Sperm proteins and cancer-testis antigens are released by the seminiferous tubules in mice and men.

Sperm develop from puberty in the seminiferous tubules, inside the blood-testis barrier to prevent their recognition as "non-self" by the immune system, and it is widely assumed that human sperm-specific proteins cannot access the circulatory or immune systems. Sperm-specific proteins aberrantly expressed in cancer, known as cancer-testis antigens (CTAs), are often pursued as cancer biomarkers and therapeutic targets based on the assumption they are neoantigens absent from the circulation in healthy men. Here, we identify a wide range of germ cell-derived and sperm-specific proteins, including multiple CTAs, that are selectively deposited by the Sertoli cells of the adult mouse and human seminiferous tubules into testicular interstitial fluid (TIF) that is "outside" the blood-testis barrier. From TIF, the proteins can access the circulatory- and immune systems. Disruption of spermatogenesis decreases the abundance of these proteins in mouse TIF, and a sperm-specific CTA is significantly decreased in TIF from infertile men, suggesting that exposure of certain CTAs to the immune system could depend on fertility status. The results provide a rationale for the development of blood-based tests useful in the management of male infertility and indicate CTA candidates for cancer immunotherapy and biomarker development that could show sex-specific and male-fertility-related responses.

GILZ-dependent modulation of mTORC1 regulates spermatogonial maintenance.

Male fertility is dependent on spermatogonial stem cells (SSCs) that self-renew and produce differentiating germ cells. Growth factors produced within the testis are essential for SSC maintenance but intrinsic factors that dictate the SSC response to these stimuli are poorly characterised. Here, we have studied the role of GILZ, a TSC22D family protein and spermatogenesis regulator, in spermatogonial function and signalling. Although broadly expressed in the germline, GILZ was prominent in undifferentiated spermatogonia and Gilz deletion in adults resulted in exhaustion of the GFRα1+ SSC-containing population and germline degeneration. GILZ loss was associated with mTORC1 activation, suggesting enhanced growth factor signalling. Expression of deubiquitylase USP9X, an mTORC1 modulator required for spermatogenesis, was disrupted in Gilz mutants. Treatment with an mTOR inhibitor rescued GFRα1+ spermatogonial failure, indicating that GILZ-dependent mTORC1 inhibition is crucial for SSC maintenance. Analysis of cultured undifferentiated spermatogonia lacking GILZ confirmed aberrant activation of ERK MAPK upstream mTORC1 plus USP9X downregulation and interaction of GILZ with TSC22D proteins. Our data indicate an essential role for GILZ-TSC22D complexes in ensuring the appropriate response of undifferentiated spermatogonia to growth factors via distinct inputs to mTORC1.

Ostracods had colonized estuaries by the late Silurian.

The fossil record of terrestrialization documents notable shifts in the environmental and physiological tolerances of many animal and plant groups. However, for certain significant components of modern freshwater and terrestrial environments, the transition out of marine settings remains largely unconstrained. Ostracod crustaceans occupy an exceptional range of modern aquatic environments and are invaluable palaeoenvironmental indicators in the fossil record. However, pre-Carboniferous records of supposed non-marine and marginal marine ostracods are sparse, and the timing of their marine to non-marine transition has proven elusive. Here, we reassess the early environmental history of ostracods in light of new assemblages from the late Silurian of Vietnam. Two, low diversity but distinct ostracod assemblages are associated with estuarine deposits. This occurrence is consistent with previous incidental reports of ostracods occupying marginal and brackish settings through the late Silurian and Devonian. Therefore, ostracods were pioneering the occupation of marginal marine and estuarine settings 60 Myr before the Carboniferous and they were a component of the early phase of transition from marine to non-marine environments.

RNA processing in the male germline: Mechanisms and implications for fertility.

Mammalian spermatogenesis is a tightly coordinated process that gives rise to mature spermatozoa capable of fertilising an ovum during sexual reproduction. A population of stem and progenitor cells known as undifferentiated spermatogonia enables continual spermatogenesis throughout life. A complex transcriptional network that balances self-renewal of spermatogonia with their timely differentiation in order to maintain constant fertility regulates this process. Importantly, post-transcriptional regulation of gene expression plays a critical role in spermatogenesis, necessitated by the profound genetic and morphological changes that occur during meiosis and sperm maturation. Pre-mRNA splicing, mRNA export, maintenance of transcript stability and translation are key RNA processing steps that are regulated in the male germline to maintain coordinated gene expression. In this review, we examine these processes in the context of mammalian spermatogenesis and provide an overview of key mediators at each step.

