JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
rpS6 regulates blood-testis barrier dynamics through Akt-mediated effects on MMP-9.
J. Cell. Sci.
PUBLISHED: 09-12-2014
Show Abstract
Hide Abstract
Mammalian target of rapamycin complex 1 (mTORC1) is an emerging regulator of blood-tissue barriers that utilizes ribosomal protein S6 (rpS6) as the downstream signaling molecule. To explore the role of rpS6 in blood-testis barrier (BTB) function, a constitutively active quadruple rpS6 phosphomimetic mutant was constructed in mammalian expression vector and overexpressed in Sertoli cells cultured in vitro that mimicked the BTB in vivo. Using this quadruple phosphomimetic mutant, phosphorylated (p)-rpS6 was shown to disrupt IGF-1/insulin signaling, thereby abolishing Akt phosphorylation, which led to an induction of MMP-9. This increase in MMP-9 secretion perturbed the Sertoli cell tight junction permeability barrier by proteolysis-mediated downregulation of tight junction proteins at the BTB. These findings were confirmed by the use of a specific MMP-9 inhibitor that blocked the disruption of the tight junction permeability barrier by the rpS6 mutant. Additionally, RNA interference (RNAi)-mediated Akt silencing was able to mimic the results of rpS6 mutant overexpression in Sertoli cells, further confirming this p-rpS6-Akt-MMP-9 signaling pathway. In conclusion, these data support a new concept of mTORC1-mediated BTB regulation, that is possibly also applicable to other blood-tissue barriers.
Related JoVE Video
Electronic medical records in clinical teaching.
Nurse Educ
PUBLISHED: 07-30-2014
Show Abstract
Hide Abstract
The purpose of the project was to provide students with experiences to develop their technology competency and examine student perceptions about an academic electronic medical record (EMR) as a learning tool. Nurse educators need to integrate EMRs into their curricula to give students practice in the use of electronic documentation and retrieval of clinical information. The findings of this study indicated that students' use of EMRs at least 5 times resulted in the development of positive perceptions about their EMR experience.
Related JoVE Video
N-wasp is required for structural integrity of the blood-testis barrier.
PLoS Genet.
PUBLISHED: 06-01-2014
Show Abstract
Hide Abstract
During spermatogenesis, the blood-testis barrier (BTB) segregates the adluminal (apical) and basal compartments in the seminiferous epithelium, thereby creating a privileged adluminal environment that allows post-meiotic spermatid development to proceed without interference of the host immune system. A key feature of the BTB is its continuous remodeling within the Sertoli cells, the major somatic component of the seminiferous epithelium. This remodeling is necessary to allow the transport of germ cells towards the seminiferous tubule interior, while maintaining intact barrier properties. Here we demonstrate that the actin nucleation promoting factor Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) provides an essential function necessary for BTB restructuring, and for maintaining spermatogenesis. Our data suggests that the N-WASP-Arp2/3 actin polymerization machinery generates branched-actin arrays at an advanced stage of BTB remodeling. These arrays are proposed to mediate the restructuring process through endocytic recycling of BTB components. Disruption of N-WASP in Sertoli cells results in major structural abnormalities to the BTB, including mis-localization of critical junctional and cytoskeletal elements, and leads to disruption of barrier function. These impairments result in a complete arrest of spermatogenesis, underscoring the critical involvement of the somatic compartment of the seminiferous tubules in germ cell maturation.
Related JoVE Video
p-FAK-Tyr(397) regulates spermatid adhesion in the rat testis via its effects on F-actin organization at the ectoplasmic specialization.
Am. J. Physiol. Endocrinol. Metab.
