ArticleLiterature Review

The dynamics and regulation of microfilament during spermatogenesis

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Abstract

Spermatogenesis is a highly complex physiological process which contains spermatogonia proliferation, spermatocyte meiosis and spermatid morphogenesis. In the past decade, actin binding proteins and signaling pathways which are critical for regulating the actin cytoskeleton in testis had been found. In this review, we summarized 5 actin-binding proteins that have been proven to play important roles in the seminiferous epithelium. Lack of them perturbs spermatids polarity and the transport of spermatids. The loss of Arp2/3 complex, Formin1, Eps8, Palladin and Plastin3 cause sperm release failure suggesting their irreplaceable role in spermatogenesis. Actin regulation relies on multiple signal pathways. The PI3K/Akt signaling pathway positively regulate the mTOR pathway to promote actin reorganization in seminiferous epithelium. Conversely, TSC1/TSC2 complex, the upstream of mTOR, is activated by the LKB1/AMPK pathway to inhibit cell proliferation, differentiation and migration. The increasing researches focus on the function of actin binding proteins (ABPs), however, their collaborative regulation of actin patterns and potential regulatory signaling networks remains unclear. We reviewed ABPs that play important roles in mammalian spermatogenesis and signal pathways involved in the regulation of microfilaments. We suggest that more relevant studies should be performed in the future.

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... Spermatogenesis is a complex process that includes spermatogonium self-renewal, spermatocyte meiosis, and spermatid development (Yang and Yang 2020). Abnormal expression of genes related to spermatogenesis may lead to spermatogenic failure (Du et al. 2021;Linn et al. 2021). ...
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... mTOR generally inhibits initiation of autophagy, which is regulated by the upstream regulator AMPK. In the presence of nutrient deficiency, AMPK is activated by high AMP levels, thereby increasing the function of tuberous sclerosis Complex 1(TSC1) and TSC2 and inhibiting the activity of GTP-binding proteins Rheb and mTOR (74). The inactivation of mTOR activates the UNC-51 like autophagy activating kinase 1 (ULK1) by dephosphorylation, the ULK1 complex (ULK1/2, ATGl3, ATG101, and FIP200) is then activated to initiate autophagy (75). ...
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... Furthermore, motor protein expression of the cytoskeleton was inhibited in TM3 cells. Actually, it has been suggested that actin binding proteins, in particular motor proteins play vital roles in spermatogenesis [34][35][36][37]. Thus, we speculated that Plcz1 plays a vital role in the maintenance of cytoskeletal motility in the reproductive organs of male mice. ...
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... Spermatogenesis is a sophisticated physiological process, which mainly includes spermatogonia differentiation and spermatid morphogenesis (Yang and Yang 2020). In most mammals and birds, spermatogenesis occurs continuously (Gribbins 2011). ...
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... Spermatogenesis is a complex network of physiological processes such as spermatogonial proliferation, spermatocyte meiosis, and spermatid morphogenesis occurring in the seminiferous tubules 27 and eventually forming mature male gametes. The differentiation of spermatogonia into spermatozoa requires the participation of multiple cell types, hormones, paracrine and autocrine factors, genes, and epigenetic regulators 28,29 . Some diseases and conditions, such as obesity, psychological factors, and environmental exposure, can affect spermatogenesis and affect the offspring 28,[30][31][32] . ...
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Physical and mental health and hormonal imbalance are associated with the problems related to infertility and reproductive disorders. The rate of infertility has increased globally over the years, due to various reasons. Given the psychosocial implications of infertility and its effects on the life of the affected people, there has been an increased focus on its treatment over the last several years. Assisted reproductive technology can only solve about 50% of the cases. Moreover, it contains significant risks and does not solve the fundamental problem of infertility. As pluripotent stem cells have the potential to differentiate into almost any type of cell, they have been widely regarded as a promising option in the development of stem cell-based fertility treatments, which could even correct genetic diseases in offspring. These advancements in reproductive biotechnology present both challenges and possibilities for solving infertility problems caused by various unexplainable factors. This review briefly presents the different types of infertility disorders and the potential applications of stem cells in the treatment of these reproductive diseases.
... Spermatogenesis is a process from SSC to mature sperm through mitosis and meiosis in the seminiferous tubules (Haseeb et al., 2019;Yang and Yang, 2020). The process involves autocrine, paracrine, and other hormonal stimuli and nutrients that are supportive of the germ cells development (Ehmcke and Schlatt, 2006;Dube and Cyr, 2012). ...
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Spermatogenesis directly determines the reproductive capacity of male animals. With the development of society, the increasing pressure on people’s lives and changes in the living environment, male fertility is declining. The leaf of Eucommia ulmoides Oliv. (Eucommiae Folium, EF) was recorded in the 2020 Chinese Pharmacopoeia and was used in traditional Chinese medicine as a tonic. In recent years, EF has been reported to improve spermatogenesis, but the mechanisms of EF remain was poorly characterized. In this study, the effect of EF ethanol extract (EFEE) on spermatogenesis was tested in mice. Chemical components related to spermatogenesis in EF were predicted by network pharmacology. The biological activity of the predicted chemical components was measured by the proliferation of C18-4 spermatogonial stem cells (SSCs) and the testosterone secretion of TM3 leydig cells. The biological activity of chlorogenic acid (CGA), the active compound in EF, was tested in vivo. The cell cycle was analysed by flow cytometry. Testosterone secretion was detected by ELISA. RNA interference (RNAi) was used to detect the effect of key genes on cell biological activity. Western blotting, qRT–PCR and immunofluorescence staining were used to analyse the molecular mechanism of related biological activities. The results showed that EFEE and CGA could improve spermatogenesis in mice. Furthermore, the main mechanism was that CGA promoted SSC proliferation, self-renewal and Leydig cell testosterone secretion by promoting the expression of SHP2 and activating the downstream signaling pathways involved in these biological processes. This study provided strong evidence for elucidating the mechanism by which EF promotes the spermatogenesis in mice and a new theoretical basis for dealing with the decrease in male reproductive capacity.
