Disorganization of claudin-11 and dysfunction of the blood-testis barrier during puberty in a cryptorchid rat model.

BACKGROUND: Cryptorchidism is known to impair spermatogenesis. The blood-testis barrier (BTB) becomes defined in seminiferous tubules around puberty and provides a suitable environment for germ cells. Little is known about the BTB in undescended testes (UDT). OBJECTIVES: To determine the role of BTB during puberty in UDT using a non-surgical cryptorchid rat model. MATERIAL AND METHODS: Unilateral cryptorchid male rats were intraperitoneally injected with non-steroidal antiandrogen during intrauterine development; the testes were harvested at 4, 5, and 6 weeks after birth. Testicular histology, expression levels of the BTB proteins (claudin-11, occludin, zonula occludens-1), and apoptotic cells were evaluated by immunohistochemistry, Western blotting, and TUNEL assay. The functionality of the BTB was investigated by electron microscopy using the lanthanum tracer method. RESULTS: The testicular histology of undescended testes 6 weeks after birth showed maturation arrest at the spermatocyte level. The BTB protein distributions were altered in the UDT, with a noticeable difference in claudin-11(CLDN11) localization from 4 to 5 weeks after birth between control and UDT samples. BTB protein levels were similar. More apoptotic germ cells were detected in the adluminal compartment of tubules in the UDT than in the control testes. Electron microscopy showed that the lanthanum tracer was limited to the BTB of control testes, whereas it penetrated the BTB of UDT. DISCUSSION: Here, loss of normal BTB function and impaired spermatogenesis were observed in UDT during puberty. CLDN11 is a pivotal tight junction protein belonging to the BTB. Tight junctions are considered as essential for normal spermatogenesis, and abnormal CLDN11 organization may cause UDT-associated male infertility. CONCLUSION: CLDN11 disorganization within the BTB may cause spermatogenic impairment, possibly by limiting the BTB function.

Responses of the proteome in testis of mice exposed chronically to environmentally relevant concentrations of Microcystin-LR.

Microcystin-LR (MC-LR), a widespread environmental contaminant, has been shown to have potent acute testicular toxicity. However, magnitudes of toxic effects, induced by MCs, depend on route and magnitude of exposure to the toxin. In the present study, male mice were orally exposed 1, 10 or 100 µg/L MC-LR for 90 or 180 days, and pathological approach and the isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics were employed with testes. Proteomics revealed that a number of differentially altered proteins may be involved in MC-LR-induced chronic testicular toxicity. The biological process analysis indicated the altered proteins played an important role in biological adhesion, cellular process, response to stimulus or rhythmic process. The cellular component analysis revealed that most of the proteins with altered expression associated with cell part, extracellular region, extracellular region part, membrane, membrane part, organelle or organelle part. The molecular function showed that these proteins were critical in molecular transducer activity. Integrity analyses provide first compelling evidence that MC-LR significantly cause dysfunction of blood-testis barrier (BTB) through affecting tight junctions and gap junctions. Moreover, phosphatidylinositol 3-kinase (PI3K)/AKT eventually contributed to injury result from chronic low-level MC-LR treatment. Identification of proteins in testis responsive to MC-LR provides insights into molecular mechanisms of chronic toxicity of MCs.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

Induction of experimental autoimmune orchitis in mice: responses to elevated circulating levels of the activin-binding protein, follistatin.

Experimental autoimmune orchitis (EAO) is a rodent model of chronic testicular inflammation that mimics the pathology observed in some types of human infertility. In a previous study, testicular expression of the inflammatory/immunoregulatory cytokine, activin A, was elevated in adult mice during the onset of EAO, indicating a potential role in the regulation of the disease. Consequently, we examined the development of EAO in mice with elevated levels of follistatin, an endogenous activin antagonist, as a potential therapeutic approach to testicular inflammation. Prior to EAO induction, mice received a single intramuscular injection of a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of the circulating form of follistatin, FST315 (FST group). Serum follistatin levels were increased 5-fold in the FST group compared with the control empty vector (EV) group at 30 and 50 days of EAO, but intra-testicular levels of follistatin or activin A were not significantly altered. Induction of EAO was reduced, but not prevented, with mild-to-severe damage in 75% of the EV group and 40% of the FST group, at 50 days following immunisation with testicular homogenate. However, the EAO damage score (based on disruption of the blood-testis barrier, apoptosis, testicular damage and fibrosis) and extent of intratesticular inflammation (expression of inflammatory mediators) were directly proportional to the levels of activin A measured in the testis at 50 days. These data implicate activin A in the progression of EAO, thereby providing a potential therapeutic target; however, elevating circulating follistatin levels were not sufficient to prevent EAO development.

Inhibition of NADPH oxidase activation reduces EAE-induced white matter damage in mice.

