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Biochemistry of Sertoli cell/germ cell junctions, germ cell transport, and spermiation in the seminiferous epithelium
Abstract
In rodents and humans, the events of spermatogenesis are divided into four distinctive series of cellular events: (i) self-renewal of spermatogonial stem cells via mitosis; (ii) mitotic renewal of spermatogonia and their differentiation into type A and type B spermatogonia; (iii) meiosis of spermatocytes (diploid, 2n), which form spermatids (haploid, 1n); and (iv) postmeiotic development of spermatids via spermiogenesis and the release of mature spermatids (i.e., spermatozoa) at spermiation. During these events, developing germ cells, most notably preleptotene spermatocytes and spermatids, must be transported across the blood–testis barrier (BTB) and the adluminal compartment, respectively. Because germ cells per se—including spermatogonia, spermatocytes, and spermatids—are immotile cells without ultrastructures of lamellipodia and filopodia that are common in motile cells (e.g., macrophages, fibroblasts, neutrophils) in mammals, they rely almost exclusively on Sertoli cells for their transport across the BTB and the remaining seminiferous epithelium. Furthermore, developing spermatids must also be transported back and forth across the seminiferous epithelium during the epithelial cycle of spermatogenesis. Thus, intriguing interactions take place at the Sertoli cell/cell and Sertoli cell/germ cell interface for germ cell transport during spermatogenesis. While the details of these cellular events and the ultrastructures involved, such as the apical and the basal ectoplasmic specialization, a testis-specific adherens junction, have been known for decades, the regulatory mechanisms and underlying regulatory molecules that define germ cell transport have only been explored since the early 2000s. Here, we critically evaluate these findings and provide an updated summary, including some thought-provoking concepts that could become the basis of investigation for the future.
... During these steps, spermatids need to be attached to Sertoli cells by adhesion structures, such as the ectoplasmic specialization and tubulolobular complex (Lie et al. 2010). Sertoli cells also need to form specific cytoskeleton networks inside them during spermiogenesis (O'Donnell et al. 2011, Yan Cheng & Mruk 2015. These structures and molecules, including focal adhesion kinase, are tightly regulated during spermiogenesis, and therefore, sperm release called spermiation is induced via these regulations. ...
... Cells that are subjected to shear force react to resist the force, by actin polymerization to strengthen their cytoplasm (Lee et al. 2006). Actin remodeling is necessary for the ectoplasmic specialization and tubulobulbar complex during spermiogenesis, both of which are known as adherent structures between the spermatozoon and Sertoli cell (Lie et al. 2010, O'Donnell et al. 2011, Yan Cheng & Mruk 2015. We found that spermatids at step 8 were located on the seminiferous epithelium, even in the inner curve. ...
... We found that spermatids at step 8 were located on the seminiferous epithelium, even in the inner curve. Although spermatids after step 8 are assumed to be attached tightly to Sertoli cells by testis-specific adherens junctions, which is called an ectoplasmic specialization (Yan Cheng & Mruk 2015), the spermatids are probably released between steps 9-16 in the inner curve. The shear force may be different between the inner and outer curves, and the different strengths of the actin filaments may cause malfunction of the adherent structures between the Sertoli cells and spermatozoa, resulting in the earlier release of spermatozoa in the inner curve of the STs. ...
Spermatozoa released from Sertoli cells must be transported to the epididymis. However, the mechanism of the luminal flow in seminiferous tubules has remained unclear to date. Therefore, in this study, we investigated luminal flow and movements in the seminiferous tubules by three-dimensional analysis and in vivo imaging. Serial 5-μm-thick mouse testicular sections at 50-µm-intervals were prepared and stained by Periodic Acid-Schiff-hematoxylin. After three-dimensional reconstruction of the seminiferous tubules, the localization of the released spermatozoa and the stages observed in the sections were recorded in each reconstructed tubule. Luminal movements in the seminiferous tubules were observed by in vivo imaging using a fluorescent-reporter mouse and two-photon excitation microscopy system. Spermatozoa without contact to the seminiferous epithelium were not accumulated toward the rete testis. Additionally, such spermatozoa were found on their way not only to the most proximal rete testis but also a more distant rete testis from any stage VIII seminiferous epithelia. In vivo imaging demonstrated that the direction of the flagella of spermatozoa attached to the seminiferous epithelium was repeatedly reversed. The epithelium at the inner curve of the seminiferous tubule was shaken more actively and had fewer spermatozoa attached compared with the epithelium at the outer curve. Our results hence suggest that the luminal flow in the seminiferous tubules is repeatedly reversed and that this physical force helps spermatozoa to be released from Sertoli cells.
In brief: Spermatozoa are released from Sertoli cells and flow in the seminiferous tubule to the rete testis. Our results suggest that the luminal flow in the tubules is repeatedly reversed and that this physical force helps spermatozoa release from the Sertoli cells.
... Sertoli cells mature for various functions. A disorder in any of its functions may damage the production of spermatozoa (Cheng & Mruk, 2015). (Şahin, 2016). ...
... These cells are closely linked to each other through the tubules external Zonula Occludens, adhesion connections sp Zonula Adherens and their communication links "Gap Junction" and thus form the blood-testicular barrier, which protects the developing spermatocytes and spermatids from autoimmune reactions and other harmful external factors. The blood-testicle barrier in seminiferous epithelium is divided into three areas; two permanent (basal and adluminal) and a temporary (intermediate) area (Cheng & Mruk, 2015). ...
... The division of these cells by spermatocyte I is very short, so they are rarely seen in the testicular sections. Spermatids occur as a result of division of spermatocytes II (Baysal, 2017;Cheng & Mruk, 2015). ...
INTRODUCTION Animals used in research and biological studies designed according to scientific rules are called experimental animals or laboratory animals. The reasons why these animals are preferred as experimental animals are; ease of cultivation, ease of performing complex genetic applications, short pregnancy duration and sexual cycles, easy feeding and care, no need for large cultivation areas and sufficient knowledge gathered over many years. According to the International Laboratory Animals Committee; 40% to 80% of the preferred laboratory animals were reported to be mice (Akman, 2007).
Laboratory animals can be listed as follows according to the frequency of experiments. 1. Mice 2. Rats 3. Rabbit 4. Guinea pigs The male reproductive system and production, nutrition and storage of haploid male sex cells is responsible for the production and secretion of male sex hormones. Male reproductive system organs include effluent channels that transmit these cells from testes that express gonad cells and secrete androgens, of the tubuli recti, rete testis, ductuli efferentes, ductus epididymis, ductus deferens, ductus ejaculatorius and urethra; and the prostate gland, seminal vesicle and bulbourethral glands and external genital penis (Bernal, Aya, De Jesus-Ayson, & Garcia, 2015).
... They play a pivotal role in spermatogenesis by providing spatial and nutritional support to developing testicular germ cells (1,2). Towards the basement membrane between adjacent Sertoli cells, there exist tight junctions and ectoplasmic specializations, which together form physical barriers to later meiotic and postmeiotic developing germ cells present in the adluminal compartments above these junctional complexes (3). Having specific transporters along the basolateral and apical membranes, Sertoli cells also act as permeability barriers regulating the movement of molecules in and out of the seminiferous tubule lumen. ...
... Having specific transporters along the basolateral and apical membranes, Sertoli cells also act as permeability barriers regulating the movement of molecules in and out of the seminiferous tubule lumen. Thus, these 2 barriers restrict the entry of blood components into the adluminal compartments and the "non-self" developing germ cells sequestered in these compartments (3,4) are protected from humoral attacks (3,5,6). In addition, a milieu advantageous for germ cell development is generated due to the property of these 2 barriers and the secretion of specific molecules by Sertoli cells, such as lactate, to be used as an energy substrate by spermatids (7,8). ...
... Having specific transporters along the basolateral and apical membranes, Sertoli cells also act as permeability barriers regulating the movement of molecules in and out of the seminiferous tubule lumen. Thus, these 2 barriers restrict the entry of blood components into the adluminal compartments and the "non-self" developing germ cells sequestered in these compartments (3,4) are protected from humoral attacks (3,5,6). In addition, a milieu advantageous for germ cell development is generated due to the property of these 2 barriers and the secretion of specific molecules by Sertoli cells, such as lactate, to be used as an energy substrate by spermatids (7,8). ...
Cultures of Sertoli cells isolated from 20-day-old mice are widely used in research as substitutes for adult Sertoli cell cultures. This practice is based on the fact that Sertoli cells cease to proliferate and become mature in vivo by days 16-20 after birth. However, it is important to verify that cultured Sertoli cells derived from 20-day-old mice do not proliferate ex vivo, and have the same properties as cultured adult Sertoli cells. Herein we described an isolation/culture method of Sertoli cells from 10-week-old adult mice with >90% purity. Properties of these cultured adult Sertoli cells were then compared with those of cultured Sertoli cells derived from 20-day-old mice (also >90% purity). By cell counting, BrdU incorporation and metaphase plate detection, we demonstrated that only adult Sertoli cells did not proliferate throughout 12 culture days. In contrast, Sertoli cells derived from 20-day-old mice still proliferated until Day 10 in culture. The morphology and profiles of intracellular lipidomics and spent medium proteomics of the two cultures were also different. Cultured adult Sertoli cells were larger in size and contained higher levels of triacylglycerols, cholesteryl esters and seminolipid, and proteins in their spent medium were mainly engaged in cellular metabolism. In contrast, proteins involved in cell division, including AMH, CDC42, collagen isoforms, were at higher levels in Sertoli cell cultures derived from 20-day-old mice. Therefore, cultured Sertoli cells derived from 10-week-old mice rather than those from 20-day-old animals should be used for studies on properties of adult Sertoli cells.
... D uring spermiogenesis, haploid spermatids derived from spermatocytes through meiosis I/II undergo extensive morphological changes, from steps 1 through 19 in rat testes (vs 1 to 16 and 1 to 6 in the mouse and human testis, respectively), which are transported across the seminiferous epithelium in the adluminal (apical) compartment (1)(2)(3)(4)(5). Thus, fully developed spermatids (i.e., spermatozoa) line up at the edge of tubule lumen to prepare for their eventual release at spermiation at stage VIII of the epithelial cycle (1,6). ...
... D uring spermiogenesis, haploid spermatids derived from spermatocytes through meiosis I/II undergo extensive morphological changes, from steps 1 through 19 in rat testes (vs 1 to 16 and 1 to 6 in the mouse and human testis, respectively), which are transported across the seminiferous epithelium in the adluminal (apical) compartment (1)(2)(3)(4)(5). Thus, fully developed spermatids (i.e., spermatozoa) line up at the edge of tubule lumen to prepare for their eventual release at spermiation at stage VIII of the epithelial cycle (1,6). Studies have shown that step 8 to 19 spermatids in the rat testis, or 8 to 16 spermatids in the mouse, are anchored onto the Sertoli epithelium by the only type of testis-specific and actin-rich anchoring device called apical ectoplasmic specialization (ES) (5,(7)(8)(9). ...
... In this context, it is of interest to note that although the morphological series of events pertinent to spermiation are known for over five decades (1,6), the role of actinand MT-based cytoskeletons and the involving molecules, in particular the regulatory proteins of actin and MT dynamics, in spermiation remain unknown. Studies have shown that treatment of adult rats with a single dose of adjudin at 50 mg/kg body weight (b.w.) by oral gavage induced extensive germ cell exfoliation, initially elongating/elongated spermatids that were readily detectable within 6.5 hours posttreatment, whereas depletion of round spermatids (i.e., step 1 to 8 spermatids) and spermatocytes were not noted until ;3 and 6 days thereafter (17,18). ...
The mechanism that regulates sperm release at spermiation is unknown. Herein, we utilized an animal model wherein rats were treated with adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, via oral gavage to induce premature release of elongating/elongated spermatid, followed by round spermatids and spermatocytes. Spermatid release mimicking spermiation occurred within 6-12 h following adjudin treatment and by 96 h, virtually all tubules were devoid of spermatids. Using this model, we tracked the organization of F-actin and microtubules (MTs) by immunofluorescence microscopy, and the association of actin or MT regulatory proteins that either promote or demolish cytoskeletal integrity through changes in the organization of actin-microfilaments or MTs by co-immunoprecipitation. Adjudin treatment induced an increase in the association of: (i) Eps8 (an actin barbed-end capping and bundling protein) or formin 1 (an actin nucleator) with actin, and (ii) EB1 (a MT stabilizing protein) with MT shortly after adjudin exposure (at 6 h), in an attempt to maintain spermatid adhesion to the Sertoli cell at the apical ectoplasmic specialization (apical ES). However, this is followed by a considerable decline of their steady-state protein levels, replacing with an increase in association of (i) Arp3 (a branched actin nucleator that converts actin filaments into a branched/unbundled network) with actin, and (ii) MARK4 (a MT destabilizing protein kinase) with MTs by 12 h after adjudin treatment. These latter changes thus promote actin and MT dis-organization, leading to apical ES disruption and the release of sperm, mimicking spermiation. In summary, spermiation is a cytoskeletal-dependent event, involving regulatory proteins that modify cytoskeletal organization.
