TiO2 nanoparticles affect spermatogenesis and adhesion junctions via the ROS-mediated mTOR signalling pathway in Eriocheir sinensis testes.

Recent findings found that TiO2 nanoparticles (TiO2-NPs) have male reproductive toxicity. However, few reports have studied the toxicity of TiO2-NPs in crustaceans. In this study, we first chose the freshwater crustacean Eriocheir sinensis (E. sinensis) to explore the male toxicity of TiO2-NP exposure and the underlying mechanisms. Three nm and 25 nm TiO2-NPs at a dose of 30 mg/kg bw induced apoptosis and damaged the integrity of the haemolymph-testis-barrier (HTB, a structure similar to the blood-testis-barrier) and the structure of the seminiferous tubule. The 3-nm TiO2-NPs caused more severe spermatogenesis dysfunction than the 25-nm TiO2-NPs. We initially confirmed that TiO2-NP exposure affected the expression patterns of adherens junctions (α-catenin and ß-catenin) and induced tubulin disorganization in the testis of E. sinensis. TiO2-NP exposure caused reactive oxygen species (ROS) generation and an imbalance of mTORC1-mTORC2 (mTORC1/rps6/Akt levels were increased, while mTORC2 activity was not changed). After using the ROS scavenger NAC to inhibit ROS generation, both the mTORC1-mTORC2 imbalance and alterations in AJs were rescued. More importantly, the mTORC1 inhibitor rapamycin abolished mTORC1/rps6/Akt hyperactivation and partially restored the alterations in AJs and tubulin. Collectively, the mTORC1-mTORC2 imbalance induced by TiO2-NPs was involved in the mechanism of AJ and HTB disruption, resulting in spermatogenesis in E. sinensis.

Crosstalk Between ß-CATENIN-Mediated Cell Adhesion and the WNT Signaling Pathway.

Cell adhesion and stable signaling regulation are fundamental ways of maintaining homeostasis. Among them, the Wnt/ß-CATENIN signaling plays a key role in embryonic development and maintenance of body dynamic homeostasis. At the same time, the key signaling molecule ß-CATENIN in the Wnt signaling can also function as a cytoskeletal linker protein to regulate tissue barriers, cell migration, and morphogenesis. Dysregulation of the balance between Wnt signaling and adherens junctions can lead to disease. How ß-CATENIN maintains the independence of these two functions, or mediates the interaction and balance of these two functions, has been explored and debated for a long time. In this study, we will focus on five aspects of ß-CATENIN chaperone molecules, phosphorylation of ß-CATENIN and related proteins, epithelial mesenchymal transition, ß-CATENIN homolog protein γ-CATENIN and disease, thus deepening the understanding of the Wnt/ß-CATENIN signaling and the homeostasis between cell adhesion and further addressing related disease problems.

Neurotoxins and pore forming toxins in sea anemones: Potential candidates for new drug development.

There are two kinds of toxins in sea anemones: neurotoxins and pore forming toxins. As a representative of the sodium channel toxin, the neurotoxin ATX II in neurotoxin mainly affects the process of action potential and the release of transmitter to affect the inactivation of the sodium channel. As the representatives of potassium channel toxins, BgK and ShK mainly affect the potassium channel current. EqTx and Sticholysins are representative of pore forming toxins, which can form specific ion channels in cell membranes and change the concentration of internal and external ions, eventually causing hemolytic effects. Based on the above mechanism, toxins such as ATX II can also cause toxic effects in tissues and organs such as heart, lung and muscle. As an applied aspect it was shown that sea anemone toxins often have strong toxic effects on tumor cells, induce cancer cells to enter the pathway of apoptosis, and can also bind to monoclonal antibodies or directly inhibit relevant channels for the treatment of autoimmune diseases.

mTORC1/C2 regulate spermatogenesis in Eriocheir sinensis via alterations in the actin filament network and cell junctions.