BACKGROUND: To evaluate the role of NADPH oxidase-mediated reactive oxygen species (ROS) production in multiple sclerosis pathogenesis, we examined the effects of apocynin, an NADPH oxidase assembly inhibitor, on experimental autoimmune encephalomyelitis (EAE). METHODS: EAE was induced by immunization with myelin oligodendrocyte glycoprotein (MOG (35-55)) in C57BL/6 female mice. Three weeks after initial immunization, the mice were analyzed for demyelination, immune cell infiltration, and ROS production. Apocynin (30 mg/kg) was given orally once daily for the entire experimental course or after the typical onset of clinical symptom (15 days after first MOG injection). RESULTS: Clinical signs of EAE first appeared on day 11 and reached a peak level on day 19 after the initial immunization. The daily clinical symptoms of EAE mice were profoundly reduced by apocynin. The apocynin-mediated inhibition of the clinical course of EAE was accompanied by suppression of demyelination, reduced infiltration by encephalitogenic immune cells including CD4, CD8, CD20, and F4/80-positive cells. Apocynin reduced MOG-induced pro-inflammatory cytokines in cultured microglia. Apocynin also remarkably inhibited EAE-associated ROS production and blood-brain barrier (BBB) disruption. Furthermore, the present study found that post-treatment with apocynin also reduced the clinical course of EAE and spinal cord demyelination. CONCLUSIONS: These results demonstrate that apocynin inhibits the clinical features and neuropathological changes associated with EAE. Therefore, the present study suggests that inhibition of NADPH oxidase activation by apocynin may have a high therapeutic potential for treatment of multiple sclerosis pathogenesis.

Specific deletion of Cdh2 in Sertoli cells leads to altered meiotic progression and subfertility of mice.

CDH2 (cadherin 2, Neural-cadherin, or N-cadherin) is the predominant protein of testicular basal ectoplasmic specializations (basal ES; a testis-specific type of adhesion junction), one of the major cell junctions composing the blood-testis barrier (BTB). The BTB is found between adjacent Sertoli cells in seminiferous tubules, which divides the tubules into basal and adluminal compartments and prevents the deleterious exchange of macromolecules between blood and seminiferous tubules. However, the exact roles of basal ES protein CDH2 in BTB function and spermatogenesis is still unknown. We thus generated mice with Cdh2 specifically knocked out in Sertoli cells by crossing Cdh2 loxP mice with Amh-Cre mice. Cdh2 deletion in Sertoli cells did not affect Sertoli cell counts, but led to compromised BTB function, delayed meiotic progression from prophase to metaphase I in testes, increased germ cell apoptosis, sloughing of meiotic cells, and, subsequently, reduced sperm counts in epididymides and subfertility of mice. However, the testes with Cdh2-specific deletion in germ cells did not show any difference from the normal control testes, and phenotypes observed in Sertoli cell and germ cell Cdh2 double-knockout mice were indistinguishable from those in mice with Cdh2 specifically knocked out only in Sertoli cells. Taken together, our data demonstrate that the adhesion junction component, Cdh2, functions just in Sertoli cells, but not in germ cells during spermatogenesis, and is essential for the integrity of BTB function, its deletion in Sertoli cells would lead to the BTB damage and subsequently meiosis and spermatogenesis failure.

Vitamin A deprivation affects the progression of the spermatogenic wave and initial formation of the blood-testis barrier, resulting in irreversible testicular degeneration in mice.

The blood testis-barrier (BTB) is essential for maintaining homeostasis in the seminiferous epithelium. Although many studies have reported that vitamin A (VA) is required for the maintenance of spermatogenesis, the relationships between the BTB, spermatogenesis and VA have not been elucidated. In this study, we analyzed BTB assembly and spermatogenesis in the testes of mice fed the VA-deficient (VAD) diet from the prepubertal period to adulthood. During the prepubertal period, no changes were observed in the initiation and progression of the first spermatogenic wave in mice fed the VAD diet. However, the numbers of preleptotene/leptotene spermatocytes derived from the second spermatogenic wave onwards were decreased, and initial BTB formation was also delayed, as evidenced by the decreased expression of mRNAs encoding BTB components and VA signaling molecules. From 60 days postpartum, mice fed the VAD diet exhibited apoptosis of germ cells, arrest of meiosis, disruption of the BTB, and dramatically decreased testis size. Furthermore, vacuolization and calcification were observed in the seminiferous epithelium of adult mice fed the VAD diet. Re-initiation of spermatogenesis by VA replenishment in adult mice fed the VAD diet rescued BTB assembly after when the second spermatogenic wave initiated from the arrested spermatogonia reached the preleptotene/leptotene spermatocytes. These results suggested that BTB integrity was regulated by VA metabolism with meiotic progression and that the impermeable BTB was required for persistent spermatogenesis rather than meiotic initiation. In conclusion, consumption of the VAD diet led to critical defects in spermatogenesis progression and altered the dynamics of BTB assembly.

