick transverse muscle sections. Stacks of images were acquired using confocal microscope. Capillary network characteristics were estimated by methods of quantitative image analysis. Muscle fibre typing was performed by histochemical analysis of myosin heavy chain isoforms on thin serial sections of skeletal muscle. Capillary length per muscle fibre length and capillary length per muscle fibre surface were increased by 27% and 23%, respectively, around small muscle fibres in obese mice, while there were no significant comparative differences around large fibres of obese and lean mice. Furthermore, the capillarization was larger around small compared to large fibres and there was a shift toward fast type myosin heavy chain isoforms, with no significant changes in muscle fibre diameters, tortuosity and anisotropy in obese mice. Overall, the results show that obese insulin-resistant mice have selective increase in capillarization around small predominantly intermediate muscle fibres, which is most likely related to the impaired glucose metabolism characteristic of type 2 diabetes.

Unequivocal imaging of aluminium in human cells and tissues by an improved method using morin Abstract Aluminium is biologically reactive and its ability to potentiate the immune response has driven its inclusion in both veterinary and human vaccines. Consequently, the need for unequivocal visualisation of aluminium in vivo has created a focused research effort to establish fluorescent molecular probes for this purpose. The most commonly used direct fluorescent labels for the detection of aluminium are morin (2′,3,4′,5,7-pentahydroxyflavone) and lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzene-1-sulphonic acid]. While the former has gained popularity in the detection of aluminium in plants and predominantly within root tips, the latter boasts greater sensitivity and selectivity for the detection of aluminium in human cells and tissues. Herein, we have developed a simplified morin staining protocol using the autofluorescence quenching agent, Sudan Black B. This modified protocol improves tissue morphology and increases analytical sensitivity, which allows intracellular aluminium to be detected in monocytes and when co-localised with senile plaques in human brain tissue of donors diagnosed with familial Alzheimer’s disease. Overall, our results demonstrate a simple approach to minimise false positives in the use of morin to unequivocally detect aluminium in vivo.

Scaffold protein Lin7 family in membrane skeletal protein complex in mouse seminiferous tubules Abstract The membrane skeletal complex, protein 4.1G–membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell–cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis.