Crystal structures of HER3 extracellular domain 4 in complex with the designed ankyrin-repeat protein D5.

The members of the human epidermal growth factor receptor (HER) family are among the most intensely studied oncological targets. HER3 (ErbB3), which had long been neglected, has emerged as a key oncogene, regulating the activity of other receptors and being involved in progression and tumor escape in multiple types of cancer. Designed ankyrin-repeat proteins (DARPins) serve as antibody mimetics that have proven to be useful in the clinic, in diagnostics and in research. DARPins have previously been selected against EGFR (HER1), HER2 and HER4. In particular, their combination into bivalent binders that separate or lock receptors in their inactive conformation has proved to be a promising strategy for the design of potent anticancer therapeutics. Here, the selection of DARPins targeting extracellular domain 4 of HER3 (HER3d4) is described. One of the selected DARPins, D5, in complex with HER3d4 crystallized in two closely related crystal forms that diffracted to 2.3 and 2.0 Šresolution, respectively. The DARPin D5 epitope comprises HER3d4 residues 568-577. These residues also contribute to interactions within the tethered (inactive) and extended (active) conformations of the extracellular domain of HER3.

Messenger RNA capture sequencing of extracellular RNA from human biofluids using a comprehensive set of spike-in controls.

Comprehensive transcriptome analysis of extracellular RNA (exRNA) purified from human biofluids is challenging because of the low RNA concentration and compromised RNA integrity. Here, we describe an optimized workflow to (1) isolate exRNA from different types of biofluids and (2) to prepare messenger RNA (mRNA)-enriched sequencing libraries using complementary hybridization probes. Importantly, the workflow includes 2 sets of synthetic spike-in RNA molecules as processing controls for RNA purification and sequencing library preparation and as an alternative data normalization strategy. For complete details on the use and execution of this protocol, please refer to Hulstaert et al. (2020).

Differential compartmentalization of BMP4/NOGGIN requires NOGGIN trans-epithelial transport.

Using self-organizing human models of gastrulation, we previously showed that (1) BMP4 initiates the cascade of events leading to gastrulation, (2) BMP4 signal reception is restricted to the basolateral domain, and (3) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically presented NOGGIN could inhibit basally delivered BMP4. Apically imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.

Induction of Extracellular Aminopeptidase Production by Peptides in Some Marine Bacterial Species.

Bacterial extracellular aminopeptidases are key enzymes in protein processing in oligotrophic seawater. To the best of our knowledge, the regulation of aminopeptidase production in microbes inhabiting seawater has not yet been reported. The present study attempted to experimentally clarify which organic materials affect bacterial extracellular aminopeptidase production by nutrient-rich and starved cells growing in artificial seawater using Photobacterium, Alteromonas, Ruegeria, and Sulfitobacter. In all four species, we found that peptides induced bacterial extracellular aminopeptidase production. Amino acids led to cell growth with markedly lower aminopeptidase production by Photobacterium and Sulfitobacter, but not by Alteromonas and Ruegeria. These results suggest that the extracellular aminopeptidases of marine bacteria are primarily produced on demand in response to the presence of relevant substrates (peptides) in seawater. Peptidyl substances may be regulatory nutrients for marine bacterial growth in aquatic environments.

Characterization and enhanced extracellular overexpression of a new salt-activated alginate lyase.

BACKGROUND: Alginate lyases (EC 4.4.2.3/4.4.2.11) have been applied to produce alginate oligosaccharides, which have physiological advantages such as prebiotic and antidiabetic effects, and are of benefit in the food and pharmaceutical industries. Extracellular production of recombinant proteins in Escherichia coli presents advantages including simplified downstream processing and high productivity; however, the presence of certain signal peptides does not always ensure successful secretion, which make the extracellular production of alginate lyase in E. coli rarely reported but of great significance. RESULTS: A PL7 family alginate lyase, Aly01, with its native signal peptide from Vibrio natriegens SK42.001, was identified, characterized, and extracellularly expressed in E. coli. The enzyme specifically released trisaccharide from alginate and was strictly NaCl activated. Green fluorescent protein (GFP) was fused with the Aly01 signal peptide and successfully secreted in E. coli to expand the feasibility of using this signal peptide to produce other heterologous proteins extracellularly. Through a synergistic strategy of utilizing Terrific Broth (TB) medium supplemented with 120 mmol L-1 glycine and 10 mmol L-1 calcium, the lag phase of protein secretion was reduced to 3 h from 12 h; meanwhile calcium remedied glycine-related cell growth impairment, leading to further enhancement of overall enzyme productivity, reaching a maximum of 4.55 U mL-1 . CONCLUSION: A new salt-activated alginate lyase, Aly01, was identified and characterized. E. coli employed its signal peptide and extracellularly expressed both Aly01 and a GFP, which indicated the signal peptide of Aly01 could be a powerful tool for extracellular production of other heterologous proteins in E. coli. © 2021 Society of Chemical Industry.