Dominant-negative Sox18 function inhibits dermal papilla maturation and differentiation in all murine hair types.

SOX family proteins SOX2 and SOX18 have been reported as being essential in determining hair follicle type; however, the role they play during development remains unclear. Here, we demonstrate that Sox18 regulates the normal differentiation of the dermal papilla of all hair types. In guard (primary) hair dermal condensate (DC) cells, we identified transient Sox18 in addition to SOX2 expression at E14.5, which allowed fate tracing of primary DC cells until birth. Similarly, expression of Sox18 was detected in the DC cells of secondary hairs at E16.5 and in tertiary hair at E18.5. Dominant-negative Sox18 mutation (opposum) did not prevent DC formation in any hair type. However, it affected dermal papilla differentiation, restricting hair formation especially in secondary and tertiary hairs. This Sox18 mutation also prevented neonatal dermal cells or dermal papilla spheres from inducing hair in regeneration assays. Microarray expression studies identified WNT5A and TNC as potential downstream effectors of SOX18 that are important for epidermal WNT signalling. In conclusion, SOX18 acts as a mesenchymal molecular switch necessary for the formation and function of the dermal papilla in all hair types.

Dielectric and Conduction Mechanisms of Parylene N at High Temperature: Phase-Transition Effect.

Dielectric and electrical properties correlated with the structure analysis have been studied on 27% semicrystalline parylene-N (-H2C-C6H4-CH2-)n thin films. Transition-phase, AC- and DC-conduction mechanisms, and the MW-interfacial polarization were identified in parylene N at high temperature by experimental and theoretical investigations. The dielectric analysis based on the dc conductivity highlights a temperature of 230 °C as a transition temperature from the α-form to the ß1-form. This structure transition is accompanied by a modification on the DC-conduction mechanisms from ionic to electronic conduction in the α-form and the ß1-form, respectively. The AC conduction mechanism is governed by the small polaron tunneling mechanism (SPTM) with WH,α = 0.23 eV and a tunneling distance of 7.71 Å in the α-form, while it becomes a correlated barrier-hopping (CBH) mechanism with a WM,ß 1 = 0.52 eV in the ß1-form. The imaginary part of the electrical modulus formalism obeys the Kohlrausch-Williams-Watt (KWW) model and shows the presence of the interfacial polarization effect. The theoretical Kohlrausch exponent (ßKWW) confirms the existence of the transition phase on the parylene N in the vicinity of the 230 °C as deduced by the DC- and the AC-conduction parameters. The correlations between the experimental results and the theoretical models are very useful knowledge and tools for diverse parylene N applications at high temperature.

Palynofloras from the upper Barremian-Aptian Nishihiro Formation (Outer Zone of southwest Japan) and the appearance of angiosperms in Japan.

Palynomorphs are reported for the first time from the Nishihiro Formation (Wakayama Prefecture, Outer Zone of southwest Japan). The Nishihiro Formation consists of brackish to shallow marine deposits, dated as late Barremian to Aptian from geological correlations. Spores prevail in the assemblage, representing Filicopsida (mainly Cyatheaceae and Anemiaceae), Marchantiopsida and Lycopsida. The pollen assemblage is dominated by Coniferales, whereas Gnetales and Bennettitales/Cycadales are only rarely observed. Moreover, we report angiosperm pollen grains of the genus Retimonocolpites for the first time in the Early Cretaceous sediments of Japan. Pollen grains of the Retimonocolpites Group are typical of early angiosperms and commonly found in assemblages from the early to mid-Cretaceous of all paleofloristic provinces. Until this paper, the oldest angiosperm fossils in Japan were represented by a single seed and a wood reported from the Albian of Hokkaido. The oldest reliable angiosperm pollen grains were reported in Hokkaido from the Cenomanian, and in Honshu from the Coniacian. Thus, Retimonocolpites pollen grains reported in the present study represent the oldest record of angiosperms in Japan. They indicate an appearance of the angiosperms in Japan older than thought until now, which is consistent with that proposed elsewhere in eastern Asia.