PUBLISHED: 07-23-2013
Show Abstract
Hide Abstract
During spermatogenesis, the molecular mechanism that confers spermatid adhesion to the Sertoli cell at the apical ectoplasmic specialization (apical ES), a testis-specific F-actin-rich adherens junction, in the rat testis remains elusive. Herein, the activated form of focal adhesion kinase (FAK), p-FAK-Tyr(397), a component of the apical ES that was expressed predominantly and stage specifically in stage VII-early stage VIII tubules, was found to be a crucial apical ES regulator. Using an FAK-Y397E phosphomimetic mutant cloned in a mammalian expression vector for its transfection vs. FAK and vector alone in adult rat testes in vivo, its overexpression was found to cause defects in spermiation. These defects in spermiation were manifested by entrapment of spermatids in the seminiferous epithelium in late stage VIII-X tubules and were mediated by a disruption on the spatiotemporal expression and/or mislocalization of actin regulatory protein actin-related protein 3, which induces branched actin polymerization, epidermal growth factor receptor pathway substrate 8 (an actin barbed end capping and bundling protein), and palladin (an actin cross-linking and bundling protein). This thus perturbed changes of F-actin organization at the apical ES to facilitate spermiation, which also led to a concomitant alteration in the distribution and upregulation of adhesion proteins nectin-2 and nectin-3 at the apical ES. As such, nectin-2 and -3 remained at the apical ES to anchor step 19 spermatids on to the epithelium, delaying spermiation. These findings illustrate a mechanistic pathway mediated by p-FAK-Tyr(397) that regulates spermatid adhesion at the apical ES in vivo.
Related JoVE Video
Regulation of blood-testis barrier (BTB) dynamics during spermatogenesis via the "Yin" and "Yang" effects of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2.
Int Rev Cell Mol Biol
PUBLISHED: 01-16-2013
Show Abstract
Hide Abstract
In mammalian testes, haploid spermatozoa are formed from diploid spermatogonia during spermatogenesis, which is a complicated cellular process. While these cellular events were reported in the 1960s and 1970s, the underlying molecular mechanism(s) that regulates these events remained unexplored until the past ?10 years. For instance, adhesion proteins were shown to be integrated components at the Sertoli cell-cell interface and/or the Sertoli-spermatid interface in the late 1980s. But only until recently, studies have demonstrated that some of the adhesion proteins serve as the platform for signal transduction that regulates cell adhesion. In this chapter, a brief summary and critical discussion are provided on the latest findings regarding these cell-adhesion proteins in the testis and their relationship to spermatogenesis. Moreover, antagonistic effects of two mammalian target of rapamycin (mTOR) complexes, known as mTORC1 and mTORC2, on cell-adhesion function in the testis are discussed. Finally, a hypothetic model is presented to depict how these two mTOR-signaling complexes having the "yin" and "yang" antagonistic effects on the Sertoli cell tight junction (TJ)-permeability barrier can maintain the blood-testis barrier (BTB) integrity during the epithelial cycle while preleptotene spermatocytes are crossing the BTB.
Related JoVE Video
Rictor/mTORC2 regulates blood-testis barrier dynamics via its effects on gap junction communications and actin filament network.
FASEB J.
PUBLISHED: 01-03-2013
Show Abstract
Hide Abstract
In the mammalian testis, coexisting tight junctions (TJs), basal ectoplasmic specializations, and gap junctions (GJs), together with desmosomes near the basement membrane, constitute the blood-testis barrier (BTB). The most notable feature of the BTB, however, is the extensive network of actin filament bundles, which makes it one of the tightest blood-tissue barriers. The BTB undergoes restructuring to facilitate the transit of preleptotene spermatocytes at stage VIII-IX of the epithelial cycle. Thus, the F-actin network at the BTB undergoes cyclic reorganization via a yet-to-be explored mechanism. Rictor, the key component of mTORC2 that is known to regulate actin cytoskeleton, was shown to express stage-specifically at the BTB in the seminiferous epithelium. Its expression was down-regulated at the BTB in stage VIII-IX tubules, coinciding with BTB restructuring at these stages. Using an in vivo model, a down-regulation of rictor at the BTB was also detected during adjudin-induced BTB disruption, illustrating rictor expression is positively correlated with the status of the BTB integrity. Indeed, the knockdown of rictor by RNAi was found to perturb the Sertoli cell TJ-barrier function in vitro and the BTB integrity in vivo. This loss of barrier function was accompanied by changes in F-actin organization at the Sertoli cell BTB in vitro and in vivo, associated with a loss of interaction between actin and ?-catenin or ZO-1. Rictor knockdown by RNAi was also found to impede Sertoli cell-cell GJ communication, disrupting protein distribution (e.g., occludin, ZO-1) at the BTB, illustrating that rictor is a crucial BTB regulator.