... Phase II denotes development of spermatocytes, such as preleptotene spermatocytes derived from type B spermatogonia, which must be transported across the blood-testis barrier (BTB) to enter the adluminal compartment while differentiating into leptotene spermatocytes, and transform into zygote, pachytene and diplotene spermatocytes to undergo meiosis I and II to generate haploid spermatids [10]. In phase III, post-meiotic haploid spermatids undergo a series of morphological, molecular, and cellular differentiation to form elongated spermatids via spermiogenesis (including steps 1-16, 1-19 and 1-6 spermatids in mouse, rat and human testes, respectively [11][12][13][14][15][16]), to be accompanied by their transport across the adluminal compartment of the seminiferous epithelium, whereas fully developed spermatids (i.e., spermatozoa) line-up at the luminal edge and are eventually released into tubule lumen at spermiation [17][18][19]. It is of interest to note that during spermiogenesis, developing spermatids exhibit a unique dual-level cell polarity. ...
... For instance, Vangl2 knockdown in Sertoli cells reduced actin bundling activity considerably with ) at the apical tubulobulbar complex (apical TBC) to support protein endocytic vesicle-mediated protein trafficking events. Studies have shown that there is a considerable reduction in the expression of Vangl2 on the apical ES in stage VII tubules, which appears to facilitate the conversion of the apical ES at the concave side of spermatid head to a transient ultrastructure known as apical TBC [19,70,85] (left panel). Apical TBC represents a giant endocytic vesicle machinery which supports endocytic-mediated protein trafficking event, facilitating protein endocytosis, transcytosis and recycling so that "old" apical ES proteins (e.g., integrins, nectins, laminins, afadin) can be recycled to assemble "new" apical ES when step 8 spermatids appear in stage VIII tubules (right panel). ...
... In humans, spermiogenesis can be disrupted, leading to: (i) late maturation arrest, manifested by an arrest in development in early spermatids with dark round nuclei, and (ii) hypospermatogenesis, with an arrest in further development of condensed oval spermatids [115]. Mature spermatozoa, once formed, are released into the tubule lumen via the final cellular process of spermatogenesis at spermiation [5,119]. While the molecular mechanism underlying spermiation remains relatively unexplored, studies in rodents have shown that spermiation is tightly regulated by the spatiotemporal expression of signaling molecules such as p-FAK-Tyr 407 , as well as actin binding/regulatory proteins (e.g., the Arp2/3 complex, Eps8), involving degeneration of apical ES and generation of laminin fragments [5,119]. ...
... Mature spermatozoa, once formed, are released into the tubule lumen via the final cellular process of spermatogenesis at spermiation [5,119]. While the molecular mechanism underlying spermiation remains relatively unexplored, studies in rodents have shown that spermiation is tightly regulated by the spatiotemporal expression of signaling molecules such as p-FAK-Tyr 407 , as well as actin binding/regulatory proteins (e.g., the Arp2/3 complex, Eps8), involving degeneration of apical ES and generation of laminin fragments [5,119]. However, this process is poorly understood in humans except that it is likely dependent on FSH and testosterone. ...
Spermatogenesis in humans is comprised of a series of highly complicated cellular events, necessary to support the production of an upward of 200 million sperm daily from puberty through the entire adulthood of a healthy man. Recent advances in the field using the techniques of cell and molecular biology, genetics, and biochemistry have unraveled many of the mysteries in spermatogenesis. In this Chapter, we highlight some recent advances in the field regarding the biology of human spermatogenesis. We also summarize and discuss recent advances regarding the regulation of spermatogenesis in humans. Due to rapid advances in our understanding of spermatogenesis and the large number of published reports in the literature in the last 2–3 decades, we focus on rapidly developing areas to stimulate the interest of our readers, in particular in areas that offer advances for the treatment of infertility in men.
... Apical and basal tubular [7,10,28,31,32] projections of the heads of spermatids into Sertoli cell cytoplasm prior to spermiation Sertoli/spermatid [l,4,5,7,19,20,24,27,30,47,48] release involving TBC, junctional complex internalization and recycling Localized with Sertoli cells [3,4,18,22,25,26,46] Supplemental Figure 3. Schematic representation of Sertoli cell (SC) proliferation in relation to endocrine and paracrine regulation of fish spermatogenesis. ...
... Apical and basal tubular [7,10,28,31,32] projections of the heads of spermatids into Sertoli cell cytoplasm prior to spermiation Sertoli/spermatid [l,4,5,7,19,20,24,27,30,47,48] release involving TBC, junctional complex internalization and recycling Localized with Sertoli cells [3,4,18,22,25,26,46] Supplemental Figure 3. Schematic representation of Sertoli cell (SC) proliferation in relation to endocrine and paracrine regulation of fish spermatogenesis. ...
It has been one and a half centuries since Enrico Sertoli published the seminal discovery of the testicular 'nurse cell', not only a key cell in the testis, but indeed one of the most amazing cells in the vertebrate body. In this review, we begin by examining the three phases of morphological research that have occurred in the study of Sertoli cells, because microscopic anatomy was essentially the only scientific discipline available for about the first 75 years after the discovery. Biochemistry and molecular biology then changed all of biological sciences, including our understanding of the functions of Sertoli cells. Immunology and stem cell biology were not even topics of science in 1865, but they have now become major issues in our appreciation of Sertoli cell's role in spermatogenesis. We end with the universal importance and plasticity of function by comparing Sertoli cells in fish, amphibians, and mammals. In these various classes of vertebrates, Sertoli cells have quite different modes of proliferation and epithelial maintenance, cystic vs. tubular formation, yet accomplish essentially the same function but in strikingly different ways.
... When Sertoli cells are cultured in vitro they are motile cells, analogous to macrophages, neutrophils and metastatic cancer cells, capable of traversing the polyester (or nitrocellulose) membrane pores on Matrigel-coated bicameral units [81]. However, Sertoli cells in vivo are relatively static but highly polarized with their nuclei and intracellular organelles (e.g., phagosomes) located exclusively to the basal compartment [82,83]. Since each Sertoli cell has to nurture ~30-50 germ cells at different stages of their development [84], such as by providing the lactate necessary to nourish germ cells [85,86], it is not likely that Sertoli cells move across the seminiferous epithelium as when they are cultured in vitro. ...
... In order to coordinate cellular events during the epithelial cycle, Sertoli cells communicate with each other and also with germ cells to support cellular events in the epithelium via gap junctions and also with distant Sertoli cells across the seminiferous epithelium via intercellular bridges (also known as tunneling nanotubes), structures that are supported by both F-actin and MT [87][88][89][90]. Furthermore, developing germ cells in particular elongating spermatids must be transported across the seminiferous epithelium so that they can line-up near the luminal edge to prepare for spermiation at stage VIII of the epithelial cycle [62,83,91,92]. Additionally, preleptotene spermatocytes transformed from type B spermatogonia residing in the basal compartment must also be transported across the immunological barrier at stage VIII of the epithelial cycle [62] so that meiosis I/II can take place in the adluminal compartment behind the BTB. ...
Sertoli cells isolated from rodents or humans and cultured in vitro are known to establish a functional tight junction (TJ)-permeability barrier that mimics the blood-testis barrier (BTB) in vivo. This model has been widely used by investigators to study the biology of the TJ and the BTB. Studies have shown that environmental toxicants (e.g., perfluorooctanesulfonate (PFOS), bisphenol A (BPA) and cadmium) that exert their disruptive effects to induce Sertoli cell injury using this in vitro model are reproducible in studies in vivo. Thus, this in vitro system provides a convenient approach to probe the molecular mechanism(s) underlying toxicant-induced testis injury but also to provide new insights in understanding spermatogenesis, such as the biology of cell adhesion, spermatid transport, and others. Herein, we provide a brief and critical review based on studies using this in vitro model of Sertoli cell cultures using primary cells isolated from rodent testes versus humans to monitor environmental toxicant-mediated Sertoli cell injury, and this information is relevant to the molecular mechanisms that regulate spermatogenesis. In short, recent findings have shown that environmental toxicants exert their effects on Sertoli cells to induce testis injury through their action on Sertoli cell actin- and/or microtubule-based cytoskeleton. These effects are mediated via their disruptive effects on actin- and/or microtubule-binding proteins. Sertoli cells also utilize differential spatiotemporal expression of these actin binding proteins to confer plasticity to the BTB to regulate germ cell transport across the BTB.
... In the testis, phagocytosis also occurs in the seminiferous epithelium, but is executed by the Sertoli cells since macrophages are usually excluded from the seminiferous epithelium. Phagocytosis is an important "self-cleaning" process in which the Sertoli cell engulfs the residual body, or excess cytoplasm of a mature spermatid, from the apical region and transported that down to the basal region where it is degraded via the lysosomal pathway [103,104]. Without MTs, the residual bodies will accumulate in the apical region as they can no longer be transported to the basal region. Vacuolization is another characteristic pathology due to MT disruption in the seminiferous epithelium [98,105,106]. ...
... The transport of germ cells across the seminiferous epithelium, such as the transport of preleptotene spermatocytes connected in clones across the BTB, and elongating/elongated spermatids across the adluminal compartment during spermiogenesis, is an intricate and tightly coordinated process. Despite decades of research on the topic of spermatogenesis [104,128], we still do not have a clear picture on how exactly germ cells are translocated in the seminiferous epithelium. It is suspected that motor proteins, such as kinesin and dynein, are responsible for the movement of non-motile germ cells. ...
In rodents and humans, testicular cells, similar to other mammalian cells, are supported by actin-, microtubule (MT)- and intermediate filament-based cytoskeletons to regulate spermatogenesis during the epithelial cycle. However, most of the published findings in the literature are limited to studies that visualize these cytoskeletons in the seminiferous epithelium during spermatogenesis. Few are focus on the underlying molecular mechanism that regulates their organization in the epithelium in response to changes in the stages of the epithelial cycle remains largely explored. Functional studies in the last decade have begun to focus on the role of binding proteins that regulate these cytoskeletons, and some interesting data have been rapidly emerging in the field. Since the actin- and intermediate-based cytoskeletons have been recently reviewed, herein we focus on the MT-based cytoskeleton for two reasons. First, besides serving as a structural support cytoskeleton, MT is known to serve as the track to support and facilitate the transport of germ cells, such as preleptotene spermatocytes connected in clones and elongating/elongated spermatids during spermiogenesis across the blood-testis barrier (BTB) and the adluminal compartment, respectively, during spermatogenesis. While these cellular events are crucial to the completion of spermatogenesis, they have been largely ignored in the past. Second, MT-based cytoskeleton is working in concert with the actin-based cytoskeleton to provide structural support to the transport of intracellular organelles across the cell cytosol, such as endosome-based vesicles, and residual bodies, phagosomes in Sertoli cells, to maintain the cellular homeostasis in the seminiferous epithelium. We critically evaluate some recent published findings herein to support a hypothesis regarding the role of MT in conferring germ cell transport in the seminiferous epithelium.
... The testis forms a unique type of adherens junction, called ectoplasmic specialization (ES), which is not found in any other epithelium and is usually considered as one of the strongest anchoring junctions in the mammalian body (Cheng and Mruk, 2015). Despite participating in the SCB (basal ES), apical ectoplasmic specialization is also found at the Sertoli cell-elongated spermatid interface, conferring spermatid adhesion and polarity (Wu et al., 2020). ...
... The actin bundles have been reported to reinforce the intercellular adhesion domains of the ES ( 7 ,8 ). As such, actin bundles in the ES must form and disassemble following the progression of a seminiferous epithelial cycle to enable rapid ES turnover (9)(10)(11). A number of actin regulatory proteins, including some required for actin assembly, have been found present at the ES (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). ...
Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of the Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that the RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of Tnik, a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics are controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.
... The blood barriers are the major physiological barriers of the body 1 that provide important biological and immunological separation of the organs from the systemic circulation. 2 The blood-testis barrier (BTB) is formed by sertoli cells, which divide the seminiferous epithelium into two compartments: basal and adluminal. Preleptotene/leptotene spermatocytes begin crossing the blood-testis barrier in late stage VIII/early stage IX, ultimately joining the adluminal compartment as zygotene spermatocytes in stage XI. ...