... We found that the expression of p-Akt protein decreased in the testes of VC rats. Previous studies showed the same results as our study of PI3K-Akt signalling pathway regulated spermatogenesis.[40][41][42] Aquila et al. study found that estradiol could enhance phosphorylation of the protein Akt, which was reg- ...
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Objectives: To evaluate the expression, potential functions and mechanisms of long noncoding RNAs (lncRNAs) in the pathogenesis of varicocele (VC)-induced spermatogenic dysfunction. Materials and methods: We established a rat model with left experimental VC and divided rats into the sham group, the VC group, and the surgical treatment group (each group, n = 10). Haematoxylin and eosin (HE) staining and sperm quality were analysed to evaluate spermatogenesis function. LncRNA expression profiles were analysed using lncRNA-Seq (each group n = 3) and validated using quantitative real-time polymerase chain reaction (each group n = 10). Correlation analysis and gene target miRNA prediction were used to construct competing endogenous RNA network. The regulated signalling pathway and spermatogenic dysfunction of differentially expressed lncRNAs (DE lncRNAs) were validated by Western blot. Results: HE detection and sperm quality analysis showed that VC could induce spermatogenic dysfunction. Eight lncRNAs were upregulated and three lncRNAs were downregulated in the VC group compared with the sham group and surgical treatment group. The lncRNA of NONRATG002949.2, NONRATG001060.2, NONRATG013271.2, NONRATG022879.2, NONRATG023424.2, NONRATG005667.2 and NONRATG010686.2 were significantly negatively related to sperm quality, while NONRATG027523.1, NONRATG017183.2 and NONRATG023747.2 were positively related to sperm quality. The lncRNAs promote spermatogenic cell apoptosis and inhibit spermatogonia and spermatocyte proliferation and meiotic spermatocytes by regulating the PI3K-Akt signalling pathway. Conclusion: DE lncRNAs may be potential biomarkers for predicting the risk of spermatogenic dysfunction in VC and the effect of surgical repair. These DE lncRNAs promote spermatogenic dysfunction by regulating the PI3K-Akt signalling pathway.
... It is widely acknowledged that spermatogenesis is a vital and complex process during the whole process of male fertility physiology (17)(18)(19). This process requires the collaboration of numerous genes, hormones, proper temperature combined with other environmental factors. ...
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Infertility is becoming much more common and affects more couples. The past years witnessed the rapid development of the diagnosis and treatment upon infertility, which give numerous coupled more opportunities become parents. Extracellular vesicles are known as nano-sized membrane vesicles to play a major role in intracellular communication. In recent years, several basic and clinical studies have tried to investigate the correlation between the reproductive health/disorder and extracellular vesicles. However, the mechanism is still unclear. In this review, we reviewed the relationship between reproductive physiology and extracellular vesicles, and then collectively focused on the recent findings on the relationship between extracellular and infertility, and its consequent influence on the novel insight regarding the therapeutic strategies for infertility in the future clinical practice.
... In mammals, the actin cytoskeleton plays an undisputed role at several key points during this process serving as a cytoskeletal track to guide exocytic vesicles from the Golgi to the acrosome or from the manchette to the centrosome/axoneme. In addition, actin filaments are crucial for the assembly and remodeling of testis-specific structures important for spermatid development, including the acrosome-acroplaxome-manchette complex, the apical ES, and the TBCs (Lie et al. 2010b;O'Donnell et al. 2011;Upadhyay et al. 2012;Qian et al. 2014a, b;Dunleavy et al. 2019;Pleuger et al., 2020;Yang and Yang 2020). Actin dynamics is spatiotemporally regulated by different actin-binding proteins (ABPs) and some of these actin regulators have been shown to be involved in mammalian spermiogenesis. ...
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Spermiogenesis is the final stage of spermatogenesis, a differentiation process during which unpolarized spermatids undergo excessive remodeling that results in the formation of sperm. The actin cytoskeleton and associated actin-binding proteins play crucial roles during this process regulating organelle or vesicle delivery/segregation and forming unique testicular structures involved in spermatid remodeling. In addition, several myosin motor proteins including MYO6 generate force and movement during sperm differentiation. MYO6 is highly unusual as it moves towards the minus end of actin filaments in the opposite direction to other myosin motors. This specialized feature of MYO6 may explain the many proposed functions of this myosin in a wide array of cellular processes in animal cells, including endocytosis, secretion, stabilization of the Golgi complex, and regulation of actin dynamics. These diverse roles of MYO6 are mediated by a range of specialized cargo-adaptor proteins that link this myosin to distinct cellular compartments and processes. During sperm development in a number of different organisms, MYO6 carries out pivotal functions. In Drosophila, the MYO6 ortholog regulates actin reorganization during spermatid individualization and male KO flies are sterile. In C. elegans, the MYO6 ortholog mediates asymmetric segregation of cytosolic material and spermatid budding through cytokinesis, whereas in mice, this myosin regulates assembly of highly specialized actin-rich structures and formation of membrane compartments to allow the formation of fully differentiated sperm. In this review, we will present an overview and compare the diverse function of MYO6 in the specialized adaptations of spermiogenesis in flies, worms, and mammals.