Related JoVE Video
A study to assess the assembly of a functional blood-testis barrier in developing rat testes.
Spermatogenesis
PUBLISHED: 08-08-2011
Show Abstract
Hide Abstract
The blood-testis barrier (BTB) is an important ultrastructure in the seminiferous tubule of the mammalian testis that segregates the events of spermatogenesis, in particular post-meiotic germ cell development, from the harmful substances in the environment including toxicants and drugs, as well as from the unwanted hormones and biomolecules in the systemic circulation. It is known that the BTB is assembled by ?15-21 days postpartum (dpp) in rats coinciding with the onset of late cell cycle progression, namely the formation of zygotene and pachytene spermatocytes by day 15-18 dpp. This is to prepare for: (1) the differentiation/transformation of pachytene spermatocytes to diplotene and dictyate spermatocytes and (2) meiosis I and II, which take place by 23-26 and 26 dpp, respectively. Recent findings have shown spermatogonia/spermatogonial stem cells (SSC) in the tubules failed to re-initiate spermatogenesis by differentiating spermatogonia beyond type A spermatogonia in the absence of a functional BTB, leading to meiotic arrest. These studies thus illustrate that a functional BTB is crucial to the initiation and/or re-initiation of spermatogenesis. Herein, we sought to examine the precise time window when a functional and intact BTB is established in the developing rat testis during the final stage of cell cycle progression and meiosis. Using the techniques of: (1) dual-labeled immunofluorescence analysis to assess the distribution of integrated proteins at the tight junction (TJ), basal ectoplasmic specialization [basal ES, a testis-specific atypical adherens junction (AJ) type] and gap junction (GJ) at the BTB, (2) functional assay to assess the BTB integrity in vivo, (3) immunoblot analysis to monitor changes in steady-state levels of adhesion proteins at the BTB, and (4) co-immunoprecipitation to assess changes in protein-protein interactions at the BTB, it was shown that a BTB was being assembled by day 15-20 dpp, but a functional BTB was not fully established until day 25 dpp in Sprague-Dawley rats, tightly associated with the onset of meiosis I and II. These findings thus illustrate the significance of the BTB on cell cycle progression and the preparation for meiosis, such as germ cell differentiation beyond type A spermatogonia.
Related JoVE Video
Interactions of laminin ?3 fragment with ?1-integrin receptor: A revisit of the apical ectoplasmic specialization-blood-testis-barrier-hemidesmosome functional axis in the testis.
Spermatogenesis
PUBLISHED: 05-06-2011
Show Abstract
Hide Abstract
Recent studies have demonstrated the presence of a functional axis that coordinates the events of spermiation and blood-testis barrier (BTB) restructuring which take place simultaneously at the opposite ends of the seminiferous epithelium at stage VIII of the epithelial cycle of spermatogenesis in the rat testis. In short, the disruption of the apical ectoplasmic specialization (apical ES) at the Sertoli cell-elongated spermatid interface, which facilitates the release of sperm at spermiation near the tubule lumen, is coordinated with restructuring at the BTB to accommodate the transit of preleptotene spermatocytes across the immunological barrier near the basement membrane. These two events are likely coordinated by a functional axis involving hemidesmosome at the Sertoli cell-basement membrane interface, and it was designated the apical ES-BTB-hemidesmosome axis. It was demonstrated that fragments of laminin chains (e.g., laminin ?3 or ?3 chains) derived from the ?6?1-integrin-laminin333 protein complex at the apical ES, which were likely generated via the action of MMP-2 (matrix metalloprotease-2, MMP2) prior to spermiation, acted as biologically active peptides to perturb the BTB permeability function by accelerating protein endocytosis (e.g., occludin) at the site, thereby destabilizing the BTB integrity to facilitate the transit of preleptotene spermatocytes. These laminin fragments also perturbed hemidesmosome function via their action on ?1-integrin, a component of hemidesmosome in the testis, which in turn, sent a signal to further destabilize the BTB function. As such, the events of spermiation and BTB restructuring are coordinated via this functional axis. Recent studies using animal models treated with toxicants, such as mono-(2-ethylhexyl) phthalate (MEHP), or adjudin, a male contraceptive under investigation, have also supported the presence of this functional axis in the mouse. In this short review, we critically evaluate the role of this local functional axis in the seminiferous epithelium in spermatogenesis. We also provide molecular modeling information on the interactions between biologically active laminin fragments and ?1-integrin, which will be important to assist in the design of more potent laminin-based peptides to disrupt this axis, thereby perturbing spermatogenesis for male contraception and to understand the underlying biology that coordinates spermiation and BTB restructuring during spermatogenesis.