The blood-testis barrier is a specialized feature within the mammalian testis, located in close proximity to the basement membrane of seminiferous tubules. This barrier serves to divide the seminiferous epithelium into distinct basal and adluminal (apical) compartments. The selectivity of the BTB to foreign particles makes it a safe haven for the virus, and the high affinity of HIV for testis might lead to the vertical transmission of the virus. In the present study, recombinant HIV1-Nef (rNef) protein was injected intravenously to examine the effect of rNef on BTB. SD male rats received 250 µg and 500 µg of rNef along with 2% Evans blue dye within 1 ml through the tail vein. After 1 hour of perfusion, the animals were sacrificed for analysis. The dye migration assay and ELISA confirmed a significant impairment in the blood-testis barrier (BTB) and the manifestation of rNef in testes tissues, respectively. Moreover, a decline in the expression of tight junction proteins, including ZO1 and Occludin, was observed during rNef-induced BTB disruption. Overall, our findings demonstrated that rNef induces BTB disruption through various signaling events. At the site of ectoplasmic specialization of the seminiferous epithelium, the localization of cadherins was found to be disrupted, making the testis a vulnerable site. In conclusion, rNef perturbs the integrity of the blood-testis barrier in rat models; hence, it can also serve as a suitable model for studying the dynamics of the blood-testis barrier.
... Therefore, interactions among testicular cells, specially between germ cells and Sertoli cells, are crucial to preserve and regulate spermatogenesis in a very coordinated and organized manner, providing all the necessary structural and nutritional support for the developing germ cells. These interactions are important to ensure the development and completion of spermatogenesis [23,25,67]. ...
One of the major concerns of the world health community is the infertility. The definition of infertility according to the World Health Organization (WHO) and the American Society for Reproductive Medicine (ASRM) is the inability of a healthy couple to achieve a conception after one year of regular, unprotected intercourse. Fertility complications affect seven percent of the male. The causes of infertility were divided to non-obstructive and obstructive. But, in almost 75% of male infertility cases are idiopathic with predominance of the genetic abnormalities. Numerical or structural chromosomal abnormalities are considered as genetic abnormalities that occur during the meiotic division in spermatogenesis. These abnormalities get transferred to the Offspring, which affects the normal and even the artificial conception. In the human reproduction, sperm cells are considered as a delivery vehicle for the male genetic material packed in chromosomes, which are composed of nearly 2-meter Deoxyribonucleic acid (DNA) molecule and their packaging proteins. This chapter points to grant a summarized description of individual components of the male reproductive system: the seminiferous tubule and spermatogenesis. Here, we describe step by step the structure of the testis seminif-erous tubule and what occurs inside these tubules like cell communication and germ cell development from spermatogonia until spermatozoon. This book chapter is very useful for the biologists and physicians working in Assisted reproduction field to understand the physiology and pathology of spermatogenesis.
... It is formed by tight junctions between Sertoli cells that divide the epithelium of the seminiferous tubules (ST) into two different compartments: basal and adluminal ( Figure 3). Although it is one of the tightest blood-tissue barriers in the mammalian body [95], it was previously reported that NPs could cross this biological barrier due to their ultra-small size [16]. In fact, in mice treated with TiO 2 [96] and Fe 2 O 3 [97], both NPs were able to penetrate the testis, despite the BTB. ...
Metal oxide nanoparticles (MONPs) are inorganic materials that have become a valuable tool for many industrial sectors, especially in healthcare, due to their versatility, unique intrinsic properties, and relatively inexpensive production cost. As a consequence of their wide applications, human exposure to MONPs has increased dramatically. More recently, their use has become somehow controversial. On one hand, MONPs can interact with cellular macromolecules, which makes them useful platforms for diagnostic and therapeutic interventions. On the other hand, research suggests that these MONPs can cross the blood–testis barrier and accumulate in the testis. Although it has been demonstrated that some MONPs have protective effects on male germ cells, contradictory reports suggest that these nanoparticles compromise male fertility by interfering with spermatogenesis. In fact, in vitro and in vivo studies indicate that exposure to MONPs could induce the overproduction of reactive oxygen species, resulting in oxidative stress, which is the main suggested molecular mechanism that leads to germ cells’ toxicity. The latter results in subsequent damage to proteins, cell membranes, and DNA, which ultimately may lead to the impairment of the male reproductive system. The present manuscript overviews the therapeutic potential of MONPs and their biomedical applications, followed by a critical view of their potential risks in mammalian male fertility, as suggested by recent scientific literature.
... Subsequently, the actin-based apical ectoplasmic specializations replace the desmosomes and characterizes the anchoring device, between Sertoli cell and elongating SPTs. The junctional complex of apical ectoplasmic specializations include Zo-1 and through the α/β catenin interact with cytoskeletal actin of Sertoli cells [86,87]. The β-catenin conditional deletion in post meiotic cells reveals its key role in survival and chromatin condensation of post-meiotic cells [88] suggesting that BPA reduced post meiotic cells by affecting β-catenin protein. ...
The objective of this work has been to characterize the estrogenic activity of bisphenol-A (BPA) and the adverse effects on the endocannabinoid system (ECS) in modulating germ cell progression. Male offspring exposed to BPA during the foetal-perinatal period at doses below the no-observed-adverse-effect-level were used to investigate the exposure effects in adulthood. Results showed that BPA accumulates specifically in epididymal fat rather than in abdominal fat and targets testicular expression of 3β-hydroxysteroid dehydrogenase and cytochrome P450 aromatase, thus promoting sustained increase of estrogens and a decrease of testosterone. The exposure to BPA affects the expression levels of some ECS components, namely type-1 (CB1) and type-2 cannabinoid (CB2) receptor and monoacylglycerol-lipase (MAGL). Furthermore, it affects the temporal progression of germ cells reported to be responsive to ECS and promotes epithelial germ cell exfoliation. In particular, it increases the germ cell content (i.e., spermatogonia while reducing spermatocytes and spermatids), accelerates progression of spermatocytes and spermatids, promotes epithelial detachment of round and condensed spermatids and interferes with expression of cell–cell junction genes (i.e., zonula occcludens protein-1, vimentin and β-catenin). Altogether, our study provides evidence that early exposure to BPA produces in adulthood sustained and site-specific BPA accumulation in epididymal fat, becoming a risk factor for the reproductive endocrine pathways associated to ECS.
... Interactions among testicular cells, in particular between Sertoli and germ cells, are crucial to maintain and regulate spermatogenesis in a very coordinated and organized manner, providing all the necessary structural and nutritional support for the developing germ cells (França et al., 2016;Cheng and Mruk, 2015;Lara et al., 2018a). Therefore, on its basal side Sertoli cells contact spermatogonia through adherens junctions, guiding their homing, niche and colonization (Lara et al., 2018a). ...
Besides having medical applications, comparative studies on reproductive biology are very useful, providing, for instance, essential knowledge for basic, conservation and biotechnological research. In order to maintain the reproductive potential and the survival of all vertebrate species, both sperm and steroid production need to occur inside the testis. From the approximately fifty thousand vertebrate species still alive, very few species are already investigated; however, our knowledge regarding Sertoli cell biology is quite good. In this regard, it is already known that since testis differentiation the Sertoli cells are the somatic cells in charge of supporting and orchestrating germ cells during development and full spermatogenesis in adult animals. In the present review, we highlight key aspects related to Sertoli cell biology in vertebrates and show that this key testis somatic cell presents huge and intrinsic plasticity, particularly when cystic (fish and amphibians) and non-cystic (reptiles, birds and mammals) spermatogenesis is compared. In particular, we briefly discuss the main aspects related to Sertoli cells functions, interactions with germ cells, Sertoli cells proliferation and efficiency, as well as those regarding spermatogonial stem cell niche regulation, which are crucial aspects responsible for the magnitude of sperm production. Most importantly, we show that we could greatly benefit from investigations using different vertebrate experimental models, mainly now that there is a big concern regarding the decline in human sperm counts caused by a multitude of factors.
... In the mammalian testis, extensive remodeling of cell junctions take place in the seminiferous epithelium during the epithelial cycle to support spermatogenesis (1)(2)(3)(4). This is necessary to accommodate the transport of preleptotene spermatocytes across the blood-testis barrier (BTB), but also developing spermatids across the seminiferous epithelium, so that spermatozoa transformed from step 19 spermatids in rat testes can be released into the tubule lumen at spermiation (5)(6)(7). ...
Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a non-hormonal male contraceptive since it effectively induces reversible male infertility without perturbing the serum concentrations of FSH, testosterone and inhibin B. Adjudin was shown to exert its effects preferentially by perturbing the testis-specific actin-rich adherens junction (AJ) at the Sertoli-spermatid interface known as apical ectoplasmic specialization (apical ES), thereby effectively induce spermatid exfoliation. Adjudin did not perturb germ cell development nor germ cell function. Also, it had no effects on Sertoli cell-cell AJ called basal ES - which together with tight junction (TJ) constitute the blood-testis barrier (BTB) - unless an acute dose of adjudin at was used. Adjudin also did not perturb the population of spermatogonial stem cells nor Sertoli cells in the testis. However, the downstream signaling protein(s) utilized by adjudin to induce transient male infertility remains unexplored. Herein, using adult rats treated with adjudin and monitored changes in the phenotypes across the seminiferous epithelium between 6 and 96 h in parallel with the steady-state protein levels of an array of signaling and cytoskeletal regulatory proteins, recently shown to be involved in apical ES, basal ES and BTB function. It was shown that adjudin exerts its contraceptive effects through changes in microtubule associated proteins (MAPs) and signaling proteins mTORC1/rpS6 and p-FAK-Y407. These findings are important to study adjudin-mediated male infertility but also the biology of spermatogenesis.
... Interactions among testicular cells, in particular between Sertoli and germ cells, are crucial to maintain and regulate spermatogenesis in a very coordinated and organized manner, providing all the necessary structural and nutritional support for the developing germ cells (França et al., 2016;Cheng and Mruk, 2015;Lara et al., 2018a). Therefore, on its basal side Sertoli cells contact spermatogonia through adherens junctions, guiding their homing, niche and colonization (Lara et al., 2018a). ...
... One area of interest since the early days of electron microscopy is characterization of the numerous junctions that Sertoli cells make with each other and with different germ cells. In the past several years, the biochemical components of many of these junctions have been characterized and have recently been reviewed [4,79]. The junctional complexes between Sertoli cells and germ cells likely provide communication between cells and are involved in the orderly movement of germ cells within the seminiferous epithelium. ...
The complex morphology of the Sertoli cells and their interactions with germ cells has been a focus of investigators since they were first described by Enrico Sertoli. In the past 50 years, information on Sertoli cells has transcended morphology alone to become increasingly more focused on molecular questions. The goal of investigators has been to understand the role of the Sertoli cells in spermatogenesis and to apply that information to problems relating to male fertility. Sertoli cells are unique in that they are a non-dividing cell population that is active for the reproductive lifetime of the animal and cyclically change morphology and gene expression. The numerous and distinctive junctional complexes and membrane specializations made by Sertoli cells provide a scaffold and environment for germ cell development. The increased focus of investigators on the molecular components and putative functions of testicular cells has resulted primarily from procedures that isolate specific cell types from the testicular milieu. Products of Sertoli cells that influence germ cell development and vice-versa have been characterized from cultured cells and from the application of transgenic technologies. Germ cell transplantation has shown that the Sertoli cells respond to cues from germ cells with regard to developmental timing and has furthered a focus on spermatogenic stem cells and the stem cell niche. Very basic and universal features of spermatogenesis such as the cycle of the seminiferous epithelium and the spermatogenic wave are initiated by Sertoli cells and maintained by Sertoli-germ cell cooperation.
... The testis forms a unique type of adherens junction, called ectoplasmic specialization (ES), which is not found in any other epithelium and is usually considered as one of the strongest anchoring junctions in the mammalian body (Cheng and Mruk, 2015). Despite participating in the SCB (basal ES), apical ectoplasmic specialization is also found at the Sertoli cell-elongated spermatid interface, conferring spermatid adhesion and polarity. ...
... Most of this work has been 93 done in primary cultures and in the context of determining how the cells regulate and produce 94 seminiferous tubule fluid, maintain pH, and initiate signaling cascades. Much less work has been 95 done to localize ion channels and pumps in morphologically differentiated Sertoli cells in vivo 96 and in the context of junction turnover; although a large number of other regulators and signaling 97 pathways are known to impact the junctions ( Cheng and Mruk 2015). One ion pump that does 98 appear specifically concentrated in the plasma membrane of Sertoli cells at apical and basal sites 99 of intercellular attachment is Na+/K+ ATPase ( Byers and Graham 1990). ...