Spermatogenesis is a finely regulated process of germ cell proliferation and differentiation that leads to the production of sperm in seminiferous tubules. Although the mammalian target of rapamycin (mTOR) signaling pathway is crucial for spermatogenesis in mammals, its functions and molecular mechanisms in spermatogenesis remain largely unknown in nonmammalian species, particularly in Crustacea. In this study, we first identified es-Raptor (the core component of mTOR complex 1) and es-Rictor (the core component of mTOR complex 2) from the testis of Eriocheir sinensis. Dynamic localization of es-Raptor and es-Rictor implied that these proteins were indispensable for the spermatogenesis of E. sinensis. Furthermore, es-Raptor and es-Rictor knockdown results showed that the mature sperm failed to be released, causing almost empty lumens in the testis. We investigated the reasons for these effects and found that the actin-based cytoskeleton was disrupted in the knockdown groups. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was impaired and affected the expression of cell junction proteins. Further study revealed that es-Raptor and es-Rictor may regulate spermatogenesis via both mTORC1- and mTORC2-dependent mechanisms that involve es-rpS6 and es-Akt/es-PKC, respectively. Moreover, to explore the testis barrier in E. sinensis, we established a cadmium chloride (CdCl2)-induced testis barrier damage model as a positive control. Morphological and immunofluorescence results were similar to those of the es-Raptor and es-Rictor knockdown groups. Altogether, es-Raptor and es-Rictor were important for spermatogenesis through maintenance of the actin filament network and cell junctions in E. sinensis.

How does retinoic acid (RA) signaling pathway regulate spermatogenesis?

Male sterility is a worldwide health problem which has troubled many unfortunate families and attracted widespread attention in the field of reproduction. Retinoic acid (RA) is a metabolite of vitamin A. Previous studies have shown that insufficient intake of vitamin A can lead to male infertility. Similarly, RA-deficiency can lead to abnormal spermatogenesis in men. RA signaling is inseparable from hormone stimulation, such as FSH, testosterone and others. It can regulate spermatogenesis as well, including the proliferation and differentiation of spermatogonia, meiosis, spermiogenesis and spermiation. To promote or inhibit spermatogenesis, RA regulates Stra8, Kit, GDNF, BMP4 and other factors in various pathways. At the self-renewal stage, RA inhibits spermatogonia renewal by directly or indirectly inhibiting DMRT, GDNF and Cyclin. At the stage of differentiation and meiosis, RA controls SSC differentiation through Kit induction and Nanos2 inhibition, and controls spermatogonia meiotic entry through up- regulation of Stra8. At the stage of spermiogenesis, RARα945;, as a key regulator, regulates spermatogenesis by up regulating Stra8 while binding with RA. Although RA plays an important role in all stages of spermatogenesis, RA signaling is more important in the early stage of spermatogonia (spg) differentiation and spermatocyte(spc) meiosis. According to the principle of RA signaling that regulates spermatogenesis, we also speculate on the future clinical application of RA, such as potential induction of SSC in vitro, contraception and restoring spermatogenesis. This paper reviews the regulatory pathways of RA, and prospects the clinical applications of RA signaling in the future.

Regulation of spermatogonial stem cell self-renewal and proliferation in mammals.

The generation of functional sperm relies on spermatogonial stem cells (SSCs) as they can maintain a stem cell pool for continuous generation of functional spermatozoa. The maintenance of SSCs is regulated by several factors. In this paper, we summarize the niche and intrinsic factors in regulating SSC self-renewal and proliferation. GDNF regulates SSC self-renewal through Ras-ERK1/2, SFC, PI3K/Akt and MEK/ERK-mTOR signaling pathways. FGF activates MAPK2K1, ERK and Akt pathways and EGF activates ERK and Akt pathways to induce SSC proliferation. Wnt ligands regulate SSC self-renewal and proliferation through both ß-catenin dependent and independent pathways. SCF1 and CXCL12 are also found to have roles in SSC maintenance. As for intrinsic factors in SSCs, ETV5, Bcl6b, Lhx1, ID4 and Nanos2 are regulated by niche factors. They act as the downstream factors of niche factors in regulating SSC self-renewal and proliferation. Transcriptional factors OCT4 and PLZF, as well as FOXO1 in SSCs can directly regulate SSC self-renewal and proliferation. Although we have identified the factors, the detailed mechanism of these factors in regulating SSC fate determination is largely unknown. Here, we summarize factors which have roles in SSC fate determination and hope it will be beneficial for further study and treatment of male infertility.