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Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell–cell and Sertoli cell–spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood–testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell–cell interface, and the apical ES at the Sertoli–spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.
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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.
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Planar cell polarity (PCP) proteins confer polarization of a field of cells (e.g., elongating/elongated spermatids) within the plane of an epithelium such as the seminiferous epithelium of the tubule during spermatogenesis. In adult rat testes, Sertoli and germ cells were found to express PCP core proteins (e.g., Van Gogh-like 2 (Vangl2)), effectors, ligands and signaling proteins. Vangl2 expressed predominantly by Sertoli cells was localized at the testis-specific actin-rich ectoplasmic specialization (ES) at the Sertoli-spermatid interface in the adluminal compartment, but also Sertoli-Sertoli interface at the blood-testis barrier (BTB), and structurally interacted with actin, N-cadherin and another PCP/polarity protein Scribble. Vangl2 knockdown (KD) by RNAi in Sertoli cells cultured in vitro with an established tight junction (TJ)-permeability barrier led to BTB tightening whereas its overexpression using a full-length cDNA construct perturbed the barrier function. These changes were mediated through an alteration on the organization actin microfilaments at the ES in Sertoli cells, involving actin regulatory proteins Eps8, Arp3 and Scribble, which in turn affected the function of adhesion protein complexes at the ES during the epithelial cycle of spermatogenesis. By using Polyplus in vivo-jetPEI reagent for Vangl2 KD in vivo with high efficacy, Vangl2 KD led to changes in F-actin organization at the ES in the epithelium, impeding spermatid and phagosome transport and polarity, meiosis and BTB dynamics. For instance, step 19 spermatids remained embedded in the epithelium alongside with step 9 and 10 spermatids in stages IX-X tubules. In summary, PCP protein Vangl2 is an ES regulator in the testis.
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Maintenance of cell polarity is essential for Sertoli cell and blood-testis barrier (BTB) function and spermatogenesis, however, the signaling mechanisms that regulate the integrity of the cytoskeleton and polarity of Sertoli cells are not fully understood. Here, we demonstrate that Rictor, a core component of mechanistic target of rapamycin complex 2 (mTORC2), was expressed in the seminiferous epithelium during testicular development, and was down-regulated in a CdCl2-induced BTB damage model. We then conditionally deleted the Rictor gene in Sertoli cells and mutant mice exhibited azoospermia and were sterile as early as 3 months old. Further study revealed that Rictor may regulate actin organization via both mTORC2-dependent and mTORC2-independent mechanisms, in which the small GTPase, Rac1, and phosphorylation of the actin filament regulatory protein, Paxillin, are involved, respectively. Loss of Rictor in Sertoli cells perturbed actin dynamics and caused microtubule disarrangement, both of which accumulatively disrupted Sertoli cell polarity and BTB integrity, accompanied by testicular developmental defects, spermiogenic arrest and excessive germ cell loss in mutant mice. Together, these findings establish the importance of Rictor/mTORC2 signaling in Sertoli cell function and spermatogenesis through the maintenance of Sertoli cell cytoskeletal dynamics, BTB integrity and cell polarity.
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Branched actin networks generated by the Arp2/3 complex provide the driving force for leading edge protrusion in migrating cells. We recently identified Arpin, a protein that inhibits the Arp2/3 complex in lamellipodia. Arpin is activated by the small GTPase Rac, which triggers lamellipodium formation, and thus Arpin renders protrusions unstable. A conserved role of Arpin is to induce migrating cells to turn in different migration models. Here we investigated the mechanism by which Arpin controls directional persistence. For this analysis, we segmented migration trajectories into alternating phases of active migration and pauses, based on a speed threshold. Regardless of the threshold value, Arpin induced more frequent pausing, during which the cell was more likely to change the direction of its migration. Arpin simultaneously acts on cell speed and directional persistence, which are strongly coupled parameters. Induction of frequent pausing by Arpin is consistent with Arpin circuitry: by inhibiting the Arp2/3 complex as a response to Rac activation, Arpin antagonizes a positive feedback loop that sustains protrusions at the leading edge and maintains active migration. We propose the 'duration of active migration' as a useful proxy to measure feedbacks associated with cell migration. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
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The mechanistic target of rapamycin complex 2 (mTORC2) regulates cell survival and cytoskeletal organization by phosphorylating its AGC kinase substrates; however, little is known about the regulation of mTORC2 itself. It was previously reported that Akt phosphorylates the mTORC2 subunit SIN1 at T86, activating mTORC2 through a positive feedback loop, though another study reported that S6K phosphorylates SIN1 at the same site, inhibiting mTORC2 activity. We performed extensive analysis of SIN1 phosphorylation upon inhibition of Akt, S6K, and mTOR under diverse cellular contexts, and we found that, in all cell lines and conditions studied, Akt is the major kinase responsible for SIN1 phosphorylation. These findings refine the activation mechanism of the Akt-mTORC2 signaling branch as follows: PDK1 phosphorylates Akt at T308, increasing Akt kinase activity. Akt phosphorylates SIN1 at T86, enhancing mTORC2 kinase activity, which leads to phosphorylation of Akt S473 by mTORC2, thereby catalyzing full activation of Akt. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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Pten plays a crucial role in the stem cell maintenance in a few organs. Pten defect also causes the premature oocytes and ovary aging. We and other groups have found that the phosphatidylinositol-3-OH kinase (PI3K)-Akt signaling regulates the proliferation and differentiation of spermatogonial stem cells (SSCs). PTEN functions as a negative regulator of the PI3K pathway. Thus, we thought that the fate of SSCs might be controlled by Pten. We report that promyelocytic leukaemia zinc finger (PLZF) and undifferentiated embryonic cell transcription factor 1 (UTF1), both of which are germ cell-specific transcriptional factors, are regulated by Pten. Conditional deletion of Pten leads to reduction in PLZF expression but induction of UTF1, which is associated with SSCs depletion and infertility in males with age. Our data demonstrate that Pten is required for the long-term maintenance of SSCs and precise regulation of spermatogenesis in mouse. The finding of a Pten-regulated GFRα1(+)/PLZF(-)/UTF1(+) progenitor population provides a new insight into the precise mechanisms controlling SSC fate.