STIM2 is involved in the regulation of apoptosis and the cell cycle in normal and malignant monocytic cells.

Calcium is a ubiquitous messenger that regulates a wide range of cellular functions, but its involvement in the pathophysiology of acute myeloid leukemia (AML) is not widely investigated. Here, we identified, from an analysis of The Cancer Genome Atlas and genotype-tissue expression databases, stromal interaction molecule 2 (STIM2) as being highly expressed in AML with monocytic differentiation and negatively correlated with overall survival. This was confirmed on a validation cohort of 407 AML patients. We then investigated the role of STIM2 in cell proliferation, differentiation, and survival in two leukemic cell lines with monocytic potential and in normal hematopoietic stem cells. STIM2 expression increased at the RNA and protein levels upon monocyte differentiation. Phenotypically, STIM2 knockdown drastically inhibited cell proliferation and induced genomic stress with DNA double-strand breaks, as shown by increased levels of phosphorylate histone H2AXγ (p-H2AXγ), followed by activation of the cellular tumor antigen p53 pathway, decreased expression of cell cycle regulators such as cyclin-dependent kinase 1 (CDK1)-cyclin B1 and M-phase inducer phosphatase 3 (CDC25c), and a decreased apoptosis threshold with a low antiapoptotic/proapoptotic protein ratio. Our study reports STIM2 as a new actor regulating genomic stability and p53 response in terms of cell cycle and apoptosis of human normal and malignant monocytic cells.

Wound-associated macrophages control collagen 1α2 transcription during the early stages of skin wound healing.

Wound-associated fibrosis is important to provide tensile strength upon wound healing but at the same time is detrimental to proper tissue regeneration. To date, there is no clear evidence of the role of macrophages and their subpopulations in the control of the kinetics of collagen production during wound healing. To evaluate in vivo the contribution of macrophages in collagen transcription, we depleted macrophages after wounding luciferase reporter mice of the collagen 1 alpha 2 (Col 1α2) promoter activity. Our data reveal that Col 1α2 starts to be transcribed at D2 after wounding, reaching a plateau after 7 days. Sustained macrophage depletion significantly reduced collagen 1α2 transcription from D4, indicating that the control of fibrosis by macrophages occurs during the early stages of the wound healing process. In conclusion, our results demonstrate an important role of wound macrophages in the control of collagen production during wound healing.

Defining Gene Function in Spermatogonial Stem Cells Through Conditional Knockout Approaches.

Mammalian male fertility is maintained throughout life by a population of self-renewing mitotic germ cells known as spermatogonial stem cells (SSCs). Much of our current understanding regarding the molecular mechanisms underlying SSC activity is derived from studies using conditional knockout mouse models. Here, we provide a guide for the selection and use of mouse strains to develop conditional knockout models for the study of SSCs, as well as their precursors and differentiation-committed progeny. We describe Cre recombinase-expressing strains, breeding strategies to generate experimental groups, and treatment regimens for inducible knockout models and provide advice for verifying and improving conditional knockout efficiency. This resource can be beneficial to those aiming to develop conditional knockout models for the study of SSC development and postnatal function.

A superior extracellular matrix binding motif to enhance the regenerative activity and safety of therapeutic proteins.

Among therapeutic proteins, cytokines and growth factors have great potential for regenerative medicine applications. However, these molecules have encountered limited clinical success due to low effectiveness and major safety concerns, highlighting the need to develop better approaches that increase efficacy and safety. Promising approaches leverage how the extracellular matrix (ECM) controls the activity of these molecules during tissue healing. Using a protein motif screening strategy, we discovered that amphiregulin possesses an exceptionally strong binding motif for ECM components. We used this motif to confer the pro-regenerative therapeutics platelet-derived growth factor-BB (PDGF-BB) and interleukin-1 receptor antagonist (IL-1Ra) a very high affinity to the ECM. In mouse models, the approach considerably extended tissue retention of the engineered therapeutics and reduced leakage in the circulation. Prolonged retention and minimal systemic diffusion of engineered PDGF-BB abolished the tumour growth-promoting adverse effect that was observed with wild-type PDGF-BB. Moreover, engineered PDGF-BB was substantially more effective at promoting diabetic wound healing and regeneration after volumetric muscle loss, compared to wild-type PDGF-BB. Finally, while local or systemic delivery of wild-type IL-1Ra showed minor effects, intramyocardial delivery of engineered IL-1Ra enhanced cardiac repair after myocardial infarction by limiting cardiomyocyte death and fibrosis. This engineering strategy highlights the key importance of exploiting interactions between ECM and therapeutic proteins for developing effective and safer regenerative therapies.