The PI3K/AKT signaling pathway: How does it regulate development of Sertoli cells and spermatogenic cells?

The PI3K/AKT signaling pathway is one of the most crucial regulatory mechanisms in animal cells, which can mainly regulate proliferation, survival and anti-apoptosis in cell lines. In the seminiferous epithelium, most studies were concentrated on the role of PI3K/AKT signaling in immature Sertoli cells (SCs) and spermatogonia stem cells (SSCs). PI3K/AKT signaling can facilitate the proliferation and anti-apoptosis of immature Sertoli cells and spermatogenic cells. Besides, in mature Sertoli cells, this pathway can disintegrate the structure of the blood-testis barrier (BTB) via regulatory protein synthesis and the cytoskeleton of Sertoli cells. All of these effects can directly and indirectly maintain and promote spermatogenesis in male testis.

Titanium dioxide nanoparticles perturb the blood-testis barrier via disruption of actin-based cell adhesive function.

As one of the most commonly used nanoparticles, titanium dioxide nanoparticles (TiO2-NPs) are widely used as coating reagents in cosmetics, medicine and other industries. The increasing risk of exposure to TiO2-NPs raises concerns about their safety. In this study, we investigated the mechanism by which TiO2-NPs cross the blood-testis barrier (BTB). TM-4 cells were selected as an in vitro Sertoli cell model of BTB. Cell viability, cell morphological changes, apoptosis, oxidative damage, and the expression levels of actin regulatory and tight junction (TJ) proteins were assessed in TM-4 cells treated with 3-nm and 24-nm TiO2-NPs. Cells treated with 3-nm TiO2-NPs exhibited increased cytotoxicity and decreased Annexin II expression, whereas cells treated with 24-nm TiO2-NPs exhibited increased Arp 3 and c-Src expression. Both TiO2-NPs induced significant oxidative stress, decreased the expression of TJ proteins (occludin, ZO-1 and claudin 5), damaged the TJ structure, and exhibited enlarged gaps between TM-4 cells. Our results indicated that both TiO2-NPs crossed the BTB by disrupting actin-based adhesive junctions of TM-4 cells; however, apoptosis was not observed. Our results provide new insights into how TiO2-NPs cross the BTB.

PIWIs maintain testis apoptosis to remove abnormal germ cells in Eriocheir sinensis.

PIWI proteins play important roles in germline development in the mammals. However, the functions of PIWIs in crustaceans remain unknown. In the present study, we identified three Piwis from the testis of Eriocheir sinensis (E. sinensis). Three Piwi genes encoded proteins with typical features of PIWI subfamilies and were highly expressed in the testis. Three PIWIs could be detected in the cytoplasm of spermatocytes and spermatids, while in spermatozoa, we could only detect PIWI1 and PIWI3 in the nucleus. The knockdown of PIWIs by dsRNA significantly affected the formation of the nuclei in spermatozoa, which resulted in deviant and irregular nuclei. PIWI defects significantly inhibited the apoptosis of abnormal germ cells through the caspase-dependent apoptosis pathway and p53 pathway. Knockdown of PIWIs inhibited the expression of caspase (Casp) 3, 7, 8, and p53 without affecting Bcl2 (B-cell lymphoma gene 2), Bax (B-cell lymphoma-2-associated X), and BaxI (B-cell lymphoma-2-associated X inhibitor), which further significantly increased abnormal spermatozoa in the knockdown-group crabs. These results show a new role of PIWI proteins in crustaceans that is different from that in mammals. In summary, PIWIs play roles in the formation of the germline nucleus and can maintain apoptosis in abnormal germ cells to remove abnormal germ cells in E. sinensis.

Inhibition of kinesin motor protein KIFC1 by AZ82 induces multipolar mitosis and apoptosis in prostate cancer cell.