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Significance The signal-induced Akt membrane recruitment is a crucial step for its activation, but the underlying mechanism is incompletely understood. We show that ubiquitin-like protein 4A (Ubl4A) is required for insulin-induced Akt membrane translocation through direct promotion of actin-related protein 2 and 3 (Arp2/3)-dependent actin branching, thereby ensuring glycogen synthesis for neonatal survival. As a novel Arp2/3-binding protein, Ubl4A may play important roles in the translocation of other actin-binding molecules controlled by a similar mechanism, as well as a broad range of cellular functions related to the actin branching network.
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The phosphatidylinositol 3-kinase (PI3K) / AKT / mammalian target of rapamycin (mTOR) signalling pathway is hyperactivated or altered in many cancer types and regulates a broad range of cellular processes including survival, proliferation, growth, metabolism, angiogenesis and metastasis. The PI3K/AKT/mTOR pathway is regulated by a wide-range of upstream signalling proteins and it regulates many downstream effectors by collaborating with various compensatory signalling pathways, primarily with RAF/MEK/ERK. Limited clinical success of the available targeted therapeutic agents and challenges mediated by tumour heterogeneity across different cancer types emphasize the importance of alterations in PI3K/AKT/mTOR pathway in the design of effective personalized treatment strategies. Here we report a comprehensive PI3K/AKT/mTOR network that represents the intricate crosstalk between compensatory pathways, which can be utilized to study AKT signalling mechanism in detail and improve the personalized combinatorial therapeutic strategies.
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The cytoskeleton plays a central part in spatial organization of the plant cytoplasm, including the endomebrane system. However, the mechanisms involved are so far only partially understood. Formins (FH2 proteins), a family of evolutionarily conserved proteins sharing the FH2 domain whose dimer can nucleate actin, mediate the co-ordination between actin and microtubule cytoskeletons in multiple eukaryotic lineages including plants. Moreover, some plant formins contain transmembrane domains and participate in anchoring cytoskeletal structures to the plasmalemma, and possibly to other membranes. Direct or indirect membrane association is well documented even for some fungal and metazoan formins lacking membrane insertion motifs, and FH2 proteins have been shown to associate with endomembranes and modulate their dynamics in both fungi and metazoans. Here we summarize the available evidence suggesting that formins participate in membrane trafficking and endomembrane, especially ER, organization also in plants. We propose that, despite some methodological pitfalls inherent to in vivo studies based on (over)expression of truncated and/or tagged proteins, formins are beginning to emerge as candidates for the so far somewhat elusive link between the plant cytoskeleton and the endomembrane system.
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The mechanism by which the drug rapamycin inhibits the mechanistic target of rapamycin (mTOR) is of intense interest because of its likely relevance in cancer biology, aging, and other age-related diseases. While rapamycin acutely and directly inhibits mTORC1, only chronic administration of rapamycin can inhibit mTORC2 in some, but not all, cell lines or tissues. The mechanism leading to cell specificity of mTORC2 inhibition by rapamycin is not understood and is especially important because many of the negative metabolic side effects of rapamycin, reported in mouse studies and human clinical trials, have been attributed recently to mTORC2 inhibition. Here, we identify the expression level of different FK506-binding proteins (FKBPs), primarily FKBP12 and FKBP51, as the key determinants for rapamycin-mediated inhibition of mTORC2. In support, enforced reduction of FKBP12 completely converts a cell line that is sensitive to mTORC2 inhibition to an insensitive cell line, and increased expression can enhance mTORC2 inhibition. Further reduction of FKBP12 in cell lines with already low FKBP12 levels completely blocks mTORC1 inhibition by rapamycin, indicating that relative FKBP12 levels are critical for both mTORC1 and mTORC2 inhibition, but at different levels. In contrast, reduction of FKBP51 renders cells more sensitive to mTORC2 inhibition. Our findings reveal that the expression of FKBP12 and FKBP51 is the rate limiting factor that determines the responsiveness of a cell line or tissue to rapamycin. These findings have implications for treating specific diseases, including neurodegeneration and cancer, as well as targeting aging in general. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
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Ezrin, radixin, moesin and merlin (ERM) proteins are highly homologous actin-binding proteins that share extensive sequence similarity with each other. These proteins tether integral membrane proteins and their cytoplasmic peripheral proteins (e.g., adaptors, nonreceptor protein kinases and phosphatases) to the microfilaments of actin-based cytoskeleton. Thus, these proteins are crucial to confer integrity of the apical membrane domain and its associated junctional complex, namely the tight junction and the adherens junction. Since ectoplasmic specialization (ES) is an F-actin-rich testis-specific anchoring junction-a highly dynamic ultrastructure in the seminiferous epithelium due to continuous transport of germ cells, in particular spermatids, across the epithelium during the epithelial cycle-it is conceivable that ERM proteins are playing an active role in these events. Although these proteins were first reported almost 25 years and have since been extensively studied in multiple epithelia/endothelia, few reports are found in the literature to examine their role in the actin filament bundles at the ES. Studies have shown that ezrin is also a constituent protein of the actin-based tunneling nanotubes (TNT) also known as intercellular bridges, which are transient cytoplasmic tubular ultrastructures that transport signals, molecules and even organelles between adjacent and distant cells in an epithelium to coordinate cell events that occur across an epithelium. Herein, we critically evaluate recent data on ERM in light of recent findings in the field in particular ezrin regarding its role in actin dynamics at the ES in the testis, illustrating additional studies are warranted to examine its physiological significance in spermatogenesis.