Impaired function of the blood-testis barrier during aging is preceded by a decline in cell adhesion proteins and GTPases.

With increasing age comes many changes in the testis, including germ cell loss. Cell junctions in the testis tether both seminiferous epithelial and germ cells together and assist in the formation of the blood-testis barrier (BTB), which limits transport of biomolecules, ions and electrolytes from the basal to the adluminal compartment and protects post-meiotic germ cells. We hypothesize that as male rats age the proteins involved in forming the junctions decrease and that this alters the ability of the BTB to protect the germ cells. Pachytene spermatocytes were isolated from Brown Norway rat testes at 4 (young) and 18 (aged) months of age using STA-PUT velocity sedimentation technique. RNA was extracted and gene expression was assessed using Affymetrix rat 230 2.0 whole rat genome microarrays. Microarray data were confirmed by q-RT-PCR and protein expression by Western blotting. Of the genes that were significantly decreased by at least 1.5 fold, 70 were involved in cell adhesion; of these, at least 20 are known to be specifically involved in junction dynamics within the seminiferous epithelium. The mRNA and protein levels of Jam2, Ocln, cdh2 (N-cadherin), ctnna (α-catenin), and cldn11 (involved in adherens junctions), among others, were decreased by approximately 50% in aged spermatocytes. In addition, the GTPases Rac1 and cdc42, involved in the recruitment of cadherins to the adherens junctions, were similarly decreased. It is therefore not surprising that with lower expression of these proteins that the BTB becomes diminished with age. We saw, using a FITC tracer, a gradual collapse of the BTB between 18 and 24 months. This provides the opportunity for harmful substances and immune cells to cross the BTB and cause the disruption of spermatogenesis that is observed with increasing age.

New perspectives on central and peripheral immune responses to acute traumatic brain injury.

Traumatic injury to the brain (TBI) results in a complex set of responses involving various symptoms and long-term consequences. TBI of any form can cause cognitive, behavioral and immunologic changes in later life, which underscores the problem of underdiagnosis of mild TBI that can cause long-term neurological deficits. TBI disrupts the blood-brain barrier (BBB) leading to infiltration of immune cells into the brain and subsequent inflammation and neurodegeneration. TBI-induced peripheral immune responses can also result in multiorgan damage. Despite worldwide research efforts, the methods of diagnosis, monitoring and treatment for TBI are still relatively ineffective. In this review, we delve into the mechanism of how TBI-induced central and peripheral immune responses affect the disease outcome and discuss recent developments in the continuing effort to combat the consequences of TBI and new ways to enhance repair of the damaged brain.

Impact of diabetes in blood-testis and blood-brain barriers: resemblances and differences.

Blood-tissue barriers prevent an uncontrolled exchange of large molecules between adjacent but metabolically separated compartments. There are several known barriers and two of the most important and tightest blood-tissue barriers are the blood-testis barrier (BTB) and the blood-brain barrier (BBB). Under normal conditions these barriers, formed by tight junctions between adjacent cells, control the entry of substances and metabolites. However, hyperglycemia and other diabetes-related complications, such as hypertension, impair the function of these biological barriers with dramatic consequences. Although both, BBB and BTB, are responsible for the maintenance of different biological processes, they have some remarkable similarities not always explored when looking at metabolic-related diseases such as diabetes. These barriers possess their own glucose sensing machinery, suffer a tied hormonal control and have specific mechanisms to counteract hyper- and hypoglycemia. In BBB and BTB the insulin signaling is also distinct from other tissues and organs thus evidencing their importance in protecting against or exacerbating the effects of diabetes on glucose metabolism. The control of glucose and lactate levels in brain and testis highlights the role of these barriers in protecting against peripheral glucose and lactate fluctuations that occur in the diabetic individual. We review the role of BBB and BTB in the control of glucose and metabolic dysfunction caused by diabetes in the brain and seminiferous epithelium. Gaining a better understanding of the molecular mechanisms through which glucose metabolism disrupts BBB and BTB function may highlight new opportunities for the treatment of diabetic complications in brain and male reproductive function.

Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders.

The neurovascular unit (NVU) comprises brain endothelial cells, pericytes or vascular smooth muscle cells, glia and neurons. The NVU controls blood-brain barrier (BBB) permeability and cerebral blood flow, and maintains the chemical composition of the neuronal 'milieu', which is required for proper functioning of neuronal circuits. Recent evidence indicates that BBB dysfunction is associated with the accumulation of several vasculotoxic and neurotoxic molecules within brain parenchyma, a reduction in cerebral blood flow, and hypoxia. Together, these vascular-derived insults might initiate and/or contribute to neuronal degeneration. This article examines mechanisms of BBB dysfunction in neurodegenerative disorders, notably Alzheimer's disease, and highlights therapeutic opportunities relating to these neurovascular deficits.