The endoplasmic reticulum (ER) forms a continuous network throughout morphologically differentiated Sertoli cells. It is an integral component of intercellular adhesion junctions in this cell type, as well as forming membrane contact sites with the plasma membrane and intracellular organelles. One of the major functions of the ER in cells generally is maintaining calcium homeostasis and generating calcium signals. In this review, we discuss what is currently known about the overall pattern of distribution of the ER in Sertoli cells and the location of calcium regulatory machinery in the various subdomains of the organelle. Current data is consistent with the hypothesis that calcium signaling by the ER of Sertoli cells may play a significant role in events related to junction remodeling that occur in the seminiferous epithelium during spermatogenesis.
... These proteins coordinate to regulate actin microfilament remodeling at the basal ES and apical ES via their stage-specific and spatiotemporal expression, which, in turn, facilitate the transport of preleptotene spermatocytes across the BTB and the transport of elongating spermatids across the adluminal compartment, respectively. The coordinated efforts of those actin regulatory proteins have recently been reviewed (Li et al. 2015a;Cheng and Mruk 2015;Su et al. 2013). Herein, we focus our discussion on drebrin, a family of proteins known to regulate the action of the Arp2/3 complex which is a barbed end nucleation protein that effectively induces branched actin polymerization, thereby converting actin microfilaments from a linear to a branched configuration. ...
Drebrin is a family of actin-binding proteins with two known members called drebrin A and E. Apart from the ability to stabilize F-actin microfilaments via their actin-binding domains near the N-terminus, drebrin also regulates multiple cellular functions due to its unique ability to recruit multiple binding partners to a specific cellular domain, such as the seminiferous epithelium during the epithelial cycle of spermatogenesis. Recent studies have illustrated the role of drebrin E in the testis during spermatogenesis in particular via its ability to recruit branched actin polymerization protein known as actin-related protein 3 (Arp3), illustrating its involvement in modifying the organization of actin microfilaments at the ectoplasmic specialization (ES) which includes the testis-specific anchoring junction at the Sertoli-spermatid (apical ES) interface and at the Sertoli cell-cell (basal ES) interface. These data are carefully evaluated in light of other recent findings herein regarding the role of drebrin in actin filament organization at the ES. We also provide the hypothetical model regarding its involvement in germ cell transport during the epithelial cycle in the seminiferous epithelium to support spermatogenesis.
... It is obvious that the precise regulatory mechanism(s) underlying these events regarding transcellular and paracellular (and active) transports in different blood-tissue barriers must be carefully evaluated in future studies. In this context, it is of interest to note that ES is also found in the apical compartment where it is designated apical ES and is restrictively expressed at the Sertoli-spermatid (steps 8-19 in the rat or 8-16 in the mouse testis) interface [77,78]. Morphologically, the apical and the basal ES share virtually similar features except that there is only a single array of actin filament bundles found in the Sertoli cell adjacent to the apposing Sertoli-spermatid plasma membranes at the apical ES vs. two arrays of actin filament bundles for basal ES (Fig. 1). ...
Signal pathways that regulate blood-tissue barriers are important for studying the biology of various blood-tissue barriers. This information, if deciphered and better understood, will provide better therapeutic management of diseases particularly in organs that are sealed by the corresponding blood-tissue barriers from systemic circulation, such as the brain and the testis. These barriers block the access of antibiotics and/or chemotherapeutical agents across the corresponding barriers. Studies in the last decade using the blood-testis barrier (BTB) in rats have demonstrated the presence of several signaling pathways that are crucial to modulate BTB function. Herein, we critically evaluate these findings and provide hypothetical models regarding the underlying mechanisms by which these signaling molecules/pathways modulate dynamics. This information should be carefully evaluated to examine their applicability in other tissue barriers which shall benefit future functional studies in the field. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.
... Furthermore, postmeiotic spermatids derive from meiosis must also be transported across the entire adluminal compartment so that fully developed spermatids (ie, spermatozoa) can line-up near the luminal edge to prepare for their release at spermiation (for reviews, see Refs. 3,4). It is envisioned that extensive remodeling takes place at the Sertoli cell-cell and Sertolispermatid interface to accommodate these events of germ cell transport. ...
Formin 1 confers actin nucleation by generating long stretches of actin microfilaments to support cell movement, cell shape, and intracellular protein trafficking. Formin 1 is likely involved in microtubule (MT) dynamics due to the presence of a MTB (microtubule binding) domain near its N-terminus. Herein, formin 1 was shown to structurally interact with α-tubulin, the building block of MT, and also EB1 (end binding protein 1, a MT plus(+)-end binding protein that stabilizes MT) in the testis. Knockdown of formin 1 in Sertoli cells with an established tight junction (TJ)-barrier was found to induce down-regulation of detyrosinated MT (a stabilized form of MT), and disorganization of MTs in which MTs were retracted from the cell cortical zone, mediated through a loss of MT polymerization and down-regulation of Ark1/2 signaling kinase. An efficient knockdown of formin 1 in the testis reduced the number of track-like structures conferred by MTs and F-actin considerably, causing defects in spermatid and phagosome transport across the seminiferous epithelium. In summary, formin1 maintains MT and F-actin track-like structures to support spermatid and phagosome transport across the seminiferous epithelium during spermatogenesis.
... While the BTB is one of the tightest blood-tissue barriers in the mammalian body, it undergoes continuous remodeling in particular at stage VIII of the epithelial cycle to accommodate the transport of preleptotene spermatocytes connected in clones across the immunological barrier so that they can differentiate into late stage spermatocytes in the adluminal compartment to prepare for meiosis (for a review, see [32]). Interestingly, the events of BTB/ basal ES remodeling coincides with the apical ES remodeling at the opposite end of the epithelium at the interface of Sertoli cells and step 19 spermatids to accommodate the release of fully developed spermatids (i.e., spermatozoa) into the tubule lumen at spermiation (for reviews, see [33,34]). The apical ES is structurally similar to the basal ES except that the actin microfilament network is confined to the Sertoli cell without any noticeable ultrastructures contributed by the spermatids. ...
In adult mammalian testes, spermatogenesis is composed of several discrete cellular events that work in tandem to support the transformation and differentiation of diploid spermatogonia to haploid spermatids during the seminiferous epithelial cycle. These include: self-renewal of spermatogonial stem cells via mitosis and their transformation into differentiated spermatogonia, meiosis I/II, spermiogenesis and the release of sperms at spermiation. Studies have shown that these cellular events are under precise and coordinated controls of multiple proteins and signaling pathways. These events are also regulated by polarity proteins that are known to confer classical apico-basal (A/B) polarity in other epithelia. Furthermore, spermatid development is likely supported by planar cell polarity (PCP) proteins since polarized spermatids are aligned across the plane of seminiferous epithelium in an orderly fashion, analogous to hair cells in the cochlea of the inner ear. Thus, the maximal number of spermatids can be packed and supported by a fixed population of differentiated Sertoli cells in adult testes in the limited space of the seminiferous epithelium. In this review, we briefly summarize recent findings regarding the role of PCP proteins in the testis. This information should be helpful in future studies to better understand the role of PCP proteins in spermatogenesis.
... The premature release of spermatids, as shown here, is not entirely unexpected because actin microfilament bundles at the apical ES are essential to confer adhesive function. It is known that the release of sperm that occurs at spermiation is a complicated cellular process, involving multiple signal proteins (2,63), and studies have shown that this event is heavily dependent on actin-based cytoskeleton, requiring the involvement of multiple actinbinding proteins and regulatory signaling protein kinases (3,64). Here, KD of plastin 3 in vivo indeed induces structural defects of the F-actin network at the apical ES as visualized by rhodamine-phalloidin staining, involving mislocalization of other actin regulatory proteins, Arp3 and Eps8. ...
Ectoplasmic specialization (ES) is an actin-rich adherens junction in the seminiferous epithelium of adult mammalian testes. ES is restricted to the Sertoli-spermatid (apical ES) interface, as well as the Sertoli cell-cell (basal ES) interface at the blood-testis barrier (BTB). ES is typified by the presence of an array of bundles of actin microfilaments near the Sertoli cell plasma membrane. These actin microfilament bundles require rapid debundling to convert them from a bundled to branched/unbundled configuration and vice versa to confer plasticity to support the transport of 1) spermatids in the adluminal compartment and 2) preleptotene spermatocytes at the BTB while maintaining cell adhesion. Plastin 3 is one of the plastin family members abundantly found in yeast, plant and animal cells that confers actin microfilaments their bundled configuration. Herein, plastin 3 was shown to be a component of the apical and basal ES in the rat testis, displaying spatiotemporal expression during the epithelial cycle. A knockdown (KD) of plastin 3 in Sertoli cells by RNA interference using an in vitro model to study BTB function showed that a transient loss of plastin 3 perturbed the Sertoli cell tight junction-permeability barrier, mediated by changes in the localization of basal ES proteins N-cadherin and β-catenin. More importantly, these changes were the result of an alteration of the actin microfilaments, converting from their bundled to branched configuration when examined microscopically, and validated by biochemical assays that quantified actin-bundling and polymerization activity. Moreover, these changes were confirmed by studies in vivo by plastin 3 KD in the testis in which mis-localization of N-cadherin and β-catenin was also detected at the BTB, concomitant with defects in the transport of spermatids and phagosomes and a disruption of cell adhesion most notably in elongated spermatids due to a loss of actin-bundling capability at the apical ES, which in turn affected localization of adhesion protein complexes at the site. In summary, plastin 3 is a regulator of actin microfilament bundles at the ES in which it dictates the configuration of the filamentous actin network by assuming either a bundled or unbundled/branched configuration via changes in its spatiotemporal expression during the epithelial cycle.-Li, N., Mruk, D. D., Wong, C. K. C., Lee, W. M., Han, D., Cheng, C. Y. Actin-bundling protein plastin 3 is a regulator of ectoplasmic specialization dynamics during spermatogenesis in the rat testis.
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Background
Restoring fertility in male cancer individuals through testicular tissue transplantation faces challenges due to hypoxia-induced loss of spermatogonial stem cells (SSCs). Hydrogel encapsulation was explored to minimize hypoxic damage in testicular tissue transplantation. For this purpose, human amnion membrane (hAM)-derived hydrogel could be an alternative.
Objective
The potential of hAM-derived hydrogel to support testis tissue grafts was evaluated.
Materials and Methods
In this experimental study, testicular tissue samples (1–3 mm³) were obtained from 16 male NMRI mice (4–5 wk, 22 ± 2 gr). These tissue fragments were either encapsulated within a hydrogel derived from a hAM or left unencapsulated (control) prior to being autologously transplanted beneath the dorsal skin of mice subjected to hemilateral or bilateral orchiectomy. The grafted testicular tissues were histologically evaluated for key parameters, including the integrity of seminiferous tubules, survival of SSCs, Sertoli cell functionality, as well as hypoxia and apoptosis on day 21.
Results
No significant differences were observed between groups regarding ST integrity, number of SSCs, Sertoli cell functionality, or the rate of hypoxia-inducible factor 1-alpha and apoptosis (p ≤ 0.05).
Conclusion
In conclusion, this study demonstrated no effect of hAM hydrogel encapsulation on the outcomes of testicular tissue transplantation.
Male infertility can be caused by chromosomal abnormalities, mutations, and epigenetic defects. Epigenetic modifiers pre-program hundreds of spermatogenic genes in spermatogonial stem cells (SSCs) for expression later in spermatids, but it remains mostly unclear whether and how those genes are involved in fertility. Here, we report that Wfdc15a, a WFDC family protease inhibitor pre-programmed by KMT2B, is essential for spermatogenesis. We found that Wfdc15a is a non-canonical bivalent gene carrying both H3K4me3 and facultative H3K9me3 in SSCs but is later activated along with the loss of H3K9me3 and acquisition of H3K27ac during meiosis. We show that Wfdc15a deficiency causes defective spermiogenesis at the beginning of spermatid elongation. Notably, depletion of Wfdc15a causes substantial disturbance of the testicular protease-antiprotease network and leads to an orchitis-like inflammatory response associated with TNFa expression in round spermatids. Together, our results reveal a unique epigenetic program regulating innate immunity crucial for fertility.
Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of Tnik , a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics is controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.
