Dietary 14C reservoir effects and the chronology of prehistoric burials at Sakhtysh, central European Russia.

We present a robust radiocarbon (14C) chronology for burials at Sakhtysh, in European Russia, where nearly 180 inhumations of Lyalovo and Volosovo pottery-using hunter-gatherer-fishers represent the largest known populations of both groups. Past dating attempts were restricted by poor understanding of dietary 14C reservoir effects (DREs). We developed a DRE correction approach that uses multiple linear regression of differences in 14C, δ13C, and δ15N between bones and teeth of the same individuals to predict DREs of up to approximately 900 years. Our chronological model dates Lyalovo burials to the early fifth millennium BCE, and Volosovo burials to the mid-fourth to early third millennium. It reveals a change in the subsistence economy at approximately 3300 BCE, coinciding with a reorientation of trade networks, and dates the final burial to the early Fatyanovo period, the regional expression of the Yamnaya/Corded Ware expansion. Our approach is applicable when freshwater 14C reservoir effects are poorly constrained and grave goods cannot be dated directly.

Effects of weaving parameters on the properties of completely biological tissue-engineered vascular grafts.

Tissue-Engineered Vascular Grafts (TEVGs) made of human textiles have been recently introduced and offer remarkable biocompatibility as well as tunable mechanical properties. The approach combines the use of Cell-Assembled extracellular Matrix (CAM) threads, produced by cultured cells in vitro, with weaving, a versatile assembly method that gives fine control over graft properties. Herein, we investigated how production parameters can modify the geometrical and mechanical properties of TEVGs to better match that of native blood vessels in order to provide long-term patency. Our goals were to decrease the mechanical strength and the luminal surface profile of our first generation of woven TEVGs, while maintaining low transmural permeability and good suture retention strength. Different TEVGs were produced by varying CAM sheet strength as well as weaving parameters such as warp count, weft ribbons width, and weft tension. An optimized design reduced the burst pressure by 35%, wall thickness by 38% and increased compliance by 269%. The improved TEVG had properties closer to that of native blood vessels, with a burst pressure of 3492 mmHg, a wall thickness of 0.69 mm, and a compliance of 4.8%/100 mmHg, while keeping excellent suture retention strength (4.7 N) and low transmural permeability (24 mL·min-1·cm-2). Moreover, the new design reduced the luminal surface profile by 48% and utilized 47% less CAM. With a comparable design, the use of decellularized CAM threads, instead of devitalized ones, led to TEVGs with much more permeable walls and higher burst pressure. The next step is to implant this optimized graft in an allogeneic sheep model of arteriovenous shunt to assess its in vivo remodeling and performance. .

The cell-assembled extracellular matrix: A focus on the storage stability and terminal sterilization of this human "bio" material.

The Cell-Assembled extracellular Matrix (CAM) is an attractive biomaterial because it provided the backbone of vascular grafts that were successfully implanted in patients, and because it can now be assembled in "human textiles". For future clinical development, it is important to consider key manufacturing questions. In this study, the impact of various storage conditions and sterilization methods were evaluated. After 1 year of dry frozen storage, no change in mechanical nor physicochemical properties were detected. However, storage at 4 °C and room temperature resulted in some mechanical changes, especially for dry CAM, but physicochemical changes were minor. Sterilization modified CAM mechanical and physicochemical properties marginally except for hydrated gamma treatment. All sterilized CAM supported cell proliferation. CAM ribbons were implanted subcutaneously in immunodeficient rats to assess the impact of sterilization on the innate immune response. Sterilization accelerated strength loss but no significant difference could be shown at 10 months. Very mild and transient inflammatory responses were observed. Supercritical CO2 sterilization had the least effect. In conclusion, the CAM is a promising biomaterial since it is unaffected by long-term storage in conditions available in hospitals (hydrated at 4 °C), and can be sterilized terminally (scCO2) without compromising in vitro nor in vivo performance. STATEMENT OF SIGNIFICANCE: In the field of tissue engineering, the use of extracellular matrix (ECM) proteins as a scaffolding biomaterial has become very popular. Recently, many investigators have focused on ECM produced by cells in vitro to produce unprocessed biological scaffolds. As this new kind of "biomaterial" becomes more and more relevant, it is critical to consider key manufacturing questions to facilitate future transition to the clinic. This article presents an extensive evaluation of long-term storage stability and terminal sterilization effects on an extracellular matrix assembled by cells in vitro. We believe that this article will be of great interest to help tissue engineers involved in so-called scaffold-free approaches to better prepare the translation from benchtop to bedside.