Construction and Validation of a New Model for the Prediction of Rupture in Patients with Intracranial Aneurysms.

BACKGROUND: Despite progress in the detection of biological molecules that contribute to intracranial aneurysm (IA) development, many pathophysiological mechanisms remain unclear, particularly with regard to predicting IA rupture. In this study, we aimed to identify hub genes and construct a new model to predict IA rupture. METHODS: Four datasets (62 ruptured IAs, 16 unruptured IAs, and 31 normal controls) were downloaded from the Gene Expression Omnibus. Differentially expressed genes (DEGs) were identified between the IAs and normal controls. All overlapping genes were analyzed using weighted gene co-expression network analysis. Functional enrichment analyses were performed using key modules. We then intersected the key module genes with DEGs. Protein-protein interaction networks were assessed to identify key hub genes. Least absolute shrinkage and selection operator logistic regression analysis was performed to construct a prediction model. A receiver operating characteristic curve was constructed to evaluate the reliability of the scoring system. RESULTS: After intersection and normalization, 433 DEGs were identified and 15,388 genes were selected for weighted gene co-expression network analysis. The black module with 1145 genes exhibited the highest correlation with IA rupture. Many potential mechanisms are involved, such as the inflammatory response, innate immune response, extracellular exosome, and extracellular space. Thirty hub genes were selected from the protein-protein interaction, and 4 independent risk genes, TNFAIP6, NCF2, OSM, and IRAK3, were identified in the least absolute shrinkage and selection operator logistic regression model. CONCLUSIONS: Our prediction model not only serves as a useful tool for assessing the risk of IA rupture, but the key genes identified herein could also serve as biomarkers and therapeutic targets.

Extracellular DNA builds and interacts with vibrio polysaccharide in the biofilm matrix formed by Vibrio cholerae.

Vibrio cholerae form biofilm, which is essential for their survival under harsh environmental conditions. The eDNA produced during biofilm formation and interaction with other components like vibrio polysaccharide is less studied in Vibrio cholerae despite its importance in biofilm structure and stability. In this study, we selected two strains of V. cholerae, which produced sufficient extracellular DNA in the biofilm, for characterization and studied its interaction with vibrio polysaccharide. Our data demonstrate that eDNA is present in the biofilm and interacts with VPS in V. cholerae. Our findings suggest that eDNA contributes to biofilm integrity by interacting with VPS and provides strength to the biofilm. Moreover, it might interact with other components of biofilm, which need further study.

Characterization of the Extracellular Fructanase FruA in Lactobacillus crispatus and Its Contribution to Fructan Hydrolysis in Breadmaking.

Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) trigger symptoms of irritable bowel syndrome (IBS). Fructan degradation during bread making reduces FODMAPs in bread while maintaining the content of dietary fiber. This study explored the presence of the fructanases FruA in lactobacilli and characterized its use in bread making. FruA was exclusively present in vertebrate-adapted lactobacilli. In Lactobacillus crispatus DSM29598, FruA was located in cell wall fractions and includes a SLAP domain. FruA hydrolyzed levan or inulin; expression of fruA was not subject to catabolite repression. Fructans in bread were reduced by less than 50% in a straight dough process; conventional sourdough fermentation reduced fructans in bread by 65-70%. Sourdough fermentation with L. crispatus reduced fructans in bread by more than 90%. In conclusion, reduction of FODMAP by sourdough fermentation may improve tolerance in many IBS patients. Fermentation with FruA-expressing L. crispatus DSM29598 produces a low FODMAP bread.