Related JoVE Video
Regulation of blood-testis barrier dynamics by desmosome, gap junction, hemidesmosome and polarity proteins: An unexpected turn of events.
Spermatogenesis
PUBLISHED: 04-04-2011
Show Abstract
Hide Abstract
The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades, the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome and gap junction that serve as a signaling platform to coordinate the "opening" and "closing" of the TJ-permeability barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or protein degradation). These events not only serve to destabilize the existing "old" BTB above preleptotene spermatocytes in transit in "clones" at the BTB, but also contribute to the assembly of "new" BTB below the transiting spermatocytes. Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring events at stage VIII of the epithelial cycle. Additionally, the findings that a gap junction at the BTB provides a possible route for the passage of toxicants [e.g., bisphenol A (BPA)] and potential male contraceptives (e.g., adjudin) across the BTB also illustrate that these coexisting junctions, while helpful to maintain the immunological barrier integrity during the transit of spermatocytes, can be the "gateway" to making the BTB so vulnerable to toxicants and/or chemicals, causing male reproductive dysfunction.
Related JoVE Video
Structural chemistry of the histone methyltransferases cofactor binding site.
J Chem Inf Model
PUBLISHED: 03-03-2011
Show Abstract
Hide Abstract
Histone methyltransferases (HMTs) transfer a methyl group from the cofactor S-adenosyl methionine to lysine or arginine residues on histone tails, thereby regulating chromatin compaction, binding of effector proteins and gene transcription. HMTs constitute an emerging target class in diverse disease areas, and selective chemical probes are necessary for target validation. Potent and selective competitors of the substrate peptide have been reported, but the chemical tractability of the cofactor binding site is poorly understood. Here, a systematic analysis of this site across structures of 14 human HMTs or close homologues was conducted. The druggability, interaction hotspots, and diversity of the cofactor binding pocket were dissected. This analysis strongly suggests that this site is chemically tractable. General principles underlying tight binding and specific guidelines to achieve selective inhibition are presented.
Related JoVE Video
Adjudin, a potential male contraceptive, exerts its effects locally in the seminiferous epithelium of mammalian testes.
Reproduction
PUBLISHED: 02-09-2011
Show Abstract
Hide Abstract
Adjudin is a derivative of 1H-indazole-3-carboxylic acid that was shown to have potent anti-spermatogenic activity in rats, rabbits, and dogs. It exerts its effects most notably locally in the apical compartment of the seminiferous epithelium, behind the blood-testis barrier, by disrupting adhesion of germ cells, most notably spermatids to the Sertoli cells, thereby inducing release of immature spermatids from the epithelium that leads to infertility. After adjudin is metabolized, the remaining spermatogonial stem cells and spermatogonia repopulate the seminiferous epithelium gradually via spermatogonial self-renewal and differentiation, to be followed by meiosis and spermiogenesis, and thus fertility rebounds. Recent studies in rats have demonstrated unequivocally that the primary and initial cellular target of adjudin in the testis is the apical ectoplasmic specialization, a testis-specific anchoring junction type restricted to the interface between Sertoli cells and elongating spermatids (from step 8 to 19 spermatids). In this review, we highlight some of the recent advances and obstacles regarding the possible use of adjudin as a male contraceptive.