Current allele distribution of the human longevity gene APOE in Europe can mainly be explained by ancient admixture.

Variation in apolipoprotein E (APOE) has been shown to have the strongest genetic effect on human longevity. The aim of this study was to unravel the evolutionary history of the three major APOE alleles in Europe by analysing ancient samples up to 12,000 years old. We detected significant allele frequency shifts between populations and over time. Our analyses indicated that selection led to large frequency differences between the earliest European populations (i.e., hunter-gatherers vs. first farmers), possibly due to changes in diet/lifestyle. In contrast, the allele distributions in populations from ~4000 BCE onward can mainly be explained by admixture, suggesting that it also played an important role in shaping current APOE variation. In any case, the resulting allele frequencies strongly influence the predisposition for longevity today, likely as a consequence of past adaptations and demographic processes.

Soybean sorting based on protein content using X-ray fluorescence spectrometry.

The purpose of this research was to evaluate performance of an energy-dispersive X-ray fluorescence (XRF) sensor to classify soybean based on protein content. The hypothesis was that sulfur signals and other XRF spectral features can be used as proxies to infer soybean protein content. Sample preparation and equipment settings to optimize detection of S and other specific emission lines were tested for this application. A logistic regression model for classifying soybean as high- or low-protein was developed based on XRF spectra and protein contents. Additionally, the model was validated with an independent set of samples. Global accuracies of the method were 0.83 (training set) and 0.81 (test set) and the corresponding kappa indices were 0.66 and 0.61, respectively. These numbers indicated satisfactory performance of the sensor, suggesting that XRF spectral features can be applied for screening protein content in soybean.

Development of a vascular substitute produced by weaving yarn made from human amniotic membrane.

Because synthetic vascular prostheses perform poorly in small-diameter revascularization, biological vascular substitutes are being developed as an alternative. Although theirin vivoresults are promising, their production involves long, complex, and expensive tissue engineering methods. To overcome these limitations, we propose an innovative approach that combines the human amniotic membrane (HAM), which is a widely available and cost-effective biological raw material, with a rapid and robust textile-inspired assembly strategy. Fetal membranes were collected after cesarean deliveries at term. Once isolated by dissection, HAM sheets were cut into ribbons that could be further processed by twisting into threads. Characterization of the HAM yarns (both ribbons and threads) showed that their physical and mechanical properties could be easily tuned. Since our clinical strategy will be to provide an off-the-shelf allogeneic implant, we studied the effects of decellularization and/or gamma sterilization on the histological, mechanical, and biological properties of HAM ribbons. Gamma irradiation of hydrated HAMs, with or without decellularization, did not interfere with the ability of the matrix to support endothelium formationin vitro. Finally, our HAM-based, woven tissue-engineered vascular grafts (TEVGs) exhibited clinically relevant mechanical properties. Thus, this study demonstrates that human, completely biological, allogeneic, small-diameter TEVGs can be produced from HAM, thereby avoiding costly cell culture and bioreactors.

Perfil clínico-epidemiológico de pacientes diagnosticados com câncer renal em hospital de alta complexidade / Clinical-epidemiological profile of patients diagnosed with kidney cancer in a high complexity hospital

RESUMO Os carcinomas de células renais (CCRs) são o sétimo tipo histológico de câncer mais comum no mundo ocidental e vêm apresentando uma tendência mantida de aumento em sua prevalência. O presente estudo teve por objetivo analisar dados clínicos, demográficos e anatomopatológicos, a partir de prontuários de pacientes diagnosticados com câncer renal, em um centro de referência de oncologia do norte gaúcho. Métodos: Trata-se de pesquisa transversal, realizada com 105 pacientes submetidos a nefrectomias, no período de janeiro de 2013 a setembro de 2018. Resultados: A nefrectomia radical foi realizada em 84,5% de amostras e o anatomopatológico indicou o carcinoma de células claras em 74,1%. Em relação ao sexo, a maioria foi do sexo masculino 64,10% e a idade média foi de 59.9 anos (DP+-11,5), variando de 31 a 81 anos. Quanto aos sintomas, 18% apresentaram a hematúria, em 13,5% dor em flanco, em 10% dor abdominal, e 6,8% dor lombar. Conclusões: O estudo mostrou que o padrão clinico-epidemiológico da neoplasia no hospital estudado está em concordância com a literatura. PALAVRAS-CHAVE: Neoplasias renais, perfil de saúde, neoplasias por tipo histológico