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As germ cells progress through spermatogenesis, they undergo a dramatic transformation, wherein a single, diploid spermatogonial stem cell ultimately produces thousands of highly specialised, haploid spermatozoa. The cytoskeleton is an integral aspect of all eukaryotic cells. It concomitantly provides both structural support and functional pliability, performing key roles in many fundamental processes including, motility, intracellular trafficking, differentiation and cell division. Accordingly, cytoskeletal dynamics underlie many key spermatogenic processes. This review summarises the organisational and functional aspects of the four major cytoskeletal components (actin, microtubules, intermediate filaments and septins) during the various spermatogenic phases in mammals. We focus on the cytoskeletal machinery of both germ cells and Sertoli cells, and thus highlight the critical importance of a dynamic and precisely regulated cytoskeleton for male fertility.
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The IGFs are the major intratesticular factors regulating immature Sertoli cell proliferation and are, therefore, critical to establish the magnitude of sperm production. However, the intratesticular source of IGF production and the downstream signaling pathway mediating IGF-dependent Sertoli cell proliferation remain unclear. Single-cell RNA sequencing on mouse embryonic testis revealed a robust expression of Igf1 and Igf2 in interstitial steroidogenic progenitors, suggesting that IGFs exert paracrine actions on immature Sertoli cells. To elucidate the intracellular signaling mechanism that underlies the proliferative effects of IGFs on immature Sertoli cells, we have generated mice with Sertoli cell-specific deletion of the Pten gene, a negative regulator of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway, alone or together with the insulin receptor (Insr) and the IGF1 receptor (Igf1r). Although ablation of Pten appears dispensable for Sertoli cell proliferation and spermatogenesis, inactivation of Pten in the absence of Insr and Igf1r rescued the Sertoli cell proliferation rate during late fetal development, testis size, and sperm production. Overall, these findings suggest that IGFs secreted by interstitial progenitor cells act in a paracrine fashion to promote the proliferation of immature Sertoli cells through the IGF/PTEN/PI3K pathway.
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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.
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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.
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Study question: Is actin capping protein (CP) β3 involved in human spermatogenesis and male infertility? Summary answer: Human CPβ3 (hCPβ3) is expressed in testis, changes its localization dynamically during spermatogenesis, and has some association with male infertility. What is known already: The testis-specific α subunit of CP (CPα3) was previously identified in human, and mutations in the cpα3 gene in mouse were shown to induce malformation of the sperm head and male infertility. However, CPβ3, which is considered to be a heterodimeric counterpart of CPα3, has been neither characterized in human nor reported in association with male infertility. Study design, size, duration: To confirm the existence of CPβ3 in human testis, fresh semen samples from proven fertile men were analyzed. To investigate protein expression during spermatogenesis, cryopreserved testis obtained from men with obstructive azoospermia were examined by immunofluorescent analysis. To assess the association of CP with male infertility, we compared protein expression of human CPα3 (hCPα3) and hCPβ3 using immunofluorescent analysis of cryopreserved sperm between men with normozoospermia (volunteers: Normo group, n = 20) and infertile men with oligozoospermia and/or asthenozoospermia (O + A group, n = 21). Participants/materials, setting, methods: The tissue-specific expression of hCPβ3 was investigated by RT-PCR and Western blot analysis. To investigate whether hCPα3 and hCPβ3 form a heterodimer, a tandem expression vector containing hcpα3 tagged with monomeric red fluorescent protein 1 and hcpβ3 tagged with enhanced green fluorescent protein in a single plasmid was constructed and analyzed by co-immunoprecipitation (Co-IP) assay. The protein expression profiles of hCPα3 and hCPβ3 during spermatogenesis were examined by immunohistochemical analysis using human spermatogenic cells. The protein expressions of hCPα3 and hCPβ3 in sperm were compared between the Normo and O + A groups by immunohistochemical analysis. Main results and the role of chance: RT-PCR showed that mRNA of hcpβ3 was expressed exclusively in testis. Western blot analysis detected hCPβ3 with anti-bovine CPβ3 antibody. Co-IP assay with recombinant protein showed that hCPα3 and hCPβ3 form a protein complex. At each step during spermatogenesis, the cellular localization of hCPβ3 changed dynamically. In spermatogonia, hCPβ3 showed a slight signal in cytoplasm. hCPβ3 expression was conspicuous mainly from spermatocytes, and hCPβ3 localization dynamically migrated from cytoplasm to the acrosomal