PRC1 directs PRC2-H3K27me3 deposition to shield adult spermatogonial stem cells from differentiation.

Spermatogonial stem cells functionality reside in the slow-cycling and heterogeneous undifferentiated spermatogonia cell population. This pool of cells supports lifelong fertility in adult males by balancing self-renewal and differentiation to produce haploid gametes. However, the molecular mechanisms underpinning long-term stemness of undifferentiated spermatogonia during adulthood remain unclear. Here, we discover that an epigenetic regulator, Polycomb repressive complex 1 (PRC1), shields adult undifferentiated spermatogonia from differentiation, maintains slow cycling, and directs commitment to differentiation during steady-state spermatogenesis in adults. We show that PRC2-mediated H3K27me3 is an epigenetic hallmark of adult undifferentiated spermatogonia. Indeed, spermatogonial differentiation is accompanied by a global loss of H3K27me3. Disruption of PRC1 impairs global H3K27me3 deposition, leading to precocious spermatogonial differentiation. Therefore, PRC1 directs PRC2-H3K27me3 deposition to maintain the self-renewing state of undifferentiated spermatogonia. Importantly, in contrast to its role in other tissue stem cells, PRC1 negatively regulates the cell cycle to maintain slow cycling of undifferentiated spermatogonia. Our findings have implications for how epigenetic regulators can be tuned to regulate the stem cell potential, cell cycle and differentiation to ensure lifelong fertility in adult males.

Biological Activities of Oleanolic Acid Derivatives from Calendula officinalis Seeds.

Phytochemical examination of butanol fraction of Calendula officinalis seeds led to the isolation of two compounds identified as 28-O-ß-D-glucopyranosyl-oleanolic acid 3-O-ß-D-glucopyranosyl (1→3)-ß-D-glucopyranosiduronic acid (CS1) and oleanolic acid 3-O-ß-D-glucopyranosyl (1→3)-ß-D-glucopyranosiduronic acid (CS2). Biological evaluation was carried out for these two compounds such as melanin biosynthesis inhibitory, hyaluronic acid production activities, anti obesity using lipase inhibition and adipocyte differentiation as well as evaluation of the protective effect against hydrogen peroxide induced neurotoxicity in neuro-2A cells. The results showed that, compound CS2 has a melanin biosynthesis stimulatory activity; however, compound CS1 has a potent stimulatory effect for the production of hyaluronic acid on normal human dermal fibroblast from adult (NHDF-Ad). Both compounds did not show any inhibitory effect on both lipase and adipocyte differentiation. Compound CS2 could protect neuro-2A cells and increased cell viability against H2 O2 . These activities (melanin biosynthesis stimulatory and protective effect against H2 O2 of CS2 and hyaluronic acid productive activities of these triterpene derivatives) have been reported for the first time. Copyright © 2016 John Wiley & Sons, Ltd.

Improved Biological Activities of Isoepoxypteryxin by Biotransformation.

Isoepoxypteryxin is the major coumarin of a Japanese medicinal plant Angelica shikokiana. This research was designed to study the effect of structural changes through fungal biotransformation on the reported biological activities of isoepoxypteryxin. Among the tested microorganisms, only Cordyceps sinensis had enzymes that could catalyze the ester hydrolysis and the reductive cleavage of the epoxide ring of isoepoxypteryxin, separately, to give two more polar metabolites (+)-cis-khellactone (P1) and a new coumarin derivative (+)-cis-3'-[(2-methyl-3-hydroxybutanoyl)oxy]-4'-acetoxy-3',4'-dihydroseselin (P2), respectively. The polar metabolite P2 showed stronger cytotoxicity and higher selectivity than isoepoxypteryxin. On the molecular level, P2 showed more in vitro inhibition of both tubulin polymerization and histone deacetylase 8 (HDAC8). Similarly, P2 showed more neuroprotection against amyloid beta fragment 1 - 42 (Aß1 - 42 )-induced neurotoxicity in human neuroblastoma cells (SH-SY5Y) and exhibited more inhibition of the in vitro aggregation of Aß1 - 42 . Both metabolites showed stronger antiplatelet aggregation by increased inhibition of thromboxane-A2 synthase (TXS) activity and thromboxane-A2 (TXA2) production. This study is the first to describe the improved cytotoxic, neuroprotective, and antiplatelet aggregation activities of isoepoxypteryxin through its biotransformation by C. sinensis.

Time-dependent change in Reishi (Ganoderma lingzhi) triterpenoids in culture with rumen fluid.

