双酚A 干扰大鼠生精过程时TJ 渗透性屏障的体外研究

目的:研究体外大鼠睾丸支持细胞紧密连接蛋白(SCJP)在类雌激素-双酚A(BPA)干扰下的损伤机制。方法:对Wistar大鼠睾丸支持细胞(Sertoli细胞)离体原代培养4-5d,通过双室培养模型建立体外紧密连接(TJ)渗透性屏障,并测量其跨上皮电阻值(TER)反应紧密连接结构的形成及BPA对紧密连接的损害程度。设溶剂(DMSO)做阴性对照,以终浓度为25μM、100μM的BPA作用于支持细胞24h,MTT法测不同浓度BPA作用的Sertoli细胞增殖活性。Western bloting观察occludin、ZO-1、Cx43表达的变化。结果:成功分离并培养Wistar大鼠睾丸支持细胞,并建立良好的体外TJ屏障模型。双室培养支持细胞上皮TER值在培养的d4达到顶峰,然后在d4-9维持相对较稳定的状态,d4以200μM,100μM,25μM BPA染毒,分别于染毒后24,48,72,96和120h测TER:与DMSO溶剂对照组相比,200μM,100μM的BPA组TER值明显下降(P<0.05),而25μM的BPA组在染毒后TER值无明显变化(P>0.05)。MTT结果显示:经不同浓度BPA作用24h后,Sertoli细胞的吸光度(OD值)随着染毒剂量的增加而逐渐降低。102、103μM浓度组与溶剂对照组有显著性差异(P<0.05),而10-2、10-1、100、101μM组和溶剂对照组无显著性差异(P>0.05)。Western blot结果显示:occludin、ZO-1、Cx43在各剂量组均有表达,与溶剂对照组相比,occludin、ZO-1表达均分别随作用剂量的增加而降低:25μM组、100μM组与溶剂对照组相比,差异均存在显著性(P<0.05);100μM组与25μM组相比,差异亦存在显著性(P<0.05)。Cx43的表达却随染毒剂量的增加而增加,与溶剂对照组相比,25μM组表达无明显增加(P>0.05),而100μM组则明显增加(P<0.05);与25μM组相比,100μM组表达明显增加(P<0.05)。结论:双酚A可通过损伤支持细胞连接蛋白正常表达,破坏了TJ屏障渗透性,从而影响正常的精子形成过程。     

细菌降解双酚类化合物的分子机制研究进展

双酚类化合物(bisphenols,BPs)作为工业原料及药物和个人护理品的重要成分之一,在自然界中广泛分布。BPs作为类雌性激素造成的生态风险已成为全球备受关注的环境问题之一。研究人员使用不同方式分离得到菌株或菌群,尝试对BPs进行无害化降解,目前已取得了一些重要的研究进展。本文对近年来细菌降解BPs的相关研究进行了系统梳理,重点关注以双酚A(bisphenol A,BPA)为典型BPs的细菌降解,总结不同路径中的关键作用基因,讨论相同路径中酶的差异及作用方式,并分析其对BPA的降解效果。本文也简要总结了双酚S(bisphenolS,BPS)和其他BPs的细菌降解研究情况。通过总结讨论现有成果,分析细菌降解BPs中尚需深入研究的内容,探寻未来BPs细菌降解机理及应用的研究方向。     

c-Yes regulates cell adhesion at the blood-testis barrier and the apical ectoplasmic specialization in the seminiferous epithelium of rat testes

During spermatogenesis, extensive junction restructuring takes place at the blood-testis barrier (BTB) and the Sertoli cell-spermatid interface known as the apical ectoplasmic specialization (apical ES, a testis-specific adherens junction) in the seminiferous epithelium. However, the mechanism(s) that regulates these critical events in the testis remains unknown. Based on the current concept in the field, changes in the phosphorylation status of integral membrane proteins at these sites can induce alterations in protein endocytosis and recycling, causing junction restructuring. Herein, c-Yes, a non-receptor protein tyrosine kinase, was found to express abundantly at the BTB and apical ES stage-specifically, coinciding with junction restructuring events at these sites during the seminiferous epithelial cycle of spermatogenesis. c-Yes also structurally associated with adhesion proteins at the BTB (e.g., occludin and N-cadherin) and the apical ES (e.g., β1-integrin, laminins β3 and γ3), possibly to regulate phosphorylation status of proteins at these sites. SU6656, a selective c-Yes inhibitor, was shown to perturb the Sertoli cell tight junction-permeability barrier in vitro, which is mediated by changes in the distribution of occludin and N-cadherin at the cell-cell interface, moving from cell surface to cytosol, thereby destabilizing the tight junction-barrier. However, this disruptive effect of SU6656 on the barrier was blocked by testosterone. Furthermore, c-Yes is crucial to maintain the actin filament network in Sertoli cells since a blockade of c-Yes by SU6656 induced actin filament disorganization. In summary, c-Yes regulates BTB and apical ES integrity by maintaining proper distribution of integral membrane proteins and actin filament organization at these sites.     

Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly

The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.     

Molecular basis of cryptorchidism-induced infertility

Artificial cryptorchidism or local testicular heat treatment can induce reversible oligospermia or azoospermia in monkeys and rats via germ cell apoptosis. Local warming of monkey testes in water at 43°C for 2 consecutive days (30 min per day) decreased the number of sperm in the semen by up to 80% on d 28, and the effect was completely reversed on d 144. Germ cells rely heavily on Sertoli cells for structural and nutritional support. Specialized junctions that play a pivotal role in spermatogenesis occur at sites of Sertoli-Sertoli and Sertoli-germ cell contact in the seminiferous epithelium. We demonstrated that expression of tight junction (TJ)-associated molecules, such as occludin and zonula occludens-1 (ZO-1), were greatly reduced 24–48 h after heat treatment, while the permeability of the blood-testis barrier (BTB) was simultaneously increased, but recovered 10 d later. These results indicate a reversible disruption of the BTB associated with transient inductions of transforming growth factor (TGF) β2 and β3 expression, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase activation, and concomitant loss of occludin and ZO-1. This suggests that expression of TJ-associated molecules and the BTB was reversibly perturbed by mild testicular hyperthermia, and that the heat-induced induction of TGF-β might be involved in downregulating TJ-associated proteins, leading to cell junction reduction. This review discusses the changes in total gene expression patterns after experimental cryptorchidism in adult mouse testes, and the cloning of several novel, physiologically significant spermatogenesis-specific genes.     

Testin regulates the blood-testis barrier via disturbing occludin/ZO-1 association and actin organization

The blood-testis barrier (BTB) separates the seminiferous epithelium into the apical and basal compartments. The BTB has to operate timely and accurately to ensure the correct migration of germ cells, meanwhile maintaining the immunological barrier. Testin was first characterized from primary Sertoli cells, it is a secretory protein and a sensitive biomarker to monitor junctions between Sertoli and germ cells. Till now, the functions of testin on BTB dynamics and the involving mechanisms are unknown. Herein, testin acts as a regulatory protein on BTB integrity. In vitro testin knockdown by RNAi caused significant damage to the Sertoli cell barrier with no apparent changes in the protein levels of several major tight junction (TJ), adhesion junction, and gap junction proteins. Also, testin RNAi caused the diffusion of two TJ structural proteins, occludin and ZO-1, diffusing away from the Sertoli cell surface into the cytoplasm. Association and colocalization between ZO-1 and occludin were decreased after testin RNAi, examined by Co-IP and coimmunofluorescent staining, respectively. Furthermore, testin RNAi induced a dramatic disruption on the arrangement of actin filament bundles and a reduced F-actin/G-actin ratio. The actin regulatory protein ARP3 appeared at the Sertoli cell interface after testin RNAi without its protein level change, whereas overexpressing testin in Sertoli cells showed no effect on TJ barrier integrity. The above findings suggest that besides as a monitor for Sertoli-germ cell junction integrity, testin is also an essential molecule to maintain Sertoli–Sertoli junctions.     

Differential effects of testosterone and TGF-β3 on endocytic vesicle-mediated protein trafficking events at the blood-testis barrier