Elevated Plasma Levels of Circulating Extracellular miR-320a-3p in Patients with Paroxysmal Atrial Fibrillation.

The potential of extracellular circulating microRNAs (miRNAs) as non-invasive biomarkers of atrial fibrillation (AF) has been confirmed by a number of recent studies. However, the current data for some miRNAs are controversial and inconsistent, probably due to pre-analytical and methodological differences. In this work, we attempted to fulfill the basic pre-analytical requirements provided for circulating miRNA studies for application to paroxysmal atrial fibrillation (PAF) research. We used quantitative PCR (qPCR) to determine the relative plasma levels of circulating miRNAs expressed in the heart or associated with atrial remodeling or fibrillation with reported altered plasma/serum levels in AF: miR-146a-5p, miR-150-5p, miR-19a-3p, miR-21-5p, miR-29b-3p, miR-320a-3p, miR-328-3p, miR-375-3p, and miR-409-3p. First, in a cohort of 90 adult outpatient clinic patients, we found that the plasma level of miR-320a-3p was elevated in PAF patients compared to healthy controls and hypertensive patients without AF. We further analyzed the impact of medication therapies on miRNA relative levels and found elevated miR-320a-3p levels in patients receiving angiotensin-converting-enzyme inhibitors (ACEI) therapy. Additionally, we found that miR-320a-3p, miR-21-5p, and miR-146a-5p plasma levels positively correlated with the CHA2DS2-Vasc score and were elevated in subjects with CHA2DS2-Vasc ≥ 2. Our results indicate that, amongst the analyzed miRNAs, miR-320a-3p may be considered as a potential PAF circulating plasma biomarker, leading to speculation as to whether this miRNA is a marker of platelet state change due to ACEI therapy.

Therapeutic antibody targeting microtubule-binding domain prevents neuronal internalization of extracellular tau via masking neuron surface proteoglycans.

Pathologically altered tau protein is a common denominator of neurodegenerative disorders including Alzheimer's disease (AD) and other tauopathies. Therefore, promising immunotherapeutic approaches target and eliminate extracellular pathogenic tau species, which are thought to be responsible for seeding and propagation of tau pathology. Tau isoforms in misfolded states can propagate disease pathology in a template-dependent manner, proposed to be mediated by the release and internalization of extracellular tau. Monoclonal antibody DC8E8, binding four highly homologous and independent epitopes in microtubule-binding domain (MTBD) of diseased tau, inhibits tau-tau interaction, discriminates between healthy and pathologically truncated tau and reduces tau pathology in animal model in vivo. Here, we show that DC8E8 antibody acts via extracellular mechanism and does not influence viability and physiological functions of neurons. Importantly, in vitro functional assays showed that DC8E8 recognises pathogenic tau proteins of different size and origin, and potently blocks their entry into neurons. Next, we examined the mechanisms by which mouse antibody DC8E8 and its humanized version AX004 effectively block the neuronal internalization of extracellular AD tau species. We determined a novel mode of action of a therapeutic candidate antibody, which potently inhibits neuronal internalization of AD tau species by masking of epitopes present in MTBD important for interaction with neuron surface Heparan Sulfate Proteoglycans (HSPGs). We show that interference of tau-heparane sulfate interaction with DC8E8 antibody via steric hindrance represents an efficient and important therapeutic approach halting tau propagation.

Exosomal and extracellular HMGB1 have opposite effects on SASH1 expression in rat astrocytes and glioma C6 cells.