The purpose of this study was to investigate the time-dependent change in Reishi (Ganoderma lingzhi) triterpenoids in rumen fluid. G. lingzhi fruiting bodies were milled and incubated in a tube with rumen fluid for 0, 4, 8, 12, 24, and 48 h at 39°C. After incubation, all the tubes were freeze-dried and extracted by ethanol. The contents of 18 triterpenoids in the ethanol extract were quantitated by liquid chromatography-mass spectrometry (LC-MS/MS). Based on the results, triterpenoids were categorized into three groups: (1) rapid decrease, indicating reductions of more than 50% within 8 h; (2) mild decrease, with reductions of more than 50% within 48 h; and (3) minimal change, even after 48 h, there was not much change. Ganoderic acid C6, DM, H, K, and TR as well as Ganoderenic acid D were classified in (1); Ganoderic acid LM2 and T-Q as well as Ganoderiol F in (2); and Ganoderic acid A, B, C1, C2, I, and TN; Gnoderenic acid C; and Ganodermanontriol in (3). In addition, a relationship between chemical structure and metabolic speed was observed in some cases. The results of this study revealed that G. lingzhi triterpenoids are digested and metabolized at different speeds in ruminant fluid.

Effect of palm kernel cake as protein source in a concentrate diet on intake, digestibility and live weight gain of goats fed Napier grass.

The effects of palm kernel cake (PKC) as a protein source in a concentrate diet (comprising 35 % crushed maize, 30 % rice bran, 32 % PKC, 2 % vitamin mineral premix and 1 % salt) were examined on intake, live weight (LW) gain and digestibility in female goats (average LW of 12.4 ± 2.6 kg). Four goats were randomly allocated to each of the four treatment diets: (a) Napier grass (Pennisetum purpureum) offered ad libitum (T1), (b) T1 + concentrate at 0.5 % of LW (T2), (c) T1 + concentrate at 1.0 % of LW (T3) and (d) T1 + concentrate at 2.0 % of LW (T4). A 7-day digestibility trial and an 82-day growth experiment were conducted. No differences were observed among diets for intakes of roughage dry matter (DM), total DM, organic matter (OM) and neutral detergent fibre (NDF). The crude protein (CP) intake increased (P < 0.05) as the level of concentrate in the diets increased. Goats fed the T2, T3 and T4 diets gained 10.2, 34.1 and 52.5 g/head/day, respectively, while the control group (T1) lost weight (-12.7 g/head/day). The apparent digestibilities of DM, OM and CP were similar (P > 0.05) among treatments. The digestibility of dietary NDF decreased (P < 0.05) with increasing levels of concentrate, but there was no significant (P > 0.05) difference between T2 and T3 diets. Supplementing a basal diet of Napier grass with PKC-based concentrate improved CP intake and LW gain. The PKC-based concentrate diet can therefore be exploited for the use of local feed resources for goat production; however, further research is required to achieve the best growth response.

Effect of seaweed feeding before calving on colostral IgA concentration in Japanese Black cows.

In this study, we investigated the effect of feeding seaweed to Japanese Black cows before calving on IgA concentrations in colostrum. Seven Japanese Black breeding cows were used as test animals, with three cows in the seaweed-fed group (seaweed group) and four in the seaweed-non-fed group (control group). Each cow was fed 6 kg of sudangrass hay and 2.5 kg of compound feed twice daily (09:00 a.m. and 04:00 p.m.) as basal diets. Both groups had free access to water. In the seaweed group, commercially available seaweed feed was fed from 2 months before calving until the day of calving. The seaweed of 150 g/head/day was added to the basal diet at the morning feeding. Colostrum collected immediately after calving was used to measure IgA concentrations by ELISA. The IgA concentration in colostrum was significantly higher in the seaweed group than in the control group (P < 0.05). This suggested that feeding seaweed to Japanese Black cows before calving may increase IgA concentration in colostrum.

Functional identification of the actual and potential stem cell compartments in mouse spermatogenesis.

To clarify the mechanisms that support the continuity of actively cycling tissues of long-lived organisms, we investigated the composition of a mouse spermatogenic stem cell system by pulse-chase of the undifferentiated spermatogonia, the population responsible for stem cell functions, in combination with transplantation and regeneration assays after pulse-labeling. We demonstrate that in addition to "actual stem cells," which are indeed self-renewing, a second population ("potential stem cells") also exists, which is capable of self-renewing but