Kinesin 14 family member KIFC1 is a mitotic kinesin which contains a C-terminal motor domain and plays a vital role for clustering the amplified centrosomes. Overexpression of KIFC1 in prostate cancer (PCa) cells showed resistance to docetaxel (DTX). The present study revealed that small KIFC1 inhibitor AZ82 suppresed the transcription and translation of KIFC1 significantly in PCa cells. AZ82 inhibited the KIFC1 expression both in the cytoplasm and nucleus of PCa cells. Inhibition of KIFC1 by AZ82 caused multipolar mitosis in PCa cells via de-clustering the amplified centrosomes and decreased the rate of cancer cell growth and proliferation. Moreover, depletion of KIFC1 reduced cells entering the cell cycle and caused PCa cells death through apoptosis by increasing the expression of Bax and Cytochrome C. Thereby, KIFC1 silencing and inhibition decreased the PCa cells survival by inducing multipolar mitosis as well as apoptosis, suggesting inhibition of KIFC1 using AZ82 might be a strategy to treat PCa by controlling the cancer cell proliferation.

Nanoparticles induce autophagy via mTOR pathway inhibition and reactive oxygen species generation.

Due to their unique physicochemical properties, nanoparticles (NPs) have been increasingly developed for use in various fields. However, there has been both growing negative concerns with toxicity and positive realization of opportunities in nanomedicine, coming from the growing understanding of the associations between NPs and the human body, particularly relating to their cellular autophagic effects. This review summarizes NP-induced autophagy via the modulation of the mTOR signaling pathway and other associated signals including AMPK and ERK and also demonstrates how reactive oxygen species generation greatly underlies the regulation processes. The perspectives in this review aim to contribute to NP design, particularly in consideration of nanotoxicity and the potential for the precise application of NPs in nanomedicine.

Bone morphogenetic protein 2 (BMP2) mediates spermatogenesis in Chinese mitten crab Eriocheir sinensis by regulating kinesin motor KIFC1 expression.

The TGF-beta superfamily is widely involved in cell events such as cell division and differentiation, while bone morphogenetic proteins (BMPs) belong to one of the subgroups. Their functions in crustacean spermatogenesis are still unknown. In this study, we first identified the bone morphogenetic protein 2 (bmp2) from Eriocheir sinensis (E. sinensis) testis. The es-BMP2 shows high expression in E. sinensis testis. We found that es-BMP2 is expressed in spermatids. The successfully knockdown of es-BMP2 through in vivo RNAi are used for functional analysis. Compared with the control group, the proportion of abnormal nuclear cup morphology in mature spermatozoa increased significantly after es-bmp2 RNAi, suggesting that es-BMP2 plays an important role in mature sperm morphogenesis. Immunofluorescence results confirm this finding. In order to study the specific mechanism of es-BMP2 involved in spermiogenesis, we tested kinesin-14 KIFC1, which functions in the nucleus formation of spermatozoa in E. sinensis. The results showed that knockdown of es-BMP2 caused a significant decrease of es-KIFC1 expression. We further performed es-bmp2 knockdown in vitro in primary cultured testis cells. es-KIFC1 expression was significantly reduced after es-bmp2 RNAi. The above results indicate that es-BMP2 participates in maintaining the spermiogenesis of E. sinensis by regulating es-KIFC1 expression.

Molecular insights into hormone regulation via signaling pathways in Sertoli cells: With discussion on infertility and testicular tumor.

Spermatogenesis is a complex and elaborate differentiation process and is critical for male fertility. The hypothalamic-pituitary-gonadal axis serves as a significant neuroendocrine system to regulate spermatogenesis. As a constitute of the hypothalamic-pituitary-gonadal axis, Sertoli cells promote spermatogenesis via protecting, nourishing, and supporting germ cells upon hormone determination. Here we clarified how the hormones in the hypothalamic-pituitary-gonadal axis, including FSH, testosterone and LH, regulate spermatogenesis via the androgen receptor, cAMP/PKA, PI3k/Akt signaling pathways in Sertoli cells. Other endogenous hormones in higher vertebrates, including ouabain, estradiol, leptin, MIS, PGD2, and thyroid hormone, also regulate spermatogenesis via the AR or cAMP/PKA signaling pathway. Among them, the dynamics of adherens junctions, gap junctions, and blood-testis barrier, glucose uptake, lactate supply and differentiation of Sertoli cells are regulated by more comprehensive hormones and signaling pathways in Sertoli cells. In infertile patients or patients with blocked spermatogenesis, the AR, cAMP/PKA and PI3k/Akt signaling pathways and related components exhibit abnormal activity or disordered content. The clinical specimens from patients with testicular cancer show similar mutated AR genes. According to the existing clinical evidence, it is valuable to study the deep mechanism of male infertility and testicular tumors from the perspective of hormones and signaling pathways in Sertoli cells.