The intricate interaction between protein endocytosis, transcytosis, recycling and endosome- or ubiquitin-mediated protein degradation determines the junction integrity in epithelial cells including Sertoli cells at the blood-testis barrier (BTB). Studies have shown that androgens and cytokines (e.g., TGF-β3) that are known to promote and disrupt BTB integrity, respectively, accelerate protein endocytosis at the BTB. We hypothesized that testosterone-induced endocytosed proteins are transcytosed and recycled back to the Sertoli cell surface, whereas cytokine-induced endocytosed proteins are degraded so that androgens and cytokines have opposing effects on BTB integrity. Herein, we report that both testosterone and TGF-β3 induced the steady-state level of clathrin, an endocytic vesicle protein. Testosterone and TGF-β3 also induced the association between internalized occludin (a BTB integral membrane protein) and clathrin, as well as early endosome antigen-1 (EEA-1). Interestingly, testosterone, but not TGF-β3, also induced the levels of proteins that regulate protein transcytosis (e.g., caveolin-1) and recycling (e.g., Rab11), and their association with internalized occludin and N-cadherin from the cell surface. In contrast, TGF-β3, but not testosterone, induced the level of ubiquitin-conjugating enzyme E2 J1 (Ube2j1), a protein crucial to the intracellular protein degradation pathway, and its association with internalized occludin. Based on these findings and recent reports in the field, we hypothesize that the concerted effects of testosterone and TGF-β3 likely facilitate the transit of preleptotene spermatocytes at the BTB while maintaining the immunological barrier in that testosterone induces the assembly of “new” tight junction (TJ)-fibrils below migrating spermatocytes via protein transcytosis and recycling before cytokines induce the disassembly of “old” TJ-fibrils above spermatocytes via endocytic vesicle-mediated degradation of internalized proteins. This thus provides a unique mechanism in the testis to facilitate the transit of preleptotene spermatocytes, many of which are connected in "clones" via cytoplasmic bridges, at the BTB while maintaining the immunological barrier during stage VIII of the seminiferous epithelial cycle of spermatogenesis.     

Regulation of blood-testis barrier dynamics by focal adhesion kinase (FAK): An unexpected turn of events

The blood-testis barrier (BTB) is conferred by co-existing tight junctions (TJs), basal ectoplasmic specialization (basal ES), desmosome-like junctions and gap junctions (GJs) between adjacent Sertoli cells near the basement membrane in the seminiferous epithelium. While the concept of the BTB has been known for more than a century and its significance to spermatogenesis discerned for more than five decades, its regulation has remained largely unknown. Recent studies, however, have demonstrated that focal adhesion kinase (FAK), a modulator of the integrin-based signaling that plays a crucial role on cell movement, apoptosis, cell survival and gene expression at the focal adhesion complex (FAC, also known as focal contact, a cell-matrix anchoring junction type), is an integrated component of the BTB, associated with the TJ-integral membrane protein occludin and its adaptor zonula occludens-1 (ZO-1). Herein, we summarize recent findings in the field regarding the significance of FAK in conferring BTB integrity based on some unexpected observations. We also critically discuss the role of FAK in regulating the timely "opening" and "closing" of the BTB to facilitate the transit of primary preleptotene spermatocytes across the BTB at stage VIII of the seminiferous epithelial cycle of spermatogenesis. Lastly, we propose a working model, which can be used to design future functional experiments to explore the involvement of FAK in BTB dynamics by investigators in the field.     

Molecular dynamics of the blood–testis barrier components during murine spermatogenesis

The blood–testis barrier (BTB) separates the seminiferous epithelium into the adluminal and basal compartments. During murine spermatogenesis, preleptotene/leptotene spermatocytes migrate from the basal to the adluminal compartment through the BTB during stages VIII–IX. In the present study, we focused on the tight junction (TJ) molecules and analyzed their spatiotemporal expression during the murine seminiferous epithelial cycle. Structural analysis revealed that the principal components of the BTB, for example, claudin‐3, claudin‐11, occludin, and zonula occludens‐1 (ZO‐1), were localized at the basal and luminal sides of the preleptotene/leptotene spermatocytes during the migration stages (VIII–IX). Although we detected claudin‐11, occludin, and ZO‐1 throughout spermatogenesis, claudin‐3 was only detected during stages VI–IX. Quantitative PCR using dissected seminiferous tubules from three stages (Early: II–VI, Middle: VII–VIII, Late: IX–I) clarified that the mRNA levels of TJ molecules were not correlated with the histoplanimetrical protein levels during spermatogenesis. Additionally, tubulobulbar complexes, considered to be involved in the internalization of TJ, were observed at the BTB site. Furthermore, a significant reduction in the mRNA levels of genes for the degradation of occludin (Itch) and endocytic recycling (Rab13) were observed during the Late and Middle stages, respectively. Therefore, we hypothesized that the lag between mRNA and protein expression of TJ molecules may be due to posttranslational modulation, for example, tubulobulbar complexes and endocytic recycling processes. In conclusion, these findings indicate that the integrity of the BTB is maintained throughout spermatogenesis, and the stage‐specific localization of claudin‐3 protein plays an important role in regulating BTB permeability. Mol. Reprod. Dev. 77: 630–639, 2010. © 2010 Wiley‐Liss, Inc.     