Renal cell carcinomas (RCCs) are the seventh most common histological type of cancer in the Western world and have been showing a sustained upward trend in their prevalence. This study aimed to analyze clinical, demographic and anatomopathological data from medical records of patients diagnosed with kidney cancer, in an oncology reference center in the north of Rio Grande do Sul. Methods: This is a cross-sectional study, carried out with 105 patients undergoing nephrectomies, from January 2013 to September 2018. Results: Radical nephrectomy was performed in 84.5% of samples and the pathological examination indicated clear cell carcinoma in 74.1%. Regarding gender, the majority were male 64.10% and the mean age was 59.9 years (SD+-11.5), ranging from 31 to 81 years. As for symptoms, 18% had hematuria, 13.5% had flank pain, 10% had abdominal pain, and 6.8% had low back pain. Conclusions: The study showed that the clinical-epidemiological pattern of RCC in the studied hospital is in agreement with the literature. KEYWORDS: Kidney neoplasms, health profile, neoplasms by histological type

Cell-assembled extracellular matrix (CAM) sheet production: Translation from using human to large animal cells.

We have created entirely biological tissue-engineered vascular grafts (TEVGs) using sheets of cell-assembled extracellular matrix (CAM) produced by human fibroblasts in vitro. A large animal TEVG would allow long-term pre-clinical studies in a clinically relevant setting (graft size and allogeneic setting). Therefore, canine, porcine, ovine, and human skin fibroblasts were compared for their ability to form CAM sheets. Serum sourcing greatly influenced CAM production in a species-dependent manner. Ovine cells produced the most homogenous and strongest animal CAM sheets but remained ≈3-fold weaker than human sheets despite variations of serum, ascorbate, insulin, or growth factor supplementations. Key differences in cell growth dynamics, tissue development, and tissue architecture and composition were observed between human and ovine. This study demonstrates critical species-to-species differences in fibroblast behavior and how they pose a challenge when attempting to substitute animal cells for human cells during the development of tissue-engineered constructs that require long-term cultures.

Significant Amplification of Instantaneous Extreme Precipitation With Convective Self-Aggregation.

This work explores the effect of convective self-aggregation on extreme rainfall intensities through an analysis at several stages of the cloud lifecycle. In addition to increases in 3-hourly extremes consistent with previous studies, we find that instantaneous rainrates increase significantly (+30%). We mainly focus on instantaneous extremes and, using a recent framework, relate their increase to increased precipitation efficiency: the local increase in relative humidity drives larger accretion efficiency and lower re-evaporation. An in-depth analysis based on an adapted scaling for precipitation extremes reveals that the dynamic contribution decreases (-25%) while the thermodynamic is slightly enhanced (+5%) with convective self-aggregation, leading to lower condensation rates. When the atmosphere is more organized into a moist convecting region and a dry convection-free region, deep convective updrafts are surrounded by a warmer environment which reduces convective instability and thus the dynamic contribution. The moister boundary-layer explains the positive thermodynamic contribution. The microphysic contribution is increased by +50% with aggregation. The latter is partly due to reduced evaporation of rain falling through a moister near-cloud environment, but also to the associated larger accretion efficiency. Thus, a potential change in convective organization regimes in a warming climate could lead to an evolution of tropical precipitation extremes significantly different than that expected from thermodynamical considerations. The relevance of self-aggregation to the real tropics is still debated. Improved fundamental understanding of self-aggregation, its sensitivity to warming and connection to precipitation extremes, is hence crucial to achieve accurate rainfall projections in a warming climate.

Macrophages Facilitate Electrical Conduction in the Heart.

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Macrophages and dendritic cells in the post-testicular environment.