Multiple signaling pathways in Sertoli cells: recent findings in spermatogenesis.

The functions of Sertoli cells in spermatogenesis have attracted much more attention recently. Normal spermatogenesis depends on Sertoli cells, mainly due to their influence on nutrient supply, maintenance of cell junctions, and support for germ cells' mitosis and meiosis. Accumulating evidence in the past decade has highlighted the dominant functions of the MAPK, AMPK, and TGF-ß/Smad signaling pathways during spermatogenesis. Among these pathways, the MAPK signaling pathway regulates dynamics of tight junctions and adherens junctions, proliferation and meiosis of germ cells, proliferation and lactate production of Sertoli cells; the AMPK and the TGF-ß/Smad signaling pathways both affect dynamics of tight junctions and adherens junctions, as well as the proliferation of Sertoli cells. The AMPK signaling pathway also regulates lactate supply. These signaling pathways combine to form a complex regulatory network for spermatogenesis. In testicular tumors or infertile patients, the activities of these signaling pathways in Sertoli cells are abnormal. Clarifying the mechanisms of signaling pathways in Sertoli cells on spermatogenesis provides new insights into the physiological functions of Sertoli cells in male reproduction, and also serves as a pre-requisite to identify potential therapeutic targets in abnormal spermatogenesis including testicular tumor and male infertility.

The dynamics and regulation of chromatin remodeling during spermiogenesis.

The functional sperm is the key factor for species continuation. The process spermatogenesis, to produce mature sperm is quite complex. It begins with the proliferation and differentiation of spermatogonia, which develop from primary spermatocytes to secondary spermatocytes and round spermatids, which eventually develop into fertile mature sperm. Spermiogenesis is the latest stage of spermatogenesis, where the round spermatids undergo a series of dramatic morphological changes and extreme condensation of chromatin to construct mature sperm with species-specific shape. During spermiogenesis, chromatin remodeling is a unique progress. It leads the nucleosome from a histone-based structure to a mostly protamine-based configuration. The main events of chromatin remodeling are the replacement of histone by histone variants, hyperacetylation, transient DNA strand breaks and repair, variants by transition proteins and finally by protamines. In this review, we synthesize and summarize the current knowledge on the progress of chromatin remodeling during spermiogenesis. We straighten out the chronological order of chromatin remodeling and illustrate the possible regulation mechanisms of each step.

KIFC1 is essential for normal spermatogenesis and its depletion results in early germ cell apoptosis in the Kuruma shrimp, Penaeus (Marsupenaeus) japonicus.

In order to explore the dynamic mechanisms during spermatogenesis of the penaeid prawns, the full length of kifc1 was cloned from testis cDNA of Penaeus japonicus through RACE. Both semi-quantitative RT-PCR and Western blot results indicated that KIFC1 was extensive expressed in different tissue of P. japonicus. Compared with other tissue, the highest expression of KIFC1 occurred in the testis. According to the immunofluorescence results, the KIFC1 protein was detected at each stage of whole process of spermatogenesis. In the spermatogonial phase, KIFC1 mainly dispersed in cytoplasm and co-localized with microtubules, while abundant KIFC1 signal was detected in the nucleus of spermatocytes. At the early stage of spermatids, KIFC1 was transported from the nucleus into the cytoplasm, and it assisted microtubule assembly onto one side of the nucleus. Finally, in mature sperm, it was weakly expressed in the acrosome. This implies that KIFC1 may participate in the mitosis of spermatogonia, meiosis of spermatocyte, and acrosome formation during spermiogenesis; it may also play functions in acrosome maintaining in mature sperm. In addition, the results of KIFC1 knockdown by dsRNA injection in vivo reveal that decreased KIFC1 expression may induce aberrant microtubule assembly, and it leads to spermatogonia and spermatocyte apoptosis.

The multiple functions of kinesin-4 family motor protein KIF4 and its clinical potential.