Exosomes are a type of extracellular vesicles derived from cells and mediators of intercellular communication. Different cell types have their own unique exosomes for exchanging information. We previously found that SASH1, a tumor suppressor, was lowly expressed or absent in glioma tissues and glioma C6 cells, but the structure and function of the corresponding exosomes had been unclear. Hence, we aimed to investigate whether exosomes generated from normal glial cells and glioma cells form different protein patterns and whether those derived from normal glial cells affect SASH1 expression in glioma cells. We collected exosomes from astrocytes and C6 cells and identified their exosomal proteins through mass spectrometry. We also performed gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses, whose results showed that both the total and unique exosomal proteins from each cell type were similar. Moreover, the KEGG analysis revealed different clusters of unique exosomal proteins in glial cells and glioma cells. In the normal glial cells, the top clusters were mainly involved in processes with RNA transcripts and proteins, whereas in glioma cells the clusters were attributed to PI3K-Akt signaling, cell adhesion, and cancer-related pathways. Western blot analysis showed that HMGB1 exists in exosomes derived from cultured astrocytes, although its expression was higher in glioma C6 cells. Furthermore, we found that exosomes extracted from astrocytes could increase SASH1 expression in C6 cells (P = 0.040), whereas those derived from HMGB1-depleted astrocytes could not (P = 0.6133). The expression levels of SASH1 decreased after the addition of extracellular recombinant HMGB1 protein, whereas that of TLR4 increased. Our study is the first to demonstrate that HMGB1 plays different roles depending on its form: as an extracellular protein, HMGB1 decreases SASH1 expression, but as an exosomal protein, HMGB1 increases SASH1 expression. Nevertheless, the mechanism, which partly depends on the TLR4 pathway, behind these opposing effects requires further study. Our novel findings on the structure-dependent roles of the cytokine HMGB1 in promoting or inhibiting cancer provide a fresh insight into the interactions of cancer cells with the microenvironment.

Diversity of extracytoplasmic function sigma (σECF ) factor-dependent signaling in Pseudomonas.

Pseudomonas bacteria are widespread and are found in soil and water, as well as pathogens of both plants and animals. The ability of Pseudomonas to colonize many different environments is facilitated by the multiple signaling systems these bacteria contain that allow Pseudomonas to adapt to changing circumstances by generating specific responses. Among others, signaling through extracytoplasmic function σ (σECF ) factors is extensively present in Pseudomonas. σECF factors trigger expression of functions required under particular conditions in response to specific signals. This manuscript reviews the phylogeny and biological roles of σECF factors in Pseudomonas, and highlights the diversity of σECF -signaling pathways of this genus in terms of function and activation. We show that Pseudomonas σECF factors belong to 16 different phylogenetic groups. Most of them are included within the iron starvation group and are mainly involved in iron acquisition. The second most abundant group is formed by RpoE-like σECF factors, which regulate the responses to cell envelope stress. Other groups controlling solvent tolerance, biofilm formation and the response to oxidative stress, among other functions, are present in lower frequency. The role of σECF factors in the virulence of Pseudomonas pathogenic species is described.

ECF σ factors with regulatory extensions: the one-component systems of the σ universe.

The σ subunit of the bacterial RNA polymerase determines promoter specificity. The extracytoplasmic function σ factors (ECFs) represent the most abundant and diverse group of alternative σ factors and are present in the vast majority of bacterial genomes. Typically, ECFs are regulated by anti-σ factors that sequester their cognate ECFs, thereby preventing their interaction with the RNA polymerase. Beyond these ECF paradigms, a number of distinct modes of regulation have been proposed and experimentally investigated. Regulatory extensions represent one such alternative mechanism of ECF regulation that can be found in 18 phylogenetically distinct ECF groups. Here, the σ factors contain additional domains that are fused to the ECF core domains and are involved in stimulus perception and modulation of σ factor activity. We will summarize the current state of knowledge on regulating ECF activity by C-terminal extensions. We will also discuss newly identified ECF groups containing either N- or C-terminal extensions and propose possible mechanisms by which these extensions have been generated and affect ECF σ factor activity. Based on their modular architecture and the resulting physical connection between stimulus perception and transcriptional output, these ECFs are analogous to one-component systems, the primary mechanism of bacterial signal transduction.

Discovery of the extracytoplasmic function σ factors.

This special issue of Molecular Microbiology marks the 25th anniversary of the discovery of the extracytoplasmic function (ECF) σ factors, proteins that subsequently emerged as the largest group of alternative σ factors and one of the three major pillars of signal transduction in bacteria, alongside one- and two-component systems. A single bacterial genome can encode > 100 ECF σ factors, and combined with their cognate anti-σ factors, they represent a modular design that primarily functions in transmembrane signal transduction. Here, we first describe the immediate events that led to the 1994 publication in the Proceeding of the National Academy of Sciences USA, and then set them in the broader context of key events in the history of σ biology research.