cap and acrosome. In mature spermatozoa, hCPβ3 accumulated in the postacrosomal region and less so at the midpiece of the tail. Double-staining analysis revealed that hCPα3 localization was identical to hCPβ3 at every step in the spermatogenic cells. Most spermatozoa from the Normo group were stained homogenously by both hCPα3 and hCPβ3. In contrast, significantly more spermatozoa in the O + A versus Normo group showed heterogeneous or lack of staining for either hCPα3 or hCPβ3 (abnormal staining) (P < 0.001). The percentage of abnormal staining was higher in the O + A group (52.4 ± 3.0%) than in the Normo group (31.2 ± 2.5%). Even by confining the observations to morphologically normal spermatozoa selected in accordance with David's criteria, the percentage of abnormal staining was still higher in the O + A group (39.9 ± 2.9%) versus the Normo group (22.5 ± 2.1%) (P < 0.001). hCPβ3 in conjunction with hCPα3 seemed to play an important role in spermatogenesis and may be associated with male infertility. Large scale data: Not applicable. Limitations reasons for caution: Owing to the difficulty of collecting fresh samples of human testis, we used cryopreserved samples from testicular sperm extraction. To examine the interaction of spermatogenic cells or localization in seminiferous tubules, fresh testis sample of healthy males are ideal. Wider implications of the findings: The altered expression of hCPα3 and hCPβ3 may not only be a cause of male infertility but also a prognostic factor for the results of ART. They may be useful biomarkers to determine the fertilization ability of human sperm in ART. Study funding/competing interests: This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JP16K20133). The authors declare no competing interests.
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Throughout the epithelial cycle of spermatogenesis, actin microfilaments arranged as bundles near the Sertoli cell plasma membrane at the Sertoli cell-cell interface that constitute the blood-testis barrier (BTB) undergo extensive re-organization by converting between bundled and unbundled/branched configuration to give plasticity to the F-actin network. This is crucial to accommodate the transport of preleptotene spermatocytes across the BTB. Herein, we sought to examine changes in the actin microfilament organization at the Sertoli cell BTB using an in vitro model since Sertoli cells cultured in vitro is known to establish a functional tight junction (TJ)-permeability barrier that mimics the BTB in vivo. Plastin 3, a known actin microfilament cross-linker and bundling protein, when overexpressed in Sertoli cells using a mammalian expression vector pCI-neo was found to perturb the Sertoli cell TJ-barrier function even though its overexpression increased the overall actin bundling activity in these cells. Furthermore, plastin 3 overexpression also perturb the proper localization and distribution of BTB-associated proteins, such as occludin-ZO1 and N-cadherin-ß-catenin, this thus destabilized the barrier function. Collectively, these data illustrate that a delicate balance of actin microfilaments between being organized in bundles vs. an unbundled/branched configuration is crucial to confer the homeostasis of the BTB and its integrity.
Article
Spermatogenesis is an extraordinary complex process. The differentiation of spermatogonia into spermatozoa requires the participation of several cell types, hormones, paracrine factors, genes and epigenetic regulators. Recent researches in animals and humans have furthered our understanding of the male gamete differentiation, and led to clinical tools for the better management of male infertility. There is still much to be learned about this intricate process. In this review, the critical steps of human spermatogenesis are discussed together with its main affecting factors.
Article
Actin remodeling is a vital process for signaling, movement and survival in all cells. In the testes, extensive actin reorganization occurs at spermatid-Sertoli cell junctions during sperm release (spermiation) and at inter Sertoli cell junctions during restructuring of the blood testis barrier (BTB). During spermiation, tubulobulbar complexes (TBCs), rich in branched actin networks, ensure recycling of spermatid-Sertoli cell junctional molecules. Similar recycling occurs during BTB restructuring around the same time as spermiation occurs. Actin related protein 2/3 complex is an essential actin nucleation and branching protein. One of its subunits, Arpc1b, was earlier found to be down-regulated in an estrogen-induced rat model of spermiation failure. Also, Arpc1b was found to be estrogen responsive through estrogen receptor beta in seminiferous tubule culture. Here, knockdown of Arpc1b by siRNA in adult rat testis led to defects in spermiation caused by failure in TBC formation. Knockdown also compromised BTB integrity and caused polarity defects of mature spermatids. Apart from these effects pertaining to Sertoli cells, Arpc1b reduction perturbed ability of germ cells to enter G2/M phase thus hindering cell division. In summary, Arpc1b, an estrogen responsive gene, is a regulator of spermiation, mature spermatid polarity, BTB integrity and cell division during adult spermatogenesis.