It is almost five decades since the discovery of the hypothalamic-pituitary-testicular axis. This refers to the hormonal axis that connects the hypothalamus, pituitary gland and testes, which in turn, regulates the production of spermatozoa through spermatogenesis in the seminiferous tubules, and testosterone through steroidogenesis by Leydig cells in the interstitium, of the testes. Emerging evidence has demonstrated the presence of a regulatory network across the seminiferous epithelium utilizing bioactive molecules produced locally at specific domains of the epithelium. Studies have shown that biologically active fragments are produced from structural laminin and collagen chains in the basement membrane. Additionally, bioactive peptides are also produced locally in non-basement membrane laminin chains at the Sertoli-spermatid interface known as apical ectoplasmic specialization (apical ES, a testis-specific actin-based anchoring junction type). These bioactive peptides are derived from structural laminins and/or collagens at the corresponding sites through proteolytic cleavage by matrix metalloproteinases (MMPs). They in turn serve as autocrine and/or paracrine factors to modulate and coordinate cellular events across the epithelium by linking the apical and basal compartments, the apical and basal ES, the blood-testis barrier (BTB), and the basement membrane of the tunica propria. The cellular events supported by these bioactive peptides/fragments include the release of spermatozoa at spermiation, remodeling of the immunological barrier to facilitate the transport of preleptotene spermatocytes across the BTB, and the transport of haploid spermatids across the epithelium to support spermiogenesis. In this review, we critically evaluate these findings. Our goal is to identify research areas that deserve attentions in future years. The proposed research also provides the much needed understanding on the biology of spermatogenesis supported by a local network of regulatory biomolecules.
mTORC1/rpS6 signaling complex promoted Sertoli blood-testis barrier (BTB) remodeling by perturbing Sertoli cell-cell adhesion site known as the basal ectoplasmic specialization (ES). mTORC1/rpS6 complex also promoted disruption of spermatid adhesion at the Sertoli-spermatid interface called apical ES. Herein, we performed analyses using the adjudin (a potential non-hormonal male contraceptive drug) model, wherein adjudin was known to perturb apical and basal ES function at high dose. Through direct administration of adjudin to the testis, adjudin at doses that failed to perturb BTB integrity per se, overexpression of an rpS6 phosphomimetic (i.e., constitutively active) mutant (i.e., p-rpS6-MT) that modified BTB function considerably potentiated adjudin efficacy. This led to disorderly spatial expression of proteins necessary to maintain the proper cytoskeletal organization of F5-actin and microtubules (MTs) across the seminiferous epithelium, leading to germ cell exfoliation and aspermatogenesis. These findings yielded important insights regarding the role of mTORC1/rpS6 signaling complex in regulating BTB homeostasis.
Sertoli cells of the mammalian seminiferous epithelium form unique subcellular actin‐related structures at intercellular junctions. The appearance of these so called “tubulobulbar complexes” (TBCs) precedes both sperm release at the apex of the epithelium and the movement of early spermatogenic cells out of the spermatogonial stem cell niche at the base of the epithelium. TBCs are considered to be part of the mechanism of junction endocytosis by Sertoli cells. The structures contain junction proteins and morphologically identifiable junctions, and are associated with markers of endocytosis. Here we review the current state of knowledge about the structure and function of TBCs. As the complexes form, they morphologically resemble and have the molecular signature of clathrin‐coated pits with extremely long necks. As they mature, the actin filament networks around the “necks” of the structures progressively disassemble and the membrane cores expand or swell into distinct “bulbs”. These bulbs acquire extensive membrane contact sites with associated cisternae of endoplasmic reticulum. Eventually the bulbs undergo scission and continue through endosomal compartments of the Sertoli cells. The morphology and composition of TBC indicates to us that the structures likely evolved from the basic clathrin‐mediated endocytosis mechanism common to cells generally, and along the way they incorporated unique features to accommodate the cyclic turnover of massive and “intact” intercellular junctions that occurs during spermatogenesis.
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The testis is one of the organs in the mammalian body that is highly sensitive to toxicants following exposure. Accumulating evidence in the field has shown that long-term exposure of the testis to many of these environmental toxicants (e.g., cadmium, 2,5-hexanedione, PFOS (perfluorooctanesulfonate) and phthalates) leads to testis dysfunction, including a declining sperm count in men due to defects in spermatogenesis. Studies have also shown that within the functional unit of the testis that support spermatogenesis, namely the seminiferous tubule, the Sertoli cell is the cell type that is highly sensitive to the disruptive effects of toxicants, leading to Sertoli cell injury. This, in turn, fails to support germ cell development in the seminiferous epithelium. Based on published findings in the literature, it is becoming clear that the Sertoli cell cytoskeleton is one of the primary target of toxicants in the testis. We also provide insightful information that underlies the molecular basis of toxicant-induced Sertoli cell injury through disruptive changes in cytoskeletal binding and regulatory proteins, as well as the involving signaling molecules.
Cell polarity is crucial to development since apico-basal polarity conferred by the three polarity protein modules (or complexes) is essential during embryogenesis, namely the Par (partition defective)-, the CRB (Crumbs)-, and the Scribble-based polarity protein modules. While these protein complexes and their component proteins have been extensively studied in Drosophila and C. elegans and also other mammalian tissues and/or cells, their presence and physiological significance in the testis remains unexplored until the first paper on the Par-based protein published in 2008. Since then, the Scribble- and the CRB-based protein complexes and their component proteins in the testis have been studied. These proteins are known to confer Sertoli and spermatid polarity in the seminiferous epithelium, and they are also integrated components of the tight junction (TJ) and the basal ectoplasmic specialization (ES) at the Sertoli cell-cell interface near the basement membrane, which in turn constitute the blood-testis barrier (BTB). These proteins are also found at the apical ES at the Sertoli-spermatid interface. Thus, these polarity proteins also play a significant role in regulating Sertoli and spermatid adhesion in the testis through their actions on actin-based cytoskeletal function. Recent studies have shown that these polarity proteins are having antagonistic effects on the BTB integrity in which the Par6- and CRB3-based polarity complexes promotes the integrity of the Sertoli cell TJ-permeability barrier, whereas the Scribble-based complex promotes restructuring/remodeling of the Sertoli TJ-barrier function. Herein, we carefully evaluate these findings and provide a hypothetic model in the context of the functions of these polarity proteins in other epithelia, so that better experiments can be designed in future studies to explore their significance in spermatogenesis.
Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES.
Germ cell transport across the seminiferous epithelium during spermatogenesis requires the intricate coordination of cell junctions, signaling proteins, and both actin- and microtubule (MT)-based cytoskeletons. Although the involvement of cytoskeletons in germ cell transport has been suggested, the precise mechanism(s) remains unknown. Based on growing evidence that actin and MT interactions underlie fundamental cellular processes, like cell motility, it is unlikely that actin- and MT-based cytoskeletons work independently to regulate germ cell transport in the testis. Using rats treated with adjudin, a potential male contraceptive that disrupts spermatid adhesion and transport in the testis, as a study model, we show herein that actin- and MT-based cytoskeletons are both necessary for transport of spermatids and residual bodies/phagosomes across the epithelium. Analysis of intratubular expression of F-actin and tubulin revealed disruption of both actin and MT networks, concomitant with misdirected spermatids and phagosomes in rats treated with adjudin. Actin regulatory proteins, Eps8 (epidermal growth factor receptor pathway substrate 8) and Arp3 (actin-related protein 3), were mis-localized and downregulated at the actin-rich anchoring junction between germ and Sertoli cells (apical ES) after adjudin treatment. Non-receptor tyrosine kinase p-FAK-Tyr(407), known to regulate F-actin nucleation via Arp3, was also mis-localized and downregulated at the apical ES, corroborating the observation of actin cytoskeleton disruption. Additionally, spatiotemporal expression of MT regulatory protein EB1 (end-binding protein 1), shown to be involved in MT-actin crosstalk herein, was also disrupted after adjudin treatment. In summary, spermatid/phagosome transport across the epithelium during spermatogenesis requires the coordination between actin- and MT-based cytoskeletons.
During spermatogenesis, developing spermatids and preleptotene spermatocytes are transported across the adluminal compartment and the blood-testis barrier (BTB), respectively, so that spermatids line-up near the luminal edge to prepare for spermiation, while preleptotene spermatocytes enter the adluminal compartment to differentiate into late spermatocytes to prepare for meiosis I/II. These cellular events involve actin microfilament re-organization at the testis-specific/actin-rich Sertoli-spermatid and Sertoli-Sertoli cell junction called apical and basal ectoplasmic specialization (ES). Formin 1, an actin nucleation protein, known to promote actin microfilament elongation and bundling, was expressed at the apical ES but limited to stage VII of the epithelial cycle, whereas its expression at the basal ES/BTB stretched from stage III-VI, diminished in VII and undetectable in VIII tubules. Using an in vitro model of studying Sertoli cell BTB function by RNAi and biochemical assays to monitor actin bundling and polymerization activity, a knockdown of formin 1 in Sertoli cells by ∼70% impeded the tight junction (TJ)-permeability function. This disruptive effect on the TJ-barrier was mediated by a loss of actin microfilament bundling and actin polymerization capability mediated by changes in the localization of branched actin-inducing protein Arp3, and actin bundling proteins Eps8 and palladin, thereby disrupting cell adhesion. Formin 1 knockdown in vivo was found to impede spermatid adhesion, transport and polarity, causing defects in spermiation in which elongated spermatids remained embedded into the epithelium in stage IX tubules, mediated by changes in the spatiotemporal expression of Arp3 and Eps8. In summary, formin 1 is a regulator of ES dynamics.
The immunosuppressants rapamycin and FK506 bind to the same intracellular protein, the immunophilin FKBP12. The FKB12-FK506 complex interacts with and inhibits the Ca(2+)-activated protein phosphatase calcineurin. The target of the FKBP12-rapamycin complex has not yet been identified. We report that a protein complex containing 245 kDa and 35 kDa components, designated rapamycin and FKBP12 targets 1 and 2 (RAFT1 and RAFT2), interacts with FKBP12 in a rapamycin-dependent manner. Sequences (330 amino acids total) of tryptic peptides derived from the 245 kDa RAFT1 reveal striking homologies to the yeast TOR gene products, which were originally identified by mutations that confer rapamycin resistance in yeast. A RAFT1 cDNA was obtained and found to encode a 289 kDa protein (2549 amino acids) that is 43% and 39% identical to TOR2 and TOR1, respectively. We propose that RAFT1 is the direct target of FKBP12-rapamycin and a mammalian homolog of the TOR proteins.
Based on enumeration of maturation-phase spermatids in testicular homogenates from adult men and rats, daily sperm production per gram of testicular parenchyma (DSP/g) was almost seven times greater in rats (21.1 ± 0.9 × 10⁶ vs 3.1 ± 0.5 × 10⁶ ; X̄ ± SEM, n = 10). The relative inefficiency of the human testis was uniformly expressed in cranial, equatorial and caudal regions. Furthermore, DSP/g was highly correlated between paired testes from individual men (r = +0.97, n = 10), and differences between paired testes were not significantly different from zero (P>0.4). Histometric analysis of glutaraldehyde-perfused testes revealed differences (P<0.05) in relative testicular compositions between humans and rats (n = 3) including higher proportions of parenchyma, seminiferous tubules, seminiferous epithelium and germinal cells in the rat testis. Humans exceeded rats only in the proportions of testis occupied by nongerminal components such as tunic, interstitium, tubule boundary tissue and Sertoli cells. Histometric determination of the proportion of the testicular parenchyma occupied by these nuclei and consideration of average volume of round spermatid nuclei yielded DSP/g estimates of 17.4 ± 1.8 × 10⁶ in rats (not significantly different from the result by the homogenization method) and 8.5 ± 1.3 × 10⁶ in humans (almost three times higher than the value obtained by the homogenization method). Although the disparity between values for human DSP/g obtained by the two methods cannot yet be explained, several lines of evidence suggest that the time divisor used for human material processed by the homogenization method is too long, thus yielding an underestimation of daily sperm production. While its absolute accuracy is questionable, the homogenization method remains a rapid and precise technique for estimating DSP/g in humans. Histometric analysis and histometric estimation of DSP/g support the concept that the human is less efficient in sperm production than the rat, but these methods suggest that the rat is only about twice as efficient per gram of testicular parenchyma as the human.
The distribution of a611 integrins at the level of cell-to-cell contacts within the rat seminiferous epithelium was investigated. Double fluorescence experiments using phalloidin staining of actin filaments and anti-integrin subunit antibodies showed that the receptor belongs to the Sertoli cell lateral domains engaged in the characteristic junctional structures known as ec-toplasmic specializations (ES), at the level both of inter-Sertoli junctions and of the contacts between Sertoli cells and elongating spermatids. In the seminiferous epithelium of aspermatogen-ic testes, obtained through X-irradiation in utero (Sertoli-cell-only testes), at the level of inter-Sertoli junctions both ES and t611 integrins are present. In order to study the dependence of et6pl receptors and ES formation upon FSH stimulation during development, 9-day-old testes were grown in organ culture in basal as well as FSH-supplemented conditions. FSH stimulation , which is necessary for the progression of spermatogenesis to early meiotic stages, appears to be required for the development of inter-Sertoli junctional structures containing ES and a6131 integrins. These observations indicate that the receptor belongs to the inter-Sertoli junctional machinery and that its expression at that level is not dependent on active spermato-genesis but requires FSH stimulation.
Laminins are heterotrimeric molecules composed of an α, a β, and a γ chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel laminin, composed of known α and β chains but containing a novel γ chain, γ3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that γ3 contains all the expected domains of a γ chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that γ3-containing laminins are likely to exist in a stable matrix.