Internal gate mutants of the GABA transporter GAT1 are capable of substrate exchange.

GAT1 is a member of the neurotransmitter:sodium: symporter family and mediates transport of GABA together with sodium and chloride in an electrogenic process enabling efficient synaptic transmission. Biochemical and modelling studies based on the structure of the bacterial homologue LeuT are consistent with a transport mechanism whereby the binding pocket is alternately accessible to either side of the membrane. This is achieved by the sequential opening and closing of extracellular and intracellular gates. The amino acid residues participating in the formation of these gates are highly conserved within the neurotransmitter:sodium: symporter family. Net flux requires that the gating mechanism is operative regardless if the binding pocket is loaded with substrate or empty. On the other hand, exchange of labelled for non-labelled substrate across the membrane only requires gating in the presence of substrate. To address the question if the gating requirements of the substrate-bound and empty transporters are similar or different, we analyzed the impact of mutation of intra- and extra-cellular gate residues on net GABA influx and on exchange by liposomes inlaid with the mutant transporters. Whereas net flux by all four internal gate mutants tested was severely abrogated, each exhibited significant levels of exchange. In contrast, two external gate mutants were impaired in both processes. Our results indicate that perturbation of the internal gate of GAT1 selectively impairs the gating mechanism of the empty transporter. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

IL-26, a Cytokine With Roles in Extracellular DNA-Induced Inflammation and Microbial Defense.

Interleukin 26 (IL-26) is the most recently identified member of the IL-20 cytokine subfamily, and is a novel mediator of inflammation overexpressed in activated or transformed T cells. Novel properties have recently been assigned to IL-26, owing to its non-conventional cationic, and amphipathic features. IL-26 binds to DNA released from damaged cells and, as a carrier molecule for extracellular DNA, links DNA to inflammation. This observation suggests that IL-26 may act both as a driver and an effector of inflammation, leading to the establishment of a deleterious amplification loop and, ultimately, sustained inflammation. Thus, IL-26 emerges as an important mediator in local immunity/inflammation. The dysregulated expression and extracellular DNA carrier capacity of IL-26 may have profound consequences for the chronicity of inflammation. IL-26 also exhibits direct antimicrobial properties. This review summarizes recent advances on the biology of IL-26 and discusses its roles as a novel kinocidin.

Direct visualization of a native Wnt in vivo reveals that a long-range Wnt gradient forms by extracellular dispersal.

Wnts are evolutionarily conserved signaling proteins with essential roles in development and disease that have often been thought to spread between cells and signal at a distance. However, recent studies have challenged this model, and whether long-distance extracellular Wnt dispersal occurs and is biologically relevant is debated. Understanding fundamental aspects of Wnt dispersal has been limited by challenges with observing endogenous ligands in vivo, which has prevented directly testing hypotheses. Here, we have generated functional, fluorescently tagged alleles for a C. elegans Wnt homolog and for the first time visualized a native, long-range Wnt gradient in a living animal. Live imaging of Wnt along with source and responding cell membranes provided support for free, extracellular dispersal. By limiting Wnt transfer between cells, we confirmed that extracellular spreading shapes a long-range gradient and is critical for neuroblast migration. These results provide direct evidence that Wnts spread extracellularly to regulate aspects of long-range signaling.

How, When, and Where Relic DNA Affects Microbial Diversity.