Related JoVE Video
Structural genomics of histone tail recognition.
Bioinformatics
PUBLISHED: 08-24-2010
Show Abstract
Hide Abstract
The structural genomics of histone tail recognition web server is an open access resource that presents within mini articles all publicly available experimental structures of histone tails in complex with human proteins. Each article is composed of interactive 3D slides that dissect the structural mechanism underlying the recognition of specific sequences and histone marks. A concise text html-linked to interactive graphics guides the reader through the main features of the interaction. This resource can be used to analyze and compare binding modes across multiple histone recognition modules, to evaluate the chemical tractability of binding sites involved in epigenetic signaling and design small molecule inhibitors.
Related JoVE Video
The crystal structure of Toxoplasma gondii pyruvate kinase 1.
PLoS ONE
PUBLISHED: 05-26-2010
Show Abstract
Hide Abstract
Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the worlds population.
Related JoVE Video
Expression and Localization of PRiMA-linked globular form acetylcholinesterase in vertebrate neuromuscular junctions.
J. Mol. Neurosci.
PUBLISHED: 07-06-2009
Show Abstract
Hide Abstract
Acetylcholinesterase (AChE) is well known to process different molecular forms via the distinct interacting partners. Proline-rich membrane anchor (PRiMA)-linked tetrameric globular AChE (G4 AChE) is mainly found in the vertebrate brain; however, recent studies from our laboratory have suggested its existence at neuromuscular junctions (nmjs). Both muscle and motor neuron express AChE at the nmjs. In muscle, the expression of PRiMA-linked AChE is down-regulated during myogenic differentiation and by motor neuron innervation. As compared with muscle, spinal cord possessed higher total AChE activity and contained PRiMA-linked AChE forms. The spinal cord expression of this form increased during development. More importantly, PRiMA-linked G4 AChE identified as aggregates localized at nmjs. These findings suggest that the restricted localization of PRiMA-linked G4 AChE at the nmjs could be contributed by the pre-synaptic motor neuron and/or the post-synaptic muscle fiber.
Related JoVE Video
Self correction of anterior crossbite: a case report.
Cases J
PUBLISHED: 03-29-2009
Show Abstract
Hide Abstract
A 9-year-old Chinese boy presented with an anterior crossbite, no treatment was performed at that time because the incisors have open root apices. The crossbite self-corrected after one year. This case demonstrated that an anterior crossbite may self-correct without treatment.
Related JoVE Video
The apical ectoplasmic specialization-blood-testis barrier functional axis is a novel target for male contraception.
Adv. Exp. Med. Biol.
Show Abstract
Hide Abstract
The blood-testis barrier (BTB), similar to other blood-tissue barriers, such as the blood-brain barrier and the blood-retinal barrier, is used to protect the corresponding organ from harmful substances (e.g., xenobiotics) including drugs and foreign compounds. More importantly, the BTB allows postmeiotic spermatid development to take place in an immune privileged site at the adluminal (or apical) compartment to avoid the production of antibodies against spermatid-specific antigens, many of which express transiently during spermiogenesis and spermiation. The BTB, however, also poses an obstacle in developing nonhormonal-based male contraceptives by sequestering drugs (e.g., adjudin) that exert their effects on germ cells in the adluminal compartment. The effects of these drugs include disruption of germ cell cycle progression and development, apoptosis, cell adhesion, metabolism and others. Recent studies have demonstrated that there is a functional axis that operates locally in the seminiferous epithelium to co-ordinate different cellular events across the Sertoli cell epithelium, such as spermiation and BTB restructuring during the seminiferous epithelial cycle of spermatogenesis. Components of this functional axis, such as the apical ectoplasmic specialization (apical ES, a testis-specific atypical anchoring junction type) and the BTB, in particular their constituent protein complexes, such as alpha6beta1-integrin and occludin at the apical ES and the BTB, respectively, can be the target of male contraception. In this chapter, we highlight recent advances regarding the likely mechanism of action of adjudin in this functional axis with emphasis on the use of molecular modeling technique to facilitate the design of better compounds in male contraceptive development.