Studies of the tissue distribution of γ3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, γ3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The γ3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, ductus deferens, and seminiferous tubules. The distribution of γ3-containing laminins on the apical surfaces of a variety of epithelial tissues is novel and suggests that they are not found within ultrastructurally defined basement membranes. It seems likely that these apical laminins are important in the morphogenesis and structural stability of the ciliated processes of these cells.
The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. However, it undergoes cyclic restructuring during the epithelial cycle of spermatogenesis in which the "old" BTB located above the preleptotene spermatocytes being transported across the immunological barrier is "disassembled" while the "new" BTB found behind these germ cells is rapidly "reassembled" mediated by endocytic vesicle-mediated protein trafficking events. Thus, the immunological barrier is maintained when preleptotene spermatocytes connected in clones via intercellular bridges are transported across the BTB. Yet the underlying mechanism(s) in particular the involving regulatory molecules that coordinate these events remain unknown. We hypothesized that c-Src and c-Yes might work in contrasting roles in endocytic vesicle-mediated trafficking, serving as molecular switches, to effectively "disassemble" and "reassemble" the "old" and the "new" BTB, respectively, to facilitate preleptotene spermatocyte transport across the BTB. Following siRNA-mediated specific knockdown of c-Src or c-Yes in Sertoli cells, we utilized biochemical assays to assess the changes in protein endocytosis, recycling, degradation and phagocytosis. c-Yes was found to promote endocytosed integral membrane BTB proteins to the pathway of transcytosis and recycling so that internalized proteins could be effectively used to assemble "new" BTB from the disassembling "old" BTB, whereas c-Src promotes endocytosed Sertoli cell BTB proteins to endosome-mediated protein degradation for the degeneration of "old" BTB. By using fluorescence beads mimicking apoptotic germ cells, Sertoli cells were found to engulf beads via c-Src-mediated phagocytosis. A hypothetical model is thus proposed which serves as the framework for future investigation.
Focal adhesion kinase (FAK), as its name implied, is an important mediator of integrin-based signaling function in mammalian cells at the focal adhesion complex (FAC, also known as focal contact) at the cell-extracellular matrix interface. FAK is intimately related to cell movement, such as in macrophages, fibroblasts and also tumor cells. In the testis, however, FAK and two of its phosphorylated forms, p-FAK-Tyr(407) and -Tyr(397), are not found at the FAC since there is no ultrastructure analogous or similar to FAC in the mammalian testis vs. other epithelia. Instead, FAK and its two phosphorylated forms are detected along the seminiferous epithelium in the rat testis at the cell-cell interface in a testis-specific adherens junction (AJ) known as the ectoplasmic specialization (ES). ES is an F-actin-rich ultrastructure in which bundles of actin filaments are sandwiched in-between plasma membrane and cisternae of endoplasmic reticulum not found in other mammalian epithelial/endothelial cells. The ES is restricted to the interface of Sertoli cells and spermatids (step 8-19) known as the apical ES, and to the Sertoli cell-cell interface known as the basal ES. Interestingly, the basal ES is also an integrated component of the blood-testis barrier (BTB), coexisting with tight junction (TJ) and gap junction (GJ), and it is conceivable that actin filament bundles at the ES undergo extensive organization, converting from their "bundled" to "de-bundled/branching" configuration to facilitate transport of germ cells across the epithelium and at the BTB during the epithelial cycle. A recent report (Lie et al. PNAS 109:12562-12567, 2012) has demonstrated that the stage-specific and spatiotemporal expression of p-FAK-Tyr(407) and -Tyr(397) are crucial to the regulation of these events via their stage-specific and spatiotemporal expression during the epithelial cycle mediated by their effects on the organization of the actin filament bundles at the ES, involving actin binding/regulatory proteins. In this Commentary, we will critically evaluate these findings in light of other recent reports in the field. While these ideas are based on studies in the BTB in the rat testis, this information should be applicable and helpful to investigators studying other tissue barriers.
Cited By (since 1996):107, Export Date: 23 March 2014, Source: Scopus
The distinctive morphology of neurons, with complex dendritic arbors and extensive axons, presents spatial challenges for intracellular signal transduction. The endosomal system provides mechanisms that enable signaling molecules initiated by extracellular cues to be trafficked throughout the expanse of the neuron, allowing intracellular signals to be sustained over long distances. Therefore endosomes are critical for many aspects of neuronal signaling that regulate cell survival, axonal growth and guidance, dendritic branching, and cell migration. An intriguing characteristic of neuronal signal transduction is that endosomal trafficking enables physiological responses that vary based on the subcellular location of signal initiation. In this review, we will discuss the specialized mechanisms and the functional significance of endosomal signaling in neurons, both during normal development and in disease.
Skeletal muscle plays a fundamental role in mobility, disease prevention, and quality of life. Skeletal muscle mass is, in part, determined by the rates of protein synthesis, and mechanical loading is a major regulator of protein synthesis and skeletal muscle mass. The mammalian/mechanistic target of rapamycin (mTOR), found in the multi-protein complex, mTORC1, is proposed to play an essential role in the regulation of protein synthesis and skeletal muscle mass. The purpose of this review is to examine the function of mTORC1 in relation to protein synthesis and cell growth, the current evidence from rodent and human studies for the activation of mTORC1 signaling by different types of mechanical stimuli, whether mTORC1 signaling is necessary for changes in protein synthesis and skeletal muscle mass that occur in response to different types of mechanical stimuli, and the proposed molecular signaling mechanisms that may be responsible for the mechanical activation of mTORC1 signaling.
The mammalian target of rapamycin (mTOR) has emerged as a potential target for drug development, particularly due to the fact that it plays such a crucial role in cancer biology. In addition, next-generation mTOR inhibitors have become available, marking an exciting new phase in mTOR-based therapy. However, the verdict on their therapeutic effectiveness remains unclear. Here we review phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR signaling as one of the primary mechanisms for sustaining tumor outgrowth and metastasis, recent advances in the development of mTOR inhibitors, and current studies addressing mTOR activation/inhibition in colorectal cancer (CRC). We will also discuss our recent comparative study of different mTOR inhibitors in a population of colon cancer stem cells (CSCs), and current major challenges for achieving individualized drug therapy using kinase inhibitors.
Endosomal signaling is emerging as one of the most important cellular events that regulate signaling function in mammalian cells or an epithelium in response to changes in environment such as the presence of stimuli mediated by cytokines, toxicants, heat, ions during growth and development, and other cellular processes such as cytokinesis and spermatogenesis. Recent studies have shown that protein endocytosis-the initial step of endosomal signaling-involves the participation of polarity proteins, such as partitioning defective protein 6 (Par6), Cdc42 and 14-3-3 (also known as Par5), which in turn is regulated by cytokines (e.g., TGF-β2, TGF-β3) and testosterone at the Sertoli cell blood-testis barrier (BTB) in the mammalian testis. In this short method paper, we provide a detailed protocol of assessing protein endocytosis, the initial and also the most critical step of endosomal signaling at the Sertoli cell BTB. This biochemical endocytosis assay summarizes our experience for the last decade, which should likely be performed in conjunction with the dual-labeled immunofluorescence analysis to assess protein endocytosis. While we are using a Sertoli cell in vitro system that mimics the BTB in vivo, this approach should be applicable to virtually all mammalian cells.
Macrophages are best known for their protective search and destroy functions against invading microorganisms. These processes are commonly known as chemotaxis and phagocytosis. Both of these processes require actin cytoskeletal remodeling to produce distinct F-actin-rich membrane structures called lamellipodia and phagocytic cups. This review will focus on the mechanisms by which macrophages regulate actin polymerization through initial receptor signaling and subsequent Arp2/3 activation by nucleation-promoting factors like the WASP/WAVE family, followed by remodeling of actin networks to produce these very distinct structures.
The role of the epididymis as a quality-control organ in preventing infertile gametes entering the ejaculate has been extensively explored, and it has been suggested that a specific region of mammalian epididymis is able to phagocytose abnormal germ cells. This study examines whether the epithelium of certain zones of the mouse epididymis can act as a selection barrier by removing immature germ cells from the lumen by phagocytosis. To detect the presence of immature germ cells in the epididymis, we generated transgenic mice expressing EGFP under the Deleted in Azoospermia Like (mDazl) promoter to easily identified immature germ cells under fluorescence microscopy. Using this technique, we observed that during the first stage of spermatogenesis in prepuberal mice, a wave of immature germ cells is released into the epididymis and that the immature epididymis is not able to react to this abnormal situation. In contrast, when immature germ cells were artificially released into the epididymis in adult mice, a phagocytic response was observed. Phagosomes appeared inside principal cells of the epididymal epithelium and were observed to contain immature germ cells at different degradation stages in different zones of the epididymis, following the main wave of immature germ cells. In this paper, we describe how the epididymal epithelium controls sperm quality by clearing immature germ cells in response to their artificially induced massive shedding into the epididymal lumen. Our observations indicate that this phenomenon is not restricted to a given epididymis region and that phagocytic capacity is gradually acquired during epididymal development.
Spatio-temporal coordination of events during cell division is crucial for animal development. In recent years, emerging data have strengthened the notion that tight coupling of cell cycle progression and cell polarity in dividing cells is crucial for asymmetric cell division and ultimately for metazoan development. Although it is acknowledged that such coupling exists, the molecular mechanisms linking the cell cycle and cell polarity machineries are still under investigation. Key cell cycle regulators control cell polarity, and thus influence cell fate determination and/or differentiation, whereas some factors involved in cell polarity regulate cell cycle timing and proliferation potential. The scope of this review is to discuss the data linking cell polarity and cell cycle progression, and the importance of such coupling for asymmetric cell division. Because studies in model organisms such as Caenorhabditis elegans and Drosophila melanogaster have started to reveal the molecular mechanisms of this coordination, we will concentrate on these two systems. We review examples of molecular mechanisms suggesting a coupling between cell polarity and cell cycle progression.
The blood-testis barrier (BTB) is an important ultrastructure for spermatogenesis. Delay in BTB formation in neonatal rats or its irreversible damage in adult rats leads to meiotic arrest and failure of spermatogonial differentiation beyond type A. While hormones, such as testosterone and FSH, are crucial to BTB function, little is known if there is a local regulatory mechanism in the seminiferous epithelium that modulates BTB function. Herein, we report that collagen α3(IV) chain, a component of the basement membrane in the rat testis, could generate a noncollagenous (NC1) domain peptide [Colα3(IV) NC1] via limited proteolysis by matrix metalloproteinase-9 (MMP-9), and that the expression of MMP-9 was upregulated by TNFα. While recombinant Colα3(IV) NC1 protein produced in E. coli failed to perturb Sertoli cell tight junction (TJ)-permeability barrier function, possibly due to the lack of glycosylation, Colα3(IV) NC1 recombinant protein produced in mammalian cells and purified to apparent homogeneity by affinity chromatography was found to reversibly perturb the Sertoli cell TJ-barrier function. Interestingly, Colα3(IV) NC1 recombinant protein did not perturb the steady-state levels of several TJ- (e.g., occludin, CAR, JAM-A, ZO-1) and basal ectoplasmic specialization- (e.g., N-cadherin, α-catenin, β-catenin) proteins at the BTB but induced changes in protein localization and/or distribution at the Sertoli cell-cell interface in which these proteins moved from the cell surface into the cell cytosol, thereby destabilizing the TJ function. These findings illustrate the presence of a local regulatory axis known as the BTB-basement membrane axis that regulates BTB restructuring during spermatogenesis.
Dendritic cell (DC) activation is essential for the induction of immune defense against pathogens, yet needs to be tightly controlled to avoid chronic inflammation and exaggerated immune responses. Here, we identify a mechanism of immune homeostasis by which adaptive immunity, once triggered, tempers DC activation and prevents overreactive immune responses. T cells, once activated, produced Protein S (Pros1) that signaled through TAM receptor tyrosine kinases in DCs to limit the magnitude of DC activation. Genetic ablation of Pros1 in mouse T cells led to increased expression of costimulatory molecules and cytokines in DCs and enhanced immune responses to T cell-dependent antigens, as well as increased colitis. Additionally, PROS1 was expressed in activated human T cells, and its ability to regulate DC activation was conserved. Our results identify a heretofore unrecognized, homeostatic negative feedback mechanism at the interface of adaptive and innate immunity that maintains the physiological magnitude of the immune response.