Experimental and theoretical study of AC electrical conduction mechanisms of semicrystalline parylene C thin films.

The electrical conduction mechanisms of semicrystalline thermoplastic parylene C (-H(2)C-C(6)H(3)Cl-CH(2)-)(n) thin films were studied in large temperature and frequency regions. The alternative current (AC) electrical conduction in parylene C is governed by two processes which can be ascribed to a hopping transport mechanism: correlated barrier hopping (CBH) model at low [77-155 K] and high [473-533 K] temperature and the small polaron tunneling mechanism (SPTM) from 193 to 413 K within the framework of the universal law of dielectric response. The conduction mechanism is explained with the help of Elliot's theory, and the Elliot's parameters are determined. From frequency- and temperature-conductivity characteristics, the activation energy is found to be 1.27 eV for direct current (DC) conduction interpreted in terms of ionic conduction mechanism. The power law dependence of AC conductivity is interpreted in terms of electron hopping with a density N(E(F)) (~10(18) eV cm(-3)) over a 0.023-0.03 eV high barrier across a distance of 1.46-1.54 Å.

Growth Factor and Cytokine Delivery Systems for Wound Healing.

Skin wound healing is a highly coordinated process involving multiple tissue-resident and recruited cell types. Cells within the wound microenvironment respond to key secreted factors such as pro-proliferative growth factors and immunomodulatory cytokines to repair the skin and promptly restore its essential barrier role. Therefore, recombinant growth factors and cytokines are promising therapeutics for skin wounds, in particular for large acute wounds such as burns, or wounds associated with underlying pathologies such as nonhealing chronic and diabetic wounds. However, translation of growth factors and cytokines into clinically effective treatments has been limited. Short half-life, poor stability, rapid diffusion, uncontrolled signaling, and systemic side effects are currently the key challenges to developing efficient growth factor- and cytokine-based therapies. To overcome these limitations, novel delivery systems have been developed to improve the regenerative potential of recombinant growth factors and cytokines. In this review, we discuss biomaterial and protein engineering strategies used to optimize the delivery of growth factor and cytokine therapeutics for skin wound treatment.

Distinctive molecular features of regenerative stem cells in the damaged male germline.

Maintenance of male fertility requires spermatogonial stem cells (SSCs) that self-renew and generate differentiating germ cells for production of spermatozoa. Germline cells are sensitive to genotoxic drugs and patients receiving chemotherapy can become infertile. SSCs surviving treatment mediate germline recovery but pathways driving SSC regenerative responses remain poorly understood. Using models of chemotherapy-induced germline damage and recovery, here we identify unique molecular features of regenerative SSCs and characterise changes in composition of the undifferentiated spermatogonial pool during germline recovery by single-cell analysis. Increased mitotic activity of SSCs mediating regeneration is accompanied by alterations in growth factor signalling including PI3K/AKT and mTORC1 pathways. While sustained mTORC1 signalling is detrimental for SSC maintenance, transient mTORC1 activation is critical for the regenerative response. Concerted inhibition of growth factor signalling disrupts core features of the regenerative state and limits germline recovery. We also demonstrate that the FOXM1 transcription factor is a target of growth factor signalling in undifferentiated spermatogonia and provide evidence for a role in regeneration. Our data confirm dynamic changes in SSC functional properties following damage and support an essential role for microenvironmental growth factors in promoting a regenerative state.

Impact of 10-Myr scale monsoon dynamics on Mesozoic climate and ecosystems.