Related JoVE Video
Role of P-glycoprotein at the blood-testis barrier on adjudin distribution in the testis: a revisit of recent data.
Adv. Exp. Med. Biol.
Show Abstract
Hide Abstract
The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in mammals including rodents and humans. It is used to sequester meiosis I and II, postmeiotic spermatid development via spermiogenesis and the release of sperm at spermiation from the systemic circulation, such that these events take place in an immune-privileged site in the adluminal (apical) compartment behind the BTB, segregated from the host immune system. Additionally, drug transporters, namely efflux (e.g., P-glycoprotein) and influx (e.g., Oatp3) pumps, many of which are integral membrane proteins in Sertoli cells at the BTB also work cooperatively to restrict the entry of drugs, toxicants, chemicals, steroids and other xenobiotics into the adluminal compartment. As such, the BTB that serves as an important physiological and selective barrier to protect germ cell development also poses a "hurdle" in male contraceptive development. For instance, adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, a potential nonhormonal male contraceptive that exerts its effects on germ cell adhesion, most notably at the Sertoli cell-spermatid interface, to induce "premature" germ cell loss from the seminiferous epithelium mimicking spermiation, has a relatively poor bioavailability largely because of the BTB. Since male contraceptives (e.g., adjudin) will be used by healthy men for an extended period of his life span after puberty, a better understanding on the BTB is necessary in order to effectively deliver drugs across this blood-tissue barrier in particular if these compounds exert their effects on developing germ cells in the adluminal compartment. This can also reduce long-term toxicity and health risk if the effective dosing can be lowered in order to widen the margin between its safety and efficacy. Herein, we summarize latest findings in this area of research, we also provide a critical evaluation on research areas that deserve attention in future studies.
Related JoVE Video
rpS6 Regulates blood-testis barrier dynamics by affecting F-actin organization and protein recruitment.
Endocrinology
Show Abstract
Hide Abstract
During spermatogenesis, preleptotene spermatocytes residing near the basement membrane of the seminiferous tubule must traverse the blood-testis barrier (BTB) at stage VIII-IX of the epithelial cycle to continue their development in the adluminal compartment. Unlike other blood-tissue barriers (e.g. the blood-brain barrier) that are created by the endothelial tight junction (TJ) barrier of capillaries, the BTB is created by specialized junctions between Sertoli cells in which TJ coexists with basal ectoplasmic specialization (basal ES, a testis-specific adherens junction). The basal ES is typified by the presence of tightly packed actin filament bundles sandwiched between cisternae of endoplasmic reticulum and the apposing plasma membranes of Sertoli cells. These actin filament bundles also confer unusual adhesive strength to the BTB. Yet the mechanisms by which these filamentous actin (F-actin) networks are regulated from the bundled to the debundled state to facilitate the transit of spermatocytes remain elusive. Herein, we provide evidence that ribosomal protein S6 (rpS6), the downstream signaling molecule of the mammalian target of rapamycin complex 1 (mTORC1) pathway, is a major regulator of F-actin organization and adhesion protein recruitment at the BTB. rpS6 is restrictively and spatiotemporally activated at the BTB during the epithelial cycle. An activation of rpS6 led to a disruption of the Sertoli cell TJ barrier and BTB integrity. Its silencing in vitro or in vivo by using small interfering RNA duplexes or short hairpin RNA was found to promote the Sertoli cell TJ permeability barrier by the recruitment of adhesion proteins (e.g. claudin-11 and occludin) to the BTB. Thus, rpS6 in the mTORC1 pathway regulates BTB restructuring via its effects on the F-actin organization and protein recruitment at the BTB.
Related JoVE Video
Focal adhesion kinase-Tyr407 and -Tyr397 exhibit antagonistic effects on blood-testis barrier dynamics in the rat.
Proc. Natl. Acad. Sci. U.S.A.