Spermatogenesis occurs within the seminiferous tubules of the testis, in close association with the somatic cells of the seminiferous epithelium, the Sertoli cells. At the completion of spermatogenesis, mature spermatids are released from the Sertoli cells into the seminiferous tubule lumen, and proceed through the excurrent duct system, known as the rete testis, until they enter the epididymis via the efferent ducts. The duration of the proliferative period and the number of Sertoli cells produced, together with the subsequent maturation period, determines the spermatogenic potential of the testis, with each Sertoli cell capable of supporting a finite number of germ cells. The endocrine regulation of spermatogenesis is accomplished via a classic negative feedback loop involving interactions between the hypothalamus, pituitary, and testis (the hypothalamic-pituitary-testis, or HPT, axis). The production of spermatozoa is dependent on stimulation by the pituitary gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), which are secreted in response to hypothalamic gonadotropinreleasing hormone (GnRH).
We have studied the presence and distribution of β 1 integrins in the seminiferous epithelium of prepubertal and adult rats. Our immunofluorescence data show that in the adult the antibody recognizes specific areas localized around the heads of elongating and maturing spermatids and above spermatogonia at stages I-VII. The following were found to be negative: a) areas adjacent to spermatogonia at stages IX-XIV and adjacent to spermatocytes and to round spermatids; b) spermiated spermatozoa. In the prepubertal rat, positive tubules are first apparent around Day 17 of age. Immunofluorescence and immunoprecipitation studies show that Sertoli cell monolayers from 3-wk-old rats express β integrins in vitro.
Potential daily sperm production per gram of testicular parenchyma (PDSP/g) based on pachytene plus diplotene primary spermatocytes was determined by two methods for 10 men aged 26 to 53 years and compared with published daily sperm production per gram parenchyma (DSP/g) based on round spermatid nuclei for these same men. The PDSP/g based on primary spermatocytes was similar (P greater than 0.05) whether determined by histometric analysis (10.0 +/- 1.1 X 10(6] or from homogenates of parenchyma (10.4 +/- 1.0 X 10(6]. When PDSP/g values were compared to DSP/g (5.9 +/- 0.9 X 10(6) for the histometric or 5.5 +/- 0.8 X 10(6) for the homogenate method), 44.9 +/- 6.7% loss of potential sperm production during postprophase of meiosis was detected by the histometric approach and 48.3 +/- 7.9% loss by the homogenate method. Similar results were obtained when testes of 15 additional men aged 26 to 53 years were evaluated by the homogenate method; PDSP/g was 10.6 +/- 0.6 X 10(6), DSP/g was 5.8 +/- 0.5 X 10(6), and the loss during postprophase of meiosis was 45.3 +/- 4.4%. For all 25 men, DSP/g was significantly (P less than 0.01) correlated with PDSP/g (r = + 0.70) and with the percentage loss of potential sperm production during postprophase of meiosis (r = 0.86). Over 73% of the variation in DSP/g could be attributed to variation in the percentage loss during postprophase of meiosis. Whether faulty meiotic divisions and/or degenerating secondary spermatocytes were responsible, almost half of the potential sperm production in these 25 men was lost during postprophase of meiosis.
Native ferritin was injected into the rete testis of rats, and seminiferous tubules infused with the tracer were collected 6 h later and prepared for electron microscopic analysis. As a result of internalization of the tracer by Sertoli cells, label was found within 12-66% of the secondary lysosomes, depending on the stage of the cycle of the seminiferous epithelium. The Zeiss MOP-3 instrument was used on selected electron microscope photographs to measure a number of morphometric parameters. Applying appropriate formulae and a computerized program, it was possible to determine the absolute numbers of labeled and unlabeled secondary lysosomes per Sertoli cell for each one of the 14 stages of the cycle. Knowing the duration of these stages, it was also possible to evaluate the turnover kinetics and life span of lysosomes for each stage of the cycle. The percentage of ferritin-labeled lysosomes, regarded as an index of the endocytic activity of Sertoli cells, remained low in stages II to VIII, increased abruptly during stage IX, stayed high during stages X to XIV, and decreased to a low level during stage I of the following cycle. Correspondingly, the turnover of secondary lysosomes was relatively slow and their life span relatively long during stages II through VIII, while the turnover of lysosomes was faster and their life span shorter during stages X through XIV-I of the cycle. During stage IX, there was a sharp drop in the number of lysosomes per Sertoli cell associated with a fast rate of disappearance and a remarkably short life span of less than 4 h for the lysosomes. These features, characteristic of stage IX, are explained by the rapid fusion of lysosomes with residual bodies, which are phagocytosed by Sertoli cells at this particular stage of the cycle. The accelerated endocytosis taking place during stages IX through XIV of the cycle may explain the reduction of the surface area of the adluminal plasma membrane of Sertoli cells as well as the reduction in volume of the tubular lumen observed during these stages. Thus, the demonstrated cyclic endocytic activity of Sertoli cells and several other cyclical events taking place within seminiferous tubules correlate well.
We have examined the effect of the distribution of anti-immunoglobulin IgG molecules on the surface of bone marrow-derived lymphocytes upon the interaction of these cells with macrophages. Lymphocytes which were diffusely coated with antibodies to surface immunoglogulin were ingested by macrophages. Lymphocytes which had the same number of anti-immunoglobulin IgG molecules redistributed to one pole of the surface bound to the macrophages' Fc receptors but were not ingested. These results confirm our previous hypothesis that ingestion of an immunologically coated particle requires the sequential, circumferential binding of specific receptors on the plasma membrane of a phagocytic cell to immunologic ligands distributed over the entire particle surface. Macrophages which had bound capped lymphocytes by the macrophages' Fc receptors removed the immune complex caps from the lymphocyte surface without destroying the lymphocytes. These lymphocytes remained attached to the macrophage surface. The finding that macrophages can phagocytize immune complexes from the surface of a cell without destroying the cell to which these complexes are attached may be important in understanding the effects of antigens and antibodies on cells participating in a humoral immune response, in identifying the mechanisms by which chronic viral infections are established, and in defining the roles of blocking antibodies in tumor immunity.
Cited By (since 1996):82, Export Date: 23 March 2014, Source: Scopus
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Cited By (since 1996):51, Export Date: 23 March 2014, Source: Scopus
In the mammalian testis, the blood–testis barrier (BTB), unlike the blood–brain and blood–retina barriers, is composed of coexisting tight junctions (TJs) and adherens junctions (AJs). Yet these junctions must open (or disassemble) to accommodate the migration of preleptotene and leptotene spermatocytes across the BTB during spermatogenesis while maintaining its integrity. In this report, we show that the BTB utilizes a unique “engagement” and “disengagement” mechanism to permit the disruption of AJ that facilitates germ cell movement without compromising the BTB integrity. For instance, both TJ (e.g., occludin and JAM-1) and AJ (e.g., N-cadherin) integral membrane proteins were colocalized to the same site at the BTB. Although these TJ- and AJ-integral membrane proteins did not physically interact with each other, they were structurally linked by means of peripheral adaptors (e.g., ZO-1 and α- and γ-catenins). As such, these proteins are structurally “engaged” under physiological conditions to reinforce the BTB. When rats were exposed to Adjudin to induce AJ restructuring that eventually led to germ cell loss from the epithelium, this structural interaction between occludin and N-cadherin by means of their adaptors became “disengaged” while their protein levels were significantly induced. In short, when the epithelium is under assault, such as by Adjudin or plausibly at the time of germ cell migration across the BTB during spermatogenesis, the TJ- and AJ-integral membrane proteins can be disengaged. Thus, this mechanism is used by the testis to facilitate AJ restructuring to accommodate germ cell migration while maintaining the BTB integrity.
• spermatogenesis
• ectoplasmic specialization
• Sertoli–germ cell interaction
Cited By (since 1996):95, Export Date: 23 March 2014, Source: Scopus
Anchoring junctions are cell adhesion apparatus present in all epithelia and endothelia. They are found at the cell-cell interface (adherens junction (AJ) and desmosome) and cell-matrix interface (focal contact and hemidesmosome). In this review, we focus our discussion on AJ in particular the dynamic changes and regulation of this junction type in normal epithelia using testis as a model. There are extensive restructuring of AJ (e.g., ectoplasmic specialization, ES, a testis-specific AJ) at the Sertoli-Sertoli cell interface (basal ES) and Sertoli-elongating spermatid interface (apical ES) during the seminiferous epithelial cycle of spermatogenesis to facilitate the migration of developing germ cells across the seminiferous epithelium. Furthermore, recent findings have shown that ES also confers cell orientation and polarity in the seminiferous epithelium, illustrating that some of the functions initially ascribed to tight junctions (TJ), such as conferring cell polarity, are also part of the inherent properties of the AJ (e.g., apical ES) in the testis. The biology and regulation based on recent studies in the testis are of interest to cell biologists in the field, in particular their regulation, which perhaps is applicable to tumorigenesis.
Polarity proteins have been implicated in regulating and maintaining tight junction (TJ) and cell polarity in epithelia. Here we report 14-3-3theta, the homolog of Caenorhabditis elegans Par5 in mammalian cells, which is known to confer cell polarity at TJ, is found at the apical ectoplasmic specialization (ES), a testis-specific adherens junction type restricted to the Sertoli cell-elongating spermatid interface, in which TJ is absent. 14-3-3theta was shown to play a critical role in conferring cell adhesion at the apical ES. A loss of 14-3-3theta expression at the apical ES was detected in the seminiferous epithelium before spermiation. Involvement of 14-3-3theta in Sertoli cell adhesion was confirmed by its knockdown by RNA interference in Sertoli cells cultured in vitro with established TJ permeability barrier that mimicked the blood-testis barrier (BTB) in vivo. Mislocalization of N-cadherin and zonula occludens-1, but not alpha- and beta-catenins, was observed after 14-3-3theta knockdown in Sertoli cells, moving from the cell-cell interface to cytosol, indicating a disruption of cell adhesion. Studies by endocytosis assay illustrated that this loss of cell adhesion was mediated by an increase in the kinetics of endocytosis of N-cadherin and junctional adhesion molecule-A at the BTB, which may represent a general mechanism by which polarity proteins regulate cell adhesion. In summary, the testis is using 14-3-3theta to regulate cell adhesion at the apical ES to facilitate spermiation and at the BTB to facilitate the transit of preleptotene spermatocytes at stages VIII-IX of the epithelial cycle. 14-3-3theta may act as a molecular switch that coordinates these two cellular events in the seminiferous epithelium during spermatogenesis.
Cited By (since 1996):20, Export Date: 23 March 2014, Source: Scopus
During spermatogenesis, spermiogenesis that releases sperm into the tubule lumen and restructuring of the blood-testis barrier (BTB) that accommodates the transit of preleptotene spermatocytes take place simultaneously, but at the opposite ends of the seminiferous epithelium. These events are tightly regulated and coordinated; however, neither the underlying mechanism(s) nor the involving molecules are known. Herein, the Scribble/Lgl (Lethal giant larvae)/Dlg (Discs large) polarity complex was shown to regulate spermatid polarity during spermiogenesis and tight junction (TJ)-permeability barrier via changes in protein distribution at the apical ectoplasmic specialization and the BTB during the epithelial cycle, respectively. Scribble, Lgl2, and Dlg1 were found to be expressed by Sertoli and germ cells. Scribble also displayed stage-specific expression at the BTB, being highest at stages VII-VIII, colocalizing with TJ proteins occludin and ZO-1. Unlike components of other polarity complex modules, such as partitioning-defective 6, the knockdown of which by RNA interference was found to impede Sertoli cell TJ barrier, a knockdown of the Scribble complex (i.e. simultaneous knockdown of Scribble, Lgl and Dlg or Lgl alone; but not Scribble or Dlg alone) both in vitro and in vivo promoted the TJ integrity. This was mediated by reorganizing actin filament network at the Sertoli cell-cell interface, which, in turn, affected changes in the localization and/or distribution of occludin and/or β-catenin at the BTB. These knockdowns also perturbed F-actin organization at the Sertoli cell-spermatid interface, thereby modulating spermatid adhesion and polarity at the apical ectoplasmic specialization. In summary, the Scribble/Lgl/Dlg complex participates in the regulation of BTB dynamics and spermatid adhesion/polarity in the testis.
Objectives
The mechanistic (mammalian) targets of rapamycin (mTOR) inhibitors with known growth inhibitory effect are currently in clinical development for treatment of human cancer. The aim of this review is to discuss current progress in incorporating these new drugs as single agents or in combination with other therapeutic modalities for treatment of more common gynecologic malignancies.
Methods
A PubMed search was conducted on” mTOR inhibitors” and “human cancer”. The relevant studies published between the year 2000 to present were reviewed .Those related to gynecologic cancer(cervical, endometrial and ovarian) were selected for this manuscript. The result of published data and their clinical application in gynecologic malignancies are presented.