Renal ischemia�Creperfusion injury causes intercalated cell-specific disruption of occludin in the collecting duct

Renal ischemic events open tight junctions and disrupt epithelial polarity. The purpose of this study was to examine the effects of ischemia-reperfusion (IR) injury on expression and distribution of the tight junction proteins, occludin and ZO-1, in the rat kidney. IR injury was induced by clamping both renal pedicles for 30 min and animals were killed at 6 h after the reperfusion. IR injury decreased blood bicarbonate level, but did not persistently alter pH, Na(+), K(+), or Cl(-). In control kidneys, occludin immunoreactivity was intense in the tight junctions in the thick ascending limb, distal convoluted tubule, and collecting duct, moderate in the thin limbs of the loop of Henle, and was not detected in the proximal tubule, glomerulus, and blood vessels. ZO-1 was expressed in the same sites in which occludin was expressed, and additionally was also expressed in the proximal tubule, glomerulus, and vascular endothelial cells. IR kidneys exhibited damaged renal tubular epithelial cells in both proximal tubule and collecting duct segments in the outer medulla. In the collecting duct, the response of intercalated cells and principal cells differed. Following IR injury, intercalated cells, but not principal cells, lost their normal epithelial polarity and were frequently extruded into the tubule lumen. Occludin, instead of being localized to tight junctions, was localized diffusely in the cytoplasm in intercalated cells of IR kidneys. Principal cells, in contrast, were not detectably affected and neither occludin nor ZO-1 expression were altered in response to IR injury. The normal localization of ZO-1 expression to tight junction sites in both the proximal tubule and collecting duct was altered in response to IR, and, instead, ZO-1 expression was present diffusely in the cytoplasm. IR injury did not alter detectably either occludin or ZO-1 localization to the tight junction of the thick ascending limb cells. The abundance of total occludin protein by immunoblot analysis was not changed with IR injury. These results demonstrate that renal IR injury causes tight junction disruptions in both the proximal tubule and the collecting duct, and that altered distribution of the tight junction protein, occludin, may play a critical role in the collecting duct dysfunction which IR induces.     

Hyperoxia disrupts pulmonary epithelial barrier in newborn rats via the deterioration of occludin and ZO-1

Background

Prolonged exposure to hyperoxia in neonates can cause hyperoxic acute lung injury (HALI), which is characterized by increased pulmonary permeability and diffuse infiltration of various inflammatory cells. Disruption of the epithelial barrier may lead to altered pulmonary permeability and maintenance of barrier properties requires intact epithelial tight junctions (TJs). However, in neonatal animals, relatively little is known about how the TJ proteins are expressed in the pulmonary epithelium, including whether expression of TJ proteins is regulated in response to hyperoxia exposure. This study determines whether changes in tight junctions play an important role in disruption of the pulmonary epithelial barrier during hyperoxic acute lung injury.

Methods

Newborn rats, randomly divided into two groups, were exposed to hyperoxia (95% oxygen) or normoxia for 1–7 days, and the severity of lung injury was assessed; location and expression of key tight junction protein occludin and ZO-1 were examined by immunofluorescence staining and immunobloting; messenger RNA in lung tissue was studied by RT-PCR; transmission electron microscopy study was performed for the detection of tight junction morphology.

Results

We found that different durations of hyperoxia exposure caused different degrees of lung injury in newborn rats. Treatment with hyperoxia for prolonged duration contributed to more serious lung injury, which was characterized by increased wet-to-dry ratio, extravascular lung water content, and bronchoalveolar lavage fluid (BALF):serum FD4 ratio. Transmission electron microscopy study demonstrated that hyperoxia destroyed the structure of tight junctions and prolonged hyperoxia exposure, enhancing the structure destruction. The results were compatible with pathohistologic findings. We found that hyperoxia markedly disrupted the membrane localization and downregulated the cytoplasm expression of the key tight junction proteins occludin and ZO-1 in the alveolar epithelium by immunofluorescence. The changes of messenger RNA and protein expression of occludin and ZO-1 in lung tissue detected by RT-PCR and immunoblotting were consistent with the degree of lung injury.

Conclusions

These data suggest that the disruption of the pulmonary epithelial barrier induced by hyperoxia is, at least in part, due to massive deterioration in the expression and localization of key TJ proteins.     