Extracellular or "relic" DNA is one of the largest pools of nucleic acids in the biosphere. Relic DNA can influence a number of important ecological and evolutionary processes, but it may also affect estimates of microbial abundance and diversity, which has implications for understanding environmental, engineered, and host-associated ecosystems. We developed models capturing the fundamental processes that regulate the size and composition of the relic DNA pools to identify scenarios leading to biased estimates of biodiversity. Our models predict that bias increases with relic DNA pool size, but only when the species abundance distributions (SADs) of relic and intact DNA are distinct from one another. We evaluated our model predictions by quantifying relic DNA and assessing its contribution to bacterial diversity using 16S rRNA gene sequences collected from different ecosystem types, including soil, sediment, water, and the mammalian gut. On average, relic DNA made up 33% of the total bacterial DNA pool but exceeded 80% in some samples. Despite its abundance, relic DNA had a minimal effect on estimates of taxonomic and phylogenetic diversity, even in ecosystems where processes such as the physical protection of relic DNA are common and predicted by our models to generate bias. Our findings are consistent with the expectation that relic DNA from different taxa degrades at a constant and equal rate, suggesting that it may not fundamentally alter estimates of microbial diversity.IMPORTANCE The ability to rapidly obtain millions of gene sequences and transcripts from a range of environments has greatly advanced understanding of the processes that regulate microbial communities. However, nucleic acids extracted from complex samples do not come only from viable microorganisms. Dead microorganisms can generate large pools of relic DNA that distort insight into the ecology and evolution of microbial systems. Here, we develop a conceptual and quantitative framework for understanding how relic DNA influences the structure of microbiomes. Our theoretical models and empirical results demonstrate that a large relic DNA pool does not automatically lead to biased estimates of microbial diversity. Rather, relic DNA effects emerge in combination with microscale processes that alter the commonness and rarity of sequences found in heterogeneous DNA pools.

Monitoring of resistance genes in Listeria monocytogenes isolates and their presence in the extracellular DNA of biofilms: a case study from the Czech Republic.

The alarming occurrence of antibiotic resistance genes in food production demands continuous monitoring worldwide. One reservoir of resistance genes is thought to be eDNA. There is currently little available information in Europe about either the extracellular DNA distribution of the bacterium or the spread of resistance genes in L. monocytogenes. Therefore, our aims were to give insight into the Listeria monocytogenes resistance situation in the Czech Republic and assess the presence of resistance genes in their extracellular DNA (eDNA). First, susceptibility tests were performed on 49 isolates of L. monocytogenes with selected antibiotics. Next, we tested DNA of suspected isolates for the presence of resistance genes in both planktonic cells and the eDNA of biofilms. Finally, fluorescent confocal microscopy was used to observe the eDNA pattern of selected isolates under conditions that mimicked the food processing environment and the human body. Susceptibility tests found isolates intermediate resistant to chloramphenicol, tetracycline, and ciprofloxacin as well as isolates resistant to ciprofloxacin. For all suspected isolates, PCR confirmed the presence of the gene lde encoding efflux pump in both types of DNA. When the biofilm was observed using confocal laser scanning microscope, the eDNA distribution patterns varied considerably according to the culture conditions. Furthermore, the food and clinical isolates varied in terms of the amount of eDNA detected. The presence of an efflux pump in both types of DNA suggests that the eDNA might serve as a reservoir of resistance genes. Surprising differences were observed in the eDNA pattern. Our results suggest that the current risk of the spread of L. monocytogenes resistance genes is low in the Czech Republic, but they also indicate the need for continuous long-term monitoring of the situation.

Germ cell-specific apoptosis by extracellular clusterin in cryptorchid dog testes.

Mammalian testes are maintained at a relatively lesser temperature than the abdominal region so that normal spermatogenesis can occur. Germ cell apoptosis has resulted in heat-damaged testes that occurs as a result of cryptorchidism, but the mechanism is not yet fully understood. To elucidate the cause of germ-cell death by cryptorchidism, cryptorchidism was surgically induced in dog testes and histological and molecular analyses were performed. Histological data indicated that the seminiferous tubules of cryptorchid testes and epididymis contained fewer germ cells. Total RNA sequencing was performed to screen for overexpressed genes in cryptorchid dog testes. Clusterin RNA was in greater abundance (approximately 12.8-fold) in cryptorchid testes than in normal testes. In addition, cleaved caspase-3 and -8 were detected in greater abundance in cryptorchid dog testes. Real time RT-PCR and western blotting analysis indicated there was a greater abundance of clusterin in cryptorchid dog testes. Furthermore, clusterin was detected in extracellular regions of cryptorchid dog testes during the 4 weeks after surgery. Thus, germ-cell specific apoptosis and expression of clusterin genes occur with a resulting presence of this protein in extracellular regions of cryptorchid dog testes. This result will facilitate further study of spermatogenesis and the specific mechanisms by which cryptorchidism results in male infertility.