Show Abstract
Hide Abstract
Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, displays phosphorylation-dependent localization in the seminiferous epithelium of adult rat testes. FAK is an integrated component of the blood-testis barrier (BTB) involved in regulating Sertoli cell adhesion via its effects on the occludin-zonula occludens-1 complex. Herein, we report that p-FAK-Tyr(407) and p-FAK-Tyr(397) display restricted spatiotemporal and almost mutually exclusive localization in the epithelium, affecting BTB dynamics antagonistically, with the former promoting and the latter disrupting the Sertoli cell tight junction-permeability barrier function. Using primary cultured Sertoli cells as an in vitro model that mimics the BTB in vivo both functionally and ultrastructurally, effects of FAK phosphorylation on BTB function were studied by expressing nonphosphorylatable and phosphomimetic mutants, with tyrosine replaced by phenylalanine (F) and glutamate (E), respectively. Compared with WT FAK, Y407E and Y397F mutations each promoted barrier function, and the promoting effect of the Y407E mutant was abolished in the Y397E-Y407E double mutant, demonstrating antagonism between Tyr(407) and Tyr(397). Furthermore, Y407E mutation induced the recruitment of actin-related protein 3 to the Sertoli cell-cell interface, where it became more tightly associated with neuronal Wiskott-Aldrich syndrome protein, promoting actin-related protein 2/3 complex activity. Conversely, Y407F mutation reduced the rate of actin polymerization at the Sertoli cell BTB. In summary, FAK-Tyr(407) phosphorylation promotes BTB integrity by strengthening the actin filament-based cytoskeleton. FAK serves as a bifunctional molecular "switch" to direct the cyclical disassembly and reassembly of the BTB during the epithelial cycle of spermatogenesis, depending on its phosphorylation status, to facilitate the transit of preleptotene spermatocytes across the BTB.
Related JoVE Video
Microtubule affinity-regulating kinase 4 (MARK4) is a component of the ectoplasmic specialization in the rat testis.
Spermatogenesis
Show Abstract
Hide Abstract
During the seminiferous epithelial cycle of spermatogenesis, the ectoplasmic specialization (ES, a testis-specific adherens junction, AJ, type) maintains the polarity of elongating/elongated spermatids and confers adhesion to Sertoli cells in the seminiferous epithelium, and known as the apical ES. On the other hand, the ES is also found at the Sertoli-Sertoli cell interface at the blood-testis barrier (BTB) known as basal ES, which together with the tight junction (TJ), maintains Sertoli cell polarity and adhesion, creating a functional barrier that limits paracellular transport of substances across the BTB. However, the apical and basal ES are segregated and restricted to the adluminal compartment and the BTB, respectively. During the transit of preleptotene spermatocytes across the BTB and the release of sperm at spermiation at stage VIII of the seminiferous epithelial cycle, both the apical and basal ES undergo extensive restructuring to facilitate cell movement at these sites. The regulation of these events, in particular their coordination, remains unclear. Studies in other epithelia have shown that the tubulin cytoskeleton is intimately related to cell movement, and MARK [microtubule-associated protein (MAP)/microtubule affinity-regulating kinase] family kinases are crucial regulators of tubulin cytoskeleton stability. Herein MARK4, the predominant member of the MARK protein family in the testis, was shown to be expressed by both Sertoli and germ cells. MARK4 was also detected at the apical and basal ES, displaying highly restrictive spatiotemporal expression at these sites, as well as co-localizing with markers of the apical and basal ES. The expression of MARK4 was found to be stage-specific during the epithelial cycle, structurally associating with ?-tubulin and the desmosomal adaptor plakophilin-2, but not with actin-based BTB proteins occludin, ?-catenin and Eps8 (epidermal growth factor receptor pathway substrate 8, an actin bundling and barbed end capping protein). More importantly, it was shown that the expression of MARK4 tightly associated with the integrity of the apical ES because a diminished expression of MARK4 associated with apical ES disruption that led to the detachment of elongating/elongated spermatids from the epithelium. These findings thus illustrate that the integrity of apical ES, an actin-based and testis-specific AJ, is dependent not only on the actin filament network, but also on the tubulin-based cytoskeleton.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.