The role of attenuated astrocyte activation in infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited neurodegenerative disorder affecting the CNS during infancy. INCL is caused by mutations in the CLN1 gene that lead to a deficiency in the lysosomal hydrolase, palmitoyl protein thioesterase 1 (PPT1). A murine model of INCL, the PPT1-deficient (PPT1(-/-)) mouse, is an accurate phenocopy of the human disease. The first pathological change observed in the PPT1(-/-) brain is regional areas of glial fibrillary acidic protein (GFAP) upregulation, which predicts future areas of neurodegeneration. We hypothesized that preventing GFAP and vimentin upregulation in reactive astrocytes will alter the CNS disease. To test this hypothesis, we generated mice simultaneously carrying null mutations in the GFAP, Vimentin, and PPT1 genes (GFAP(-/-)Vimentin(-/-)PPT1(-/-)). Although the clinical and pathological features of the GFAP(-/-)Vimentin(-/-)PPT1(-/-) mice are similar to INCL, the disease appears earlier and progresses more rapidly. One mechanism underlying this accelerated phenotype is a profound neuroinflammatory response within the CNS. Thus, our data identify a protective role for intermediate filament upregulation during astrocyte activation in INCL, a model of chronic neurodegeneration.

Brain metastases of mouse mammary adenocarcinoma is increased by acute stress.

Brain metastases from mammary adenocarcinoma constitute the chief cause of morbidity and mortality. Some evidence suggests that stress may contribute to disease progression and metastases. Here we show that acute restraint stress (30 min) induces statistically significant increase in brain metastases of systemically administered luciferase-tagged 4T1-BR-3P mouse mammary adenocarcinoma cells as evidenced by the total brain-associated photons from 5.6 × 10(7) photons in unstressed controls to 1.7 × 10(8) photons in C57BL/6 (p = 0.0018) and from 7.6 × 10(7) to 2.1 × 10(7) photons in BALB/c (p = 0.004) mice. Acute stress may increase metastases by disrupting the blood-brain-barrier (BBB), through release of corticotropin-releasing-hormone (CRH) activating perivascular brain mast cells.

[High power microwave radiation damages blood-testis barrier in rats].

OBJECTIVE: To determine the effect of high power microwave (HPM) radiation on the structure and function of blood-testis barrier (BTB) in rats. METHODS: One hundred and sixty-six male Wistar rats were treated by heart perfusion of lanthanum-glutaraldehyde solution and tail vein injection of evans blue (EB) at 6 h, 1, 3, 7 and 14 d after exposed to 0, 10, 30 and 100 mW/cm2 HPM radiation for 5 minutes, the structural change of BTB and distribution of lanthanum or EB observed through the light microscope, electron microscope and laser scanning confocal microscopy (LSCM). RESULTS: Testicular interstitial edema, vascular congestion or hyperemia with accumulation of plasma proteins and red blood cells in the inner compartment of seminiferous tubules were observed after exposure to HPM. The above-mentioned pathological changes were aggravated at 1-7 d and relieved at 14 d after radiation, obviously more severe in the 30 and 100 mW/cm2 exposure groups than in the 10 mW/cm2. Both lanthanum precipitation and EB were deposited in the inner compartment. CONCLUSION: HPM radiation may damage the structure and increase the permeability of BTB.

Altered expression of ZO-1 and ZO-2 in Sertoli cells and loss of blood-testis barrier integrity in testicular carcinoma in situ.

Carcinoma in situ (CIS) is the noninvasive precursor of most human testicular germ cell tumors. In normal seminiferous epithelium, specialized tight junctions between Sertoli cells constitute the major component of the blood-testis barrier. Sertoli cells associated with CIS exhibit impaired maturation status, but their functional significance remains unknown. The aim was to determine whether the blood-testis barrier is morphologically and/or functionally altered. We investigated the expression and distribution pattern of the tight junction proteins zonula occludens (ZO) 1 and 2 in normal seminiferous tubules compared to tubules showing CIS. In normal tubules, ZO-1 and ZO-2 immunostaining was observed at the blood-testis barrier region of adjacent Sertoli cells. Within CIS tubules, ZO-1 and ZO-2 immunoreactivity was reduced at the blood-testis barrier region, but spread to stain the Sertoli cell cytoplasm. Western blot analysis confirmed ZO-1 and ZO-2, and their respective mRNA were shown by RT-PCR. Additionally, we assessed the functional integrity of the blood-testis barrier by lanthanum tracer study. Lanthanum permeated tight junctions in CIS tubules, indicating disruption of the blood-testis barrier. In conclusion, Sertoli cells associated with CIS show an altered distribution of ZO-1 and ZO-2 and lose their blood-testis barrier function.