Results
mTOR is directly involved in many cell signaling pathways, and mTOR inhibitors have demonstrated anti-tumor activity against a variety of human malignancies, including gynecologic cancers. Combinations of mTOR inhibitors with other treatment modalities, e.g. cytotoxic chemotherapy, hormonal therapies, and other targeted molecular agents, have shown encouraging results particularly in endometrial and ovarian cancer.
Conclusions
Patients with advanced or recurrent gynecologic cancers who have failed initial treatment are in need of new treatment modalities. There is strong evidence that mTOR inhibitors limit tumor proliferation and progression. The PI3k/AKT/mTOR pathway is often deregulated in gynecologic cancer,. Patients with PIK3CA mutations are more responsive to PI3K/AKT/mTOR inhibitors than patients without these mutations. Routine screening for PIK3CA mutations warrants further investigation when PI3K/AKT/mTOR inhibitors are considered in treatment of patients with gynecologic cancer.
Statins are currently able to stabilize atherosclerotic plaques by lowering plasma cholesterol and pleiotropic effects, but a residual risk for atherosclerotic disease remains. Therefore, effective prevention of atherosclerosis and treatment of its complications is still a major clinical challenge. A large body of evidence indicates that mammalian target of rapamycin (mTOR) inhibitors such as rapamycin or everolimus have pleiotropic anti-atherosclerotic effects so that these drugs can be used as add-on therapy to prevent or delay the pathogenesis of atherosclerosis. Moreover, bioresorbable scaffolds eluting everolimus trigger a healing process in the vessel wall, both in pigs and humans, that results in late lumen enlargement and plaque regression. At present, this phenomenon of atheroregression is poorly understood. However, given that mTOR inhibitors suppress cell proliferation and trigger autophagy, a cellular survival pathway and a process linked to cholesterol efflux, we hypothesize that these compounds can inhibit (or reverse) the basic mechanisms that control plaque growth and destabilization. Unfortunately, adverse effects associated with mTOR inhibitors such as dyslipidemia and hyperglycemia have recently been identified. Dyslipidemia is manageable via statin treatment, while the anti-diabetic drug metformin would prevent hyperglycemia. Because metformin has beneficial macrovascular effects, this drug in combination with an mTOR inhibitor might have significant promise to treat patients with unstable plaques. Moreover, both statins and metformin are known to inhibit mTOR via AMPK activation so that they would fully exploit the beneficial effects of mTOR inhibition in atherosclerosis.
Laminins play a fundamental role in basement membrane architecture and function in human skin. The C-terminal laminin G domain-like (LG) modules of laminin α chains are modified by proteolysis to generate LG1-3 and secreted LG4-5 tandem modules. In this study, we provide evidence that skin-derived cells process and secrete biologically active peptides from the LG4-5 module of the laminin α3, α4 and α5 chain in vitro and in vivo. We show enhanced expression and processing of the LG4-5 module of laminin α3 in keratinocytes after infection and in chronic wounds in which the level of expression and further processing of the LG4-5 module correlated with the speed of wound healing. Furthermore, bacterial or host-derived proteases promote processing of laminin α3 LG4-5. On a functional level, we show that LG4-5-derived peptides play a role in wound healing. Moreover, we demonstrate that LG4-derived peptides from the α3, α4 and α5 chains have broad antimicrobial activity and possess strong chemotactic activity to mononuclear cells. Thus, the data strongly suggest a novel multifunctional role for laminin LG4-5-derived peptides in human skin and its involvement in physiological processes and pathological conditions such as inflammation, chronic wounds and skin infection. © 2014 S. Karger AG, Basel.
Tumor microenvironment is a complex system composed of a largely altered extracellular matrix with different cell types that determine angiogenic responses and tumor progression. Upon the influence of hypoxia, tumor cells secrete cytokines that activate stromal cells to produce proteases and angiogenic factors. In addition to stromal ECM breakdown, proteases exert various pro- or anti-tumorigenic functions and participate in the release of various ECM fragments, named matrikines or matricryptins, capable to act as endogenous angiogenesis inhibitors and to limit tumor progression.
We will focus on the matrikines derived from the NC1 domains of the different constitutive chains of basement membrane-associated collagens and mainly collagen IV. The putative targets of the matrikine control are the proliferation and invasive properties of tumor or inflammatory cells, and the angiogenic and lymphangiogenic responses. Collagen-derived matrikines such as canstatin, tumstatin or tetrastatin for example, decrease tumor growth in various cancer models. Their anti-cancer activities comprise anti-proliferative effects on tumor or endothelial cells by induction of apoptosis or cell cycle blockade and the induction of a loss of their migratory phenotype. They were used in various preclinical therapeutic strategies: i) induction of their overexpression by cancer cells or by the host cells, ii) use of recombinant proteins or synthetic peptides or structural analogues designed from the structure of the active sequences, iii) used in combined therapies with conventional chemotherapy or radiotherapy.
Collagen-derived matrikines strongly inhibited tumor growth in many preclinical cancer models in mouse. They constitute a new family of anticancer agents able to limit cancer progression.
Cells possess adaptive biosynthetic systems to maintain cellular energy levels for survival under adverse environmental conditions. Autophagy is an evolutionarily conserved cellular catabolic process that breaks down and recycles cytosolic material including macromolecules and organelles through lysosomal degradation. This catabolic process, represented by macroautophagy, is induced by a variety of cellular stresses such as nutrient starvation, which causes a shortage of cellular energy for cells to maintain cellular homeostasis and essential biological activities. In contrast, upon nutrient availability, cells stimulate anabolic processes. The mechanistic/mammalian target rapamycin (mTOR), a serine/threonine protein kinase, is a key player in stimulating cellular anabolism in response to nutrients and growth factors, and plays a crucial role in suppressing autophagy activity. Growing evidence has suggested that autophagy activity is required for the maintenance and physiological functions of renal cells including proximal tubular cells and podocytes. In this section, we will discuss recent progresses in the regulation of autophagy by the mTOR signaling.
Endocytosis entails selective packaging of cell-surface proteins, such as receptors for cytokines and adhesion components, in cytoplasmic vesicles (endosomes). The series of sorting events that determines the fate of internalized proteins, either degradation in lysosomes or recycling back to the plasma membrane, relies on intrinsic sequence motifs, posttranslational modifications (e.g., phosphorylation and ubiquitination), and transient assemblies of both Rab GTPases and phosphoinositide-binding proteins. This multicomponent process is enhanced and skewed in cancer cells; we review mechanisms enabling both major drivers of cancer, p53 and Ras, to bias recycling of integrins and receptor tyrosine kinases (RTKs). Likewise, cadherins and other junctional proteins of cancer cells are constantly removed from the cell surface, thereby disrupting tissue polarity and instigating motile phenotypes. Mutant forms of RTKs able to evade Cbl-mediated ubiquitination, along with overexpression of the wild-type forms and a variety of defective feedback regulatory loops, are frequently detected in tumors. Finally, we describe pharmacological attempts to harness the peculiar endocytic system of cancer, in favor of effective patient treatment.
Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progresses in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis. Expected final online publication date for the Annual Review of Physiology Volume 76 is February 10, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
The intracellular trafficking machinery contributes to the spatial and temporal control of signaling by receptor tyrosine kinases (RTKs). The primary role in this process is played by endocytic trafficking, which regulates the localization of RTKs and their downstream effectors, as well as the duration and the extent of their activity. The key regulatory points along the endocytic pathway are internalization of RTKs from the plasma membrane, their sorting to degradation or recycling, and their residence in various endosomal compartments. Here I will review factors and mechanisms that modulate RTK signaling by (1) affecting receptor internalization, (2) regulating the balance between degradation and recycling of RTK, and (3) compartmentalization of signals in endosomes and other organelles. Cumulatively, these mechanisms illustrate a multilayered control of RTK signaling exerted by the trafficking machinery.
Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality in humans. However, the underlying mechanism(s) remains unknown. Herein, PFOS at 10-20 μM (∼5-10 μg/ml) was found to be more potent than BPA (100 μM) in perturbing blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction (TJ)-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43 (Cx43), a GJ integral membrane protein. Third, p-FAK-Tyr(407) was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific miRNA, miR-135b, to knockdown the expression of p-FAK-Tyr(407) was found to worsen PFOS-mediated Sertoli cell TJ disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating p-FAK-Tyr(407) and Cx43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mis-localization of actin-regulatory and adhesion proteins at the BTB.
Epithelial cell carcinogenesis involves the loss of cell polarity, alteration of polarized protein presentation, dynamic cell morphology changes, increased proliferation, and increased cell motility and invasion. Membrane vesicle trafficking underlies all of these processes. Specific membrane trafficking regulators, including RAB small GTPases, through the coordinated dynamics of intracellular trafficking along cytoskeletal pathways, determine the cell surface presentation of proteins and the overall function of both differentiated and neoplastic cells. Although mutations in vesicle trafficking proteins may not be direct drivers of transformation, components of the machinery of vesicle movement have crucial roles in the phenotypes of neoplastic cells. Therefore, the regulators of membrane vesicle trafficking decisions are essential mediators of the full range of cell physiologies that drive cancer cell biology, including initial loss of cell polarity, invasion and metastasis. Targeting of these fundamental intracellular processes may permit the manipulation of cancer cell behaviour.
Spermatogenesis, the process of spermatozoa production, is regulated by several endocrine factors, including testosterone, follicle stimulating hormone, luteinizing hormone and estradiol 17β. For spermatogenesis to reach completion, developing germ cells must traverse the seminiferous epithelium while remaining transiently attached to Sertoli cells. If germ cell adhesion were to be compromised for a period of time longer than usual, germ cells would slough the seminiferous epithelium and infertility would result. Presently, Sertoli-germ cell adhesion is known to be mediated largely by classical and desmosomal cadherins. More recent studies, however, have begun to expand long-standing concepts and to examine the roles of other proteins such as intercellular adhesion molecules. In this review, we focus on the biology of intercellular adhesion molecules in the mammalian testis, hoping that this information is useful in the design of future studies.
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-Tyr397, a component of the apical ES that expressed 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 versus FAK in adult rat testes in vivo, its overexpression was found to cause defects in spermiation. These defects in spermiation was 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 mis-localization of actin regulatory proteins, Arp3 (actin-related protein 3 that induces branched actin polymerization), Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and palladin (an actin cross-linking and bundling protein); as well as adhesion proteins nectin-2 and nectin-3. 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 up-regulation of 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 to the epithelium via F-actin to confer adhesion, delaying spermiation. These findings illustrate a mechanistic pathway mediated by p-FAK-Tyr397 that regulates spermatid adhesion at the apical ES in vivo.
Cells internalize extracellular solutes, ligands, and proteins and lipids in the plasma membrane (PM) by endocytosis. The removal of membrane from the PM is counteracted by endosomal recycling pathways that return the endocytosed proteins and lipids back to the PM. Recycling to the PM can occur from early endosomes (EEs). However, many cells have a distinct subpopulation of endosomes that have a mildly acidic pH of 6.5 and are involved in the endosomal recycling. These endosomes are dubbed recycling endosomes (REs). In recent years, studies have begun to reveal that function of REs is not limited to the endosomal recycling. In this review, I summarize the nature of membrane trafficking pathways that pass through REs and the cell biological roles of these pathways.
Recently it has become clear that trafficking of integrins to late endosomes is key to the regulation of integrin expression and function during cell migration. Here we discuss the molecular machinery that dictates whether integrins are sorted to recycling endosomes or are targeted to late endosomes and lysosomes. Integrins and other receptors that are sorted to late endosomes are not necessarily degraded and, under certain circumstances, can be spared destruction and returned to the cell surface to drive cell migration and invasion. We will discuss how the exchange of adhesion receptors and other key regulators of cell migration between late endosomes/lysosomes and the plasma membrane can promote dynamic turnover of adhesions during cell migration.
Within tumor microenvironment, a lot of growth factors such as hepatocyte growth factor and epidermal growth factor may induce similar signal cascade down stream of receptor tyrosine kinase (RTK) and trigger tumor metastasis synergistically. In the past decades, the intimate relationship of RTK-mediated receptor endocytosis with signal transduction was well established. In general, most RTK undergoes clathrin-dependent endocytosis and/or clathrin-independent endocytosis. The internalized receptors may sustain the signaling within early endosome, recycling to plasma membrane for subsequent ligand engagement or sorting to late endosomes/lysosome for receptor degradation. Moreover, receptor endocytosis influences signal transduction in a temporal and spatial manner for periodical and polarized cellular processes such as cell migration. The endosomal signalings triggered by various metastatic factors are quite similar in some critical points, which are essential for triggering cell migration and tumor progression. There are common regulators for receptor endocytosis including dynamin, Rab4, Rab5, Rab11 and Cbl. Moreover, many critical regulators within the RTK signal pathway such as Grb2, p38, PKC and Src were also modulators of endocytosis. In the future, these may constitute a new category of targets for prevention of tumor metastasis.