Article
The serine/threonine kinase mTOR forms two distinct complexes, mTORC1 and mTORC2, and controls a number of biological processes, including proliferation, survival and autophagy. Although the function of mTORC1 has been extensively studied, the mTORC2 signaling pathway largely remains to be elucidated. Here, we have shown that mTORC2 phosphorylates filamin A, an actin cross-linking protein, at serine 2152 (S2152) both in vivo and in living cells. Treatment of HeLa cells with Torin1 (an mTORC1/mTORC2 inhibitor), but not rapamycin (an mTORC1 inhibitor), suppressed the phosphorylation of filamin A, which decreased the binding of filamin A with β7-integrin cytoplasmic tail. Torin1 also inhibited focal adhesion formation and cell migration in A7 filamin A-replete melanoma cells but not in M2 filamin A-deficient cells, suggesting a pivotal role for mTORC2 in filamin A function. Finally, reduced focal adhesion formation in M2 cells was significantly rescued by expressing wild type but not S2152A nonphosphorylatable mutant of filamin A. Taken together, our results indicate that mTORC2 regulates filamin A-dependent focal adhesions and cell migration.
Article
mTOR (mammalian target of rapamycin) is one of the most important signaling molecules in mammalian cells which regulates an array of cellular events, ranging from cell metabolism to cell proliferation. Based on the association of mTOR with the core component proteins, such as Raptor or Rictor, mTOR can become the mTORC1 (mammalian target of rapamycin complex 1) or mTORC2, respectively. Studies have shown that during the epithelial cycle of spermatogenesis, mTORC1 promotes remodeling and restructuring of the blood-testis barrier (BTB) in vitro and in vivo, making the Sertoli cell tight junction (TJ)-permeability barrier "leaky"; whereas mTORC2 promotes BTB integrity, making the Sertoli cell TJ-barrier "tighter". These contrasting effects, coupled with the spatiotemporal expression of the core signaling proteins at the BTB that confer the respective functions of mTORC1 vs. mTORC2 thus provide a unique mechanism to modulate BTB dynamics, allowing or disallowing the transport of biomolecules and also preleptotene spermatocytes across the immunological barrier. More importantly, studies have shown that these changes to BTB dynamics conferred by mTORC1 and mTORC2 are mediated by changes in the organization of the actin microfilament networks at the BTB, and involve gap junction (GJ) intercellular communication. Since GJ has recently been shown to be crucial to reboot spermatogenesis and meiosis following toxicant-induced aspermatogenesis, these findings thus provide new insightful information regarding the integration of mTOR and GJ to regulate spermatogenesis.
Chapter
A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others.This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study.
Article
The xenoestrogen 4-nonylphenol (NP) induces reproductive dysfunction of male rats, but the fundamental mechanism of this phenomenon is largely unexplored. Sertoli cells (SCs) are pivotal for spermatogenesis and male fertility. The involvement of autophagy in NP-induced apoptotic and necrotic death of SCs was investigated. In this study, 24-h exposure of SCs to 20–30 μM NP decreased cell viability, caused G2/M arrest, triggered Δym loss, increased ROS production and induced caspase-dependent apoptsis, necrosis as well as autophagosome formation. NP-induced autophagy was confirmed by monodansylcadaverine-staining and LC3-I/LC3-II conversion. Furthermore, NP up-regulated the Thr172p-AMPK/AMPK and Thr183/185p-JNK/JNK ratios. This was followed by the down-regulation of Ser473p-Akt/Akt, Thr1462p-TSC2/TSC2, Ser2448p-mTOR/mTOR, Thr389p-p70S6 K/p70S6 K and Thr37/45p-4EBP1/4EBP1. Intriguingly, NP-induced apoptosis, autophagy and necrosis could be inhibited through blocking ROS generation by N-acetylcysteine. Autophagy inhibitor 3-MA enhanced NP-induced apoptosis and necrosis. Moreover, The activation of AMPK/mTOR/p70s6k/4EBP1 and JNK signalling pathways induced by NP could be efficiently reversed by pretreatment of N-acetylcysteine or 3-MA. Collectively, our findings provide the first evidence that NP promotes apoptosis, autophagy and necrosis simultaneously in SCs and that this process may involve ROS-dependent JNK- and Akt/AMPK/mTOR pathways. Modulation of autophagy induced by NP may serve as a survival mechanism against apoptosis and necrosis.
Article
The blood-testis barrier (BTB) is an important ultrastructure in the testis since the onset of spermatogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule (MT)-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases.
Article
Spermatogenesis is a critical process for maintaining male fertility. Sustained spermatogonial stem cell self-renewal and differentiation ensures constant spermatogenesis, and several signalling pathways regulate this process. An increasing number of studies have suggested that the mammalian target of rapamycin (mTOR) signalling pathway plays an important role in spermatogenesis; however, the mechanism remains unknown. Our study showed that mTOR was positively related with spermatogenesis by detecting mTOR expression and the expression of its target p70s6k, rps6 and 4e-bp1 at different developmental stages. Phosphorylated p70s6k, rps6 and 4ebp1 levels were independently and gradually down-regulated with age. Subsequently, we showed in vivo and in vitro that, upon mTOR inactivation by rapamycin, the number of sperm significantly decreased (P < 0.05) and spermatogonia proliferation was blocked. Phosphorylated p70s6k and rps6 levels were down-regulated, but the levels of phosphorylated 4e-bp1 did not change. Spermatogonia were treated with the specific PI3K inhibitor LY294002, and p70s6k, rps6 and 4ebp1 phosphorylation overtly decreased. Therefore, we suggest that mTOR plays an important role in spermatogenesis by regulating p70s6k activation and that 4e-bp1 is either directly or indirectly regulated by PI3K.