Human KIF4 is a member of Kinesin-4 kinesin family. The highly conserved structure contains an N-terminal motor region, coiled-coil region and C-terminal loading region. KIF4 plays important roles in DNA repair and DNA replication, which maintains genetic stability. KIF4 is also essential for regulation of mitosis and meiosis. KIF4 cooperates with condensin I and TopoIIα to help with chromosomal condensation, and binds to a plethora of cell-cycle proteins to regulate spindle organization and cytokinesis. Additionally, KIF4 plays roles in germ plasm aggregation and radial order in germ cells. In neuronal cells, KIF4 promotes proper axon growth by transporting substrates P0 and L1 to their proper location. Interestingly, KIF4 is abnormally expressed in a variety of cancers, where KIF4 is often up-regulated but can also be down-regulated in some cancers. This suggests distinctive regulatory mechanisms for different cancers. Recent studies support important roles for KIF4 in cancers such as the promotion of drug resistance or inhibition of apoptosis. Previous studies showed that by inhibiting or enhancing the expression of KIF4, the proliferation of cancer cells can be significantly reduced. Therefore KIF4 has potential as a therapeutic target for cancer therapy. Moreover, the misregulation of KIF4 is related to viral infection and neural system diseases like Alzheimer. We believe better understanding of this protein will help us develop better therapies for the diseases mentioned above. Here, we summarize KIF4 functions in normal cells and in various cancers, and provide an overview on the association between KIF4 disorders and disease progression.

Gene expression pattern of KIFC3 during spermatogenesis of the skink Eumeces chinensis.

Kinesin superfamily is a class of microtubule-dependent motors that play crucial roles in acrosome biogenesis, nuclear reshaping and flagellum formation during spermiogenesis. We have cloned kinesin-like gene kifc3 (termed ec-kifc3) from the total RNA of the testis of the skink Eumeces chinensis. The cDNA sequence of ec-kifc3 had a full-length of 3033bp, including a 260bp 5'-untranslated region (5'UTR), a 445bp 3'-untranslated region (3'UTR) and an open reading frame that encoded a 775-amino-acid protein. Additionally, the calculated molecular weight of the putative ec-KIFC3 was 87kDa and its estimated isoelectric point was 6.18. Structurally, the putative ec-KIFC3 had three domains: head domain, neck domain and tail domain. Protein alignment demonstrated that ec-KIFC3 had 47.2%, 67.8%, 68.8%, 69.3% and 76.8% identity with its homologues in Xenopus laevis, Mus musculus, Cricetulus griseus, Homo sapiens, and Gallus gallus. The phylogenetic analysis showed that ec-KIFC3 was more related to KIFC3 in vertebrates than invertebrates. Tissue expression results showed the presence of ec-KIFC3 in various tissues with its highest expression in the testis. In situ hybridization demonstrated that ec-KIFC3 mRNA was distributed around the nucleus in early and middle stage spermatids and expressed in the nucleus in the elongating spermatids during spermiogenesis. Besides, the ec-KIFC3 mRNA was expressed in the acrosome of the developmental spermatids. From the results of in situ hybridization and previous researches, we speculated that ec-KIFC3 may play a role in nuclear morphogenesis and acrosome formation during spermiogenesis of E. chinensis.

Microwave-assisted one-step extraction-derivatization for rapid analysis of fatty acids profile in herbal medicine by gas chromatography-mass spectrometry.

A rapid and practical microwave-assisted one-step extraction-derivatization (MAED) method was developed for gas chromatography-mass spectrometry analysis of fatty acids profile in herbal medicine. Several critical experimental parameters for MAED, including reaction temperature, microwave power and the amount of derivatization reagent (methanol), were optimized with response surface methodology. The results showed that the chromatographic peak areas of total fatty acids and total unsaturated fatty acids content obtained with MAED were markedly higher than those obtained by the conventional Soxhlet or microwave extraction and then derivatization method. The investigation of kinetics and thermodynamics of the derivatization reaction revealed that microwave assistance could reduce activation energy and increase the Arrhenius pre-exponential factor. The MAED method simplified the sample preparation procedure, shortened the reaction time, but improved the extraction and derivatization efficiency of lipids and reduced ingredient losses, especially for the oxidization and isomerization of unsaturated fatty acids. The simplicity, speed and practicality of this method indicates great potential for high throughput analysis of fatty acids in natural medicinal samples.