Cytokines and junction restructuring during spermatogenesis--a lesson to learn from the testis

In the mammalian testis, preleptotene and leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) at late stage VIII through early stage IX of the epithelial cycle during spermatogenesis, entering the adluminal compartment for further development. However, until recently the regulatory mechanisms that regulate BTB dynamics remained largely unknown. We provide a critical review regarding the significance of cytokines in regulating the 'opening' and 'closing' of the BTB. We also discuss how cytokines may be working in concert with adaptors that selectively govern the downstream signaling pathways. This process, in turn, regulates the dynamics of either Sertoli-Sertoli tight junction (TJ), Sertoli-germ cell adherens junction (AJ), or both junction types in the epithelium, thereby permitting TJ opening without compromising AJs, and vice versa. We also discuss how adaptors alter their protein-protein association with the integral membrane proteins at the cell-cell interface via changes in their phosphorylation status, thereby altering adhesion function at AJ. These findings illustrate that the testis is a novel in vivo model to study the biology of junction restructuring. Furthermore, a molecular model is presented regarding how cytokines selectively regulate TJ/AJ restructuring in the epithelium during spermatogenesis.     

Zonula Occludens-1, Occludin and E-cadherin Expression and Organization in Salivary Glands with Sj?gren’s Syndrome

Sjögren’s syndrome (SS) is a chronic inflammatory autoimmune disorder that causes secretory dysfunction of the salivary glands leading to dry mouth. Previous studies reported that tight junction (TJ) proteins are down-regulated and lose polarity in human minor salivary glands with SS, suggesting that TJ structure is compromised in SS patients. In this paper, we utilized the NOD/ShiLtJ mouse with the main goal of evaluating this model for future TJ research. We found that the organization of apical proteins in areas proximal and distal to lymphocytic infiltration remained intact in mouse and human salivary glands with SS. These areas looked comparable to control glands (i.e., with no lymphocytic infiltration). TJ staining was absent in areas of lymphocytic infiltration coinciding with the loss of salivary epithelium. Gene expression studies show that most TJs are not significantly altered in 20-week-old NOD/ShiLtJ mice as compared with age-matched C57BL/6 controls. Protein expression studies revealed that the TJ proteins, zonula occludens-1 (ZO-1), occludin, claudin-12, as well as E-cadherin, do not significantly change in NOD/ShiLtJ mice. Our results suggest that ZO-1, occludin and E-cadherin are not altered in areas without lymphocytic infiltration. However, future studies will be necessary to test the functional aspect of these results.     

Polyamines are necessary for synthesis and stability of occludin protein in intestinal epithelial cells

Occludin is an integral membrane protein that forms the sealing element of tight junctions and is critical for epithelial barrier function. Polyamines are implicated in multiple signaling pathways driving different biological functions of intestinal epithelial cells (IEC). The present study determined whether polyamines are involved in expression of occludin and play a role in intestinal epithelial barrier function. Studies were conducted in stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) associated with a highly differentiated phenotype. Polyamine depletion by alpha-difluoromethylornithine (DFMO) decreased levels of occludin protein but failed to affect expression of its mRNA. Other tight junction proteins, zonula occludens (ZO)-1, ZO-2, claudin-2, and claudin-3, were also decreased in polyamine-deficient cells. Decreased levels of tight junction proteins in DFMO-treated cells were associated with dysfunction of the epithelial barrier, which was overcome by exogenous polyamine spermidine. Decreased levels of occludin in polyamine-deficient cells was not due to the reduction of intracellular-free Ca(2+) concentration ([Ca(2+)](cyt)), because either increased or decreased [Ca(2+)](cyt) did not alter levels of occludin in the presence or absence of polyamines. The level of newly synthesized occludin protein was decreased by approximately 70% following polyamine depletion, whereas its protein half-life was reduced from approximately 120 min in control cells to approximately 75 min in polyamine-deficient cells. These findings indicate that polyamines are necessary for the synthesis and stability of occludin protein and that polyamine depletion disrupts the epithelial barrier function, at least partially, by decreasing occludin.     

Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO₂} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.     