Earth's orbital variations on timescales of 104-105 years, known as Milankovitch cycles, have played a critical role in pacing climate change and ecosystem dynamics, through glacial and/or monsoon dynamics. However, the climatic and biotic consequences of these cycles on much longer (~ 107 years) timescales remain unclear, due to a lack of long proxy records with precise age constraints. Here, we show ~ 10-Myr scale variations in early Mesozoic (250-180 Ma) records of lake-level, desert distribution, biogenic-silica burial flux, atmospheric CO2 levels (pCO2), and sea-surface-temperature (SST). Their phase relationships, coupled with carbon cycle modeling results, suggest that orbitally-paced summer monsoon dynamics modulates changes in terrestrial weatherability by ~ 20%, affecting changes in pCO2 of up to 500-1,000 ppmv and 3-7 °C SST. We also infer that these ~ 10-Myr scale climatic variations could have been causally linked to biotic turnover, size variations in dinosaur footprints, and tetrapod dispersal, potentially through spatio-temporal variations in resource availability and arid-hot climatic barriers at low-middle latitudes.

Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein-Protein Interactions.

Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein-protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.

DDX5 plays essential transcriptional and post-transcriptional roles in the maintenance and function of spermatogonia.

Mammalian spermatogenesis is sustained by mitotic germ cells with self-renewal potential known as undifferentiated spermatogonia. Maintenance of undifferentiated spermatogonia and spermatogenesis is dependent on tightly co-ordinated transcriptional and post-transcriptional mechanisms. The RNA helicase DDX5 is expressed by spermatogonia but roles in spermatogenesis are unexplored. Using an inducible knockout mouse model, we characterise an essential role for DDX5 in spermatogonial maintenance and show that Ddx5 is indispensable for male fertility. We demonstrate that DDX5 regulates appropriate splicing of key genes necessary for spermatogenesis. Moreover, DDX5 regulates expression of cell cycle genes in undifferentiated spermatogonia post-transcriptionally and is required for cell proliferation and survival. DDX5 can also act as a transcriptional co-activator and we demonstrate that DDX5 interacts with PLZF, a transcription factor required for germline maintenance, to co-regulate select target genes. Combined, our data reveal a critical multifunctional role for DDX5 in regulating gene expression programmes and activity of undifferentiated spermatogonia.

Identification of dynamic undifferentiated cell states within the male germline.

The role of stem cells in tissue maintenance is appreciated and hierarchical models of stem cell self-renewal and differentiation often proposed. Stem cell activity in the male germline is restricted to undifferentiated A-type spermatogonia (Aundiff); however, only a fraction of this population act as stem cells in undisturbed testis and Aundiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult Aundiff fractions and dissect Aundiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing Aundiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1. Importantly, we find that PDX1 marks a population with potent stem cell capacity unique to mature, homeostatic testis and demonstrate dynamic interconversion between PDX1+ and PDX1- Aundiff states upon transplant and culture. We conclude that Aundiff exist in a series of dynamic cell states with distinct function and provide evidence that stability of such states is dictated by niche-derived cues.

Germline Stem Cell Activity Is Sustained by SALL4-Dependent Silencing of Distinct Tumor Suppressor Genes.

Sustained spermatogenesis in adult males and fertility recovery following germ cell depletion are dependent on undifferentiated spermatogonia. We previously demonstrated a key role for the transcription factor SALL4 in spermatogonial differentiation. However, whether SALL4 has broader roles within spermatogonia remains unclear despite its ability to co-regulate genes with PLZF, a transcription factor required for undifferentiated cell maintenance. Through development of inducible knockout models, we show that short-term integrity of differentiating but not undifferentiated populations requires SALL4. However, SALL4 loss was associated with long-term functional decline of undifferentiated spermatogonia and disrupted stem cell-driven regeneration. Mechanistically, SALL4 associated with the NuRD co-repressor and repressed expression of the tumor suppressor genes Foxl1 and Dusp4. Aberrant Foxl1 activation inhibited undifferentiated cell growth and survival, while DUSP4 suppressed self-renewal pathways. We therefore uncover an essential role for SALL4 in maintenance of undifferentiated spermatogonial activity and identify regulatory pathways critical for germline stem cell function.