Article
The mTOR complex 2, mTORC2, is a critical downstream effector of PI3K that stimulates AGC kinase members, including AKT, PKC, and SGK. Liu and colleagues reported that the pleckstrin homology domain of SIN1, an essential component of mTORC2, directly binds the PI3K product PtdIns(3,4,5)P3 to promote mTORC2 kinase activation and membrane localization, thereby revealing a mechanistic link between PI3K and mTORC2. Cancer Discov; 5(11); 1127–9. ©2015 AACR. See related article by Liu and colleagues, p. 1194.
Article
Arp2/3 complex plays a fundamental role in the nucleation of actin filaments (AFs) in yeasts, plants, and animals. In plants, the aberrant shaping and elongation of several types of epidermal cells observed in Arp2/3 complex knockout plant mutants suggest the importance of Arp2/3-mediated actin nucleation for various morphogenetic processes. Here we show that ARPC2, a core Arp2/3 complex subunit, interacts with both actin filaments (AFs) and microtubules (MTs). Plant GFP-ARPC2 expressed in Nicotiana tabacum BY-2 cells, leaf epidermal cells of Nicotiana benthamiana and root epidermal cells of Arabidopsis thaliana decorated MTs. The interaction with MTs was demonstrated by pharmacological approach selectively interfering with either AFs or MTs dynamics as well as by the in vitro co-sedimentation assays. A putative MT-binding domain of tobacco NtARPC2 protein was identified using the co-sedimentation of several truncated NtARPC2 proteins with MTs. Newly identified MT-binding ability of ARPC2 subunit of Arp2/3 complex may represent a new molecular mechanism of AFs and MTs interaction.
Article
Formins are a growing class of actin nucleation proteins that promote the polymerization of actin microfilaments, forming long stretches of actin microfilaments to confer actin filament bundling in mammalian cells. As such, microfilament bundles can be formed in specific cellular domains, in particular in motile mammalian cells, such as filopodia. Since ectoplasmic specialization (ES), a testis-specific adherens junction (AJ), at the Sertoli cell-cell and Sertoli-spermatid interface is constituted by arrays of actin microfilament bundles, it is likely that formins are playing a significant physiological role on the homeostasis of ES during the epithelial cycle of spermatogenesis. In this Commentary, we provide a timely discussion on formin 1 which was recently shown to be a crucial regulator of actin microfilaments at the ES in the rat testis (Li N et al. Endocrinology, 2015, in press; DOI: 10.1210/en.2015-1161, PMID:25901598). We also highlight research that is needed to unravel the functional significance of formins in spermatogenesis.
Article
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. © FASEB.
Article
Testicular histological alterations following Sertoli cell cytoskeleton disruption are numerous. The Sertoli cell cytoskeleton is comprised of intermediate filaments, microtubules, microfilaments and their direct interacting proteins and performs essential functions including structural support of the seminiferous epithelium, apicobasal movement of elongate spermatids, and release of elongate spermatids from the seminiferous epithelium during spermiation. This review summarizes the histological changes occurring after disruption of the Sertoli cell cytoskeleton, including the signature lesion of seminiferous epithelium sloughing. By presenting examples of histological changes after exposure to toxins or toxicants directly affecting the Sertoli cell cytoskeleton or genetic manipulations of this cytoskeleton, the toxicologist observing similar histological changes associated with exposure to novel compounds can use this information to generate hypotheses about a potential mode of action.
Article
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.
Article
The present review examines the role of actin binding proteins (ABPs) on blood-testis barrier (BTB), an androgen-dependent ultrastructure in the testis, in particular their involvement on BTB remodeling during spermatogenesis. The BTB divides the seminiferous epithelium into the basal and the adluminal compartments. The BTB is constituted by coexisting actin-based tight junction, basal ectoplasmic specialization, and gap junction, and also intermediate filament-based desmosome between Sertoli cells near the basement membrane. Junctions at the BTB undergo continuous remodeling to facilitate the transport of preleptotene spermatocytes residing in the basal compartment across the immunological barrier during spermatogenesis. Thus, meiosis I/II and postmeiotic spermatid development take place in the adluminal compartment behind the BTB. BTB remodeling also regulates exchanges of biomolecules between the two compartments. As tight junction, basal ectoplasmic specialization, and gap junction use F-actin for attachment, actin microfilaments rapidly convert between their bundled and unbundled/branched configuration to confer BTB plasticity. The events of actin reorganization are regulated by two major classes of ABPs that convert actin microfilaments between their bundled and branched/unbundled configuration. We provide a model on how ABPs regulate BTB remodeling, shedding new light on unexplained male infertility, such as environmental toxicant-induced reproductive dysfunction since the testis, in particular the BTB, is sensitive to environmental toxicants, such as cadmium, bisphenol A, phthalates, and PFOS (perfluorooctanesulfonic acid or perfluorooctane sulfonate).
Article
In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ)-proteins via re-organization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1, participates in this process via a yet-to-be defined mechanism. Herein, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240 and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ-barrier mimicking the BTB in vivo, it perturbed the TJ-permeability by down-regulating and re-distributing TJ-proteins at the cell-cell interface. These changes are mediated by a re-organization of actin microfilaments, which was triggered by a re-distribution of activated Arp3 as well as changes in Arp3-N-WASP interaction. This in turn induced re-organization of actin microfilaments, converting them from a "bundled" to an "unbundled/branched" configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt since an Akt knockdown by RNAi was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which rpS6/mTORC1 signal complex regulates BTB function by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue-barriers.