Pre- and postnatal bisphenol A treatment results in persistent deficits in the sexual behavior of male rats, but not female rats, in adulthood

Perinatal administration of the endocrine disruptor bisphenol A (BPA) reportedly inhibits the sexual behavior of sexually naïve adult male rats. In order to evaluate the effects of BPA administration during early development on later reproductive behavior, we administered one of five doses of bisphenol A daily to pregnant female rats throughout gestation and lactation, and quantified the appetitive and consummatory sexual behaviors of the resultant male and female offspring over multiple sexual encounters in adulthood. Males receiving low dose perinatal BPA (50 μg/kg bw/day) showed persistent deficits in sexual behavior in adulthood. Males receiving the highest dose (5 mg/kg bw/day), however, were indistinguishable from controls with respect to consummatory sexual behaviors but showed decreased latencies to engage in those behaviors when sexually naïve, with significant non-linear, or U-shaped, dose-response relationships observed on the first and last day of testing. Adult female sexual behavior was not affected by early BPA administration at any dose tested. These results are consistent with previous reports that BPA exerts behavioral effects especially at low doses, and further indicates that BPA can cause lasting impairment of sexual behavior in males, but does not alter the normal development of female appetitive or consummatory sexual behaviors. To our knowledge, this is the first report indicating that adult sexual performance is impaired in sexually experienced animals following perinatal exposure to bisphenol A.     

Cholesterol efflux stimulates metalloproteinase-mediated cleavage of occludin and release of extracellular membrane particles containing its C-terminal fragments

That changes in membrane lipid composition alter the barrier function of tight junctions illustrates the importance of the interactions between tetraspan integral tight junction proteins and lipids of the plasma membrane. Application of methyl-β-cyclodextrin to both apical and basolateral surfaces of MDCK cell monolayers for 2 h, results in an ∼80% decrease in cell cholesterol, a fall in transepithelial electrical resistance, and a 30% reduction in cell content of occludin, with a smaller reduction in levels of claudins-2, -3, and -7. There were negligible changes in levels of actin and the two non-tight junction membrane proteins GP-135 and caveolin-1. While in untreated control cells breakdown of occludin, and probably other tight junction proteins, is mediated by intracellular proteolysis, our current data suggest an alternative pathway whereby in a cholesterol-depleted membrane, levels of tight junction proteins are decreased via direct release into the intercellular space as components of membrane-bound particles. Occludin, along with two of its degradation products and several claudins, increases in the basolateral medium after incubation with methyl-β-cyclodextrin for 30 min. In contrast caveolin-1 is detected only in the apical medium after adding methyl-β-cyclodextrin. Release of occludin and its proteolytic fragments continues even after removal of methyl-β-cyclodextrin. Sedimentation and ultrastructural studies indicate that the extracellular tight junction proteins are associated with the membrane-bound particles that accumulate between adjacent cells. Disruption of the actin filament network by cytochalasin D did not diminish methyl-β-cyclodextrin-induced release of tight junction proteins into the medium, suggesting that the mechanism underlying their formation is not actin-dependent. The 41- and  48-kDa C-terminal occludin fragments formed during cholesterol depletion result from the action of a GM6001-sensitive metalloproteinase(s) at some point in the path leading to release of the membrane particles.     

Oxidized phospholipids mediate occludin expression and phosphorylation in vascular endothelial cells

Oxidized l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified LDL, induces production of proinflammatory cytokines and development of atherosclerotic lesions. We tested the hypothesis that OxPAPC alters expression, phosphorylation, and localization of tight junction (TJ) proteins, particularly occludin, a transmembrane TJ protein. OxPAPC reduced total occludin protein and increased occludin phosphorylation dose dependently (10-50 microg/ml) and time dependently in bovine aortic endothelial cells. OxPAPC decreased occludin mRNA and reduced the immunoreactivity of zonula occludens-1 at the cell-cell contacts. Furthermore, OxPAPC increased the diffusive flux of 10-kDa dextran in a dose-dependent manner. O2-* production by bovine aortic endothelial cells increased nearly twofold after exposure to OxPAPC. Also, enzymatic generation of O2-* by xanthine oxidase-lumazine and H2O2 by glucose oxidase-glucose increased occludin phosphorylation, implicating reactive oxygen species as modulators of the OxPAPC effects on occludin phosphorylation. Superoxide dismutase and/or catalase blocked the effects of OxPAPC on occludin protein content and phosphorylation, occludin mRNA, zonula occludens-1 immunoreactivity, and diffusive flux of 10-kDa dextran. These findings suggest that changes in TJ proteins are potential mechanisms by which OxPAPC compromises the barrier properties of the vascular endothelium. OxPAPC-induced disruption of TJs, which likely facilitates transmigration of LDL and inflammatory cells into the subendothelial layers, may be mediated by reactive oxygen species.