Macrophages (MΦ) and dendritic cells (DCs) are heterogeneous families of functionally and developmentally related immune cells that play crucial roles in tissue homeostasis and the regulation of immune responses. During the past 5 years, immunologists have generated a considerable amount of data that challenge dogmas about the ontogeny and functions of these highly versatile cells. The male excurrent duct system plays a critical role in the establishment of fertility by allowing sperm maturation, transport and storage. In addition, it is challenged by pathogens and must establish a protective and tolerogenic environment for a continuous flow of autoantigenic spermatozoa. The post-testicular environment and, in particular, the epididymis contain an intricate network of DCs and MΦ; however, the immunophysiology of this intriguing and highly specialized mucosal system is poorly understood. This review summarizes the current trends in mouse MΦ and DC biology and speculates about their roles in the steady-state epididymis. Unraveling immune cell functions in the male reproductive tract is an essential prerequisite for the design of innovative strategies aimed at controlling male fertility and treating infertility.

Exploring the role of mononuclear phagocytes in the epididymis.

The onslaught of foreign antigens carried by spermatozoa into the epididymis, an organ that has not demonstrated immune privilege, a decade or more after the establishment of central immune tolerance presents a unique biological challenge. Historically, the physical confinement of spermatozoa to the epididymal tubule enforced by a tightly interwoven wall of epithelial cells was considered sufficient enough to prevent cross talk between gametes and the immune system and, ultimately, autoimmune destruction. The discovery of an intricate arrangement of mononuclear phagocytes (MPs) comprising dendritic cells and macrophages in the murine epididymis suggests that we may have underestimated the existence of a sophisticated mucosal immune system in the posttesticular environment. This review consolidates our current knowledge of the physiology of MPs in the steady state epididymis and speculates on possible interactions between auto-antigenic spermatozoa, pathogens and the immune system by drawing on what is known about the immune system in the intestinal mucosa. Ultimately, further investigation will provide valuable information regarding the origins of pathologies arising as a result of autoimmune or inflammatory responses in the epididymis, including epididymitis and infertility.

Macrophages retain hematopoietic stem cells in the spleen via VCAM-1.

Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)(+) macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE(-/-) mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques.

Role of testicular luminal factors on Basal cell elongation and proliferation in the mouse epididymis.

A subset of basal cells (BCs) in the initial segment (IS) of the mouse epididymis has a slender body projection between adjacent epithelial cells. We show here that these projections occasionally cross the apical tight junctions and are in contact with the luminal environment. Luminal testicular factors are critical for the establishment of the IS epithelium, and we investigated their role in the regulation of this luminal sensing property. Efferent duct ligation (EDL) was performed to block luminal flow from the testis without affecting blood flow. Cytokeratin 5 (KRT5) labeling showed a time-dependent reduction of the percentage of BCs with intercellular projections from 1 to 5 days after EDL, compared to controls. Double labeling for caspase-3 and KRT5 showed that a subset of BCs undergoes apoptosis 1 day after EDL. Ki67/KRT5 double labeling showed a low rate of BC proliferation under basal conditions. However, EDL induced a marked increase in the proliferation rate of a subset of BCs 2 days after EDL. A 2-wk treatment with the androgen receptor antagonist flutamide did not affect the number of BCs with intercellular projections, but reduced BC proliferation. Flutamide treatment also reduced the increase in BC proliferation induced 2 days after EDL. We conclude that, in the adult mouse IS, 1) luminal testicular factors play an important role in the ability of BCs to extend their body projection towards the lumen, and are essential for the survival of a subset of BCs; 2) androgens play an important role in the proliferation of some of the BCs that survive the initial insult induced by EDL; and 3) the formation and elongation of BC intercellular projections do not depend on androgens.

CFTR interacts with ZO-1 to regulate tight junction assembly and epithelial differentiation through the ZONAB pathway.

Mutations in CFTR lead to dysfunction of tubular organs, which is currently attributed to impairment of its conductive properties. We now show that CFTR regulates tight junction assembly and epithelial cell differentiation through modulation of the ZO-1-ZONAB pathway. CFTR colocalizes with ZO-1 at the tight junctions of trachea and epididymis, and is expressed before ZO-1 in Wolffian ducts. CFTR interacts with ZO-1 through the CTFR PDZ-binding domain. In a three-dimensional (3D) epithelial cell culture model, CFTR regulates tight junction assembly and is required for tubulogenesis. CFTR inhibition or knockdown reduces ZO-1 expression and induces the translocation of the transcription factor ZONAB (also known as YBX3) from tight junctions to the nucleus, followed by upregulation of the transcription of CCND1 and downregulation of ErbB2 transcription. The epididymal tubules of cftr(-/-) and cftr(ΔF508) mice have reduced ZO-1 levels, increased ZONAB nuclear expression, and decreased epithelial cell differentiation, illustrated by the reduced expression of apical AQP9 and V-ATPase. This study provides a new paradigm for the etiology of diseases associated with CFTR mutations, including cystic fibrosis.

Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.

RATIONALE: Macrophages populate the steady-state myocardium. Previously, all macrophages were thought to arise from monocytes; however, it emerged that, in several organs, tissue-resident macrophages may self-maintain through local proliferation. OBJECTIVE: Our aim was to study the contribution of monocytes to cardiac-resident macrophages in steady state, after macrophage depletion in CD11b(DTR/+) mice and in myocardial infarction. METHODS AND RESULTS: Using in vivo fate mapping and flow cytometry, we estimated that during steady state the heart macrophage population turns over in ≈1 month. To explore the source of cardiac-resident macrophages, we joined the circulation of mice using parabiosis. After 6 weeks, we observed blood monocyte chimerism of 35.3±3.4%, whereas heart macrophages showed a much lower chimerism of 2.7±0.5% (P<0.01). Macrophages self-renewed locally through proliferation: 2.1±0.3% incorporated bromodeoxyuridine 2 hours after a single injection, and 13.7±1.4% heart macrophages stained positive for the cell cycle marker Ki-67. The cells likely participate in defense against infection, because we found them to ingest fluorescently labeled bacteria. In ischemic myocardium, we observed that tissue-resident macrophages died locally, whereas some also migrated to hematopoietic organs. If the steady state was perturbed by coronary ligation or diphtheria toxin-induced macrophage depletion in CD11b(DTR/+) mice, blood monocytes replenished heart macrophages. However, in the chronic phase after myocardial infarction, macrophages residing in the infarct were again independent from the blood monocyte pool, returning to the steady-state situation. CONCLUSIONS: In this study, we show differential contribution of monocytes to heart macrophages during steady state, after macrophage depletion or in the acute and chronic phase after myocardial infarction. We found that macrophages participate in the immunosurveillance of myocardial tissue. These data correspond with previous studies on tissue-resident macrophages and raise important questions on the fate and function of macrophages during the development of heart failure.

Epithelial basal cells are distinct from dendritic cells and macrophages in the mouse epididymis.

The epithelium that lines the epididymal duct establishes the optimal milieu in which spermatozoa mature, acquire motility, and are stored. This finely tuned environment also protects antigenic sperm against pathogens and autoimmunity, which are potential causes of transient or permanent infertility. The epididymal epithelium is pseudostratified and contains basal cells (BCs) that are located beneath other epithelial cells. Previous studies showed that in the mouse epididymis, BCs possess macrophage-like characteristics. However, we previously identified a dense population of cells belonging to the mononuclear phagocyte (MP) system (comprised of macrophages and dendritic cells) in the basal compartment of the mouse epididymis and showed that a subset of MPs express the macrophage marker F4/80. In the present study, we evaluate the distribution of BCs and MPs in the epididymis of transgenic CD11c-EYFP mice, in which EYFP is expressed exclusively in MPs, using antibodies against the BC marker keratin 5 (KRT5) and the macrophage marker F4/80. Immunofluorescence labeling for laminin, a basement membrane marker, showed that BCs and most MPs are located in the basal region of the epithelium. Confocal microscopy showed that in the initial segment, both BCs and MPs project intraepithelial extensions and establish a very intricate network. Flow cytometry experiments demonstrated that epididymal MPs and BCs are phenotypically distinct. BCs do not express F4/80, and MPs do not express KRT5. Therefore, despite their proximity and some morphological similarities with peritubular macrophages and dendritic cells, BCs do not belong to the MP system.

ATP secretion in the male reproductive tract: essential role of CFTR.

Extracellular ATP is essential for the function of the epididymis and spermatozoa, but ATP release in the epididymis remains uncharacterized. We investigated here whether epithelial cells release ATP into the lumen of the epididymis, and we examined the role of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) and HCO(3)(-) conducting ion channel known to be associated with male fertility, in this process. Immunofluorescence labelling of mouse cauda epididymidis showed expression of CFTR in principal cells but not in other epithelial cells. CFTR mRNA was not detectable in clear cells isolated by fluorescence-activated cell sorting (FACS) from B1-EGFP mice, which express enhanced green fluorescent protein (EGFP) exclusively in these cells in the epididymis. ATP release was detected from the mouse epididymal principal cell line (DC2) and increased by adrenaline and forskolin. Inhibition of CFTR with CFTR(inh172) and transfection with CFTR-specific siRNAs in DC2 cells reduced basal and forskolin-activated ATP release. CFTR-dependent ATP release was also observed in primary cultures of mouse epididymal epithelial cells. In addition, steady-state ATP release was detected in vivo in mice, by measuring ATP concentration in a solution perfused through the lumen of the cauda epididymidis tubule and collected by cannulation of the vas deferens. Luminal CFTR(inh172) reduced the ATP concentration detected in the perfusate. This study shows that CFTR is involved in the regulation of ATP release from principal cells in the cauda epididymidis. Given that mutations in CFTR are a leading cause of male infertility, we propose that defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with these mutations.

Establishment of cell-cell cross talk in the epididymis: control of luminal acidification.

Male infertility is often caused by sperm that have low motility and interact poorly with the oocyte. Spermatozoa acquire these crucial functions in the epididymis. A low luminal bicarbonate (HCO(3)(-)) concentration and low pH keep sperm quiescent during their maturation and storage in this organ. This review describes how epididymal epithelial cells work in a concerted manner, together with spermatozoa, to establish and maintain this acidic luminal environment. Clear cells express the proton-pumping ATPase (V-ATPase) in their apical membrane and actively secrete protons. HCO(3)(-) induces V-ATPase accumulation in apical microvilli in clear cells via HCO(3)(-)-sensitive adenylyl cyclase-dependent cAMP production. HCO(3)(-) is secreted from principal cells following basolateral stimulation, to transiently "prime" spermatozoa before ejaculation. Luminal ATP and adenosine also induce V-ATPase apical accumulation in clear cells via activation of P2 and P1 receptors, respectively. ATP is released into the lumen from sperm and principal cells and is then metabolized into adenosine by local nucleotidases. In addition, the V-ATPase is regulated by luminal angiotensin II via activation of basal cells, which can extend narrow body projections that cross the tight junction barrier. Basal cells then secrete nitric oxide, which diffuses out to stimulate proton secretion in clear cells via activation of the cGMP pathway. Thus, an elaborate communication network is present between principal cells and clear cells, and between basal cells and clear cells, to control luminal acidification. Monitoring and decoding these "intercellular conversations" will help define pathophysiologic mechanisms underlying male infertility.

Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells.

Luminal acidification in the epididymis is critical for sperm maturation and storage. Clear cells express the vacuolar H(+)-ATPase (V-ATPase) in their apical membrane and are major contributors to proton secretion. We showed that this process is regulated via recycling of V-ATPase-containing vesicles. We now report that RhoA and its effector ROCKII are enriched in rat epididymal clear cells. In addition, cortical F-actin was detected beneath the apical membrane and along the lateral membrane of "resting" clear cells using a pan-actin antibody or phalloidin-TRITC. In vivo luminal perfusion of the cauda epididymal tubule with the ROCK inhibitors Y27632 (10-30 µM) and HA1077 (30 µM) or with the cell-permeable Rho inhibitor Clostridium botulinum C3 transferase (3.75 µg/ml) induced the apical membrane accumulation of V-ATPase and extension of V-ATPase-labeled microvilli in clear cells. However, these newly formed microvilli were devoid of ROCKII. In addition, Y27632 (30 µM) or HA1077 (30 µM) decreased the ratio of F-actin to G-actin detected by Western blot analysis in epididymal epithelial cells, and Y27632 also decreased the ratio of F-actin to G-actin in clear cells isolated by fluorescence activated cell sorting from B1-enhanced green fluorescence protein (EGFP) transgenic mice. These results provide evidence that depolymerization of the cortical actin cytoskeleton via inhibition of RhoA or its effector ROCKII favors the recruitment of V-ATPase from the cytosolic compartment into the apical membrane in clear cells. In addition, our data suggest that the RhoA-ROCKII pathway is not locally involved in the elongation of apical microvilli. We propose that inhibition of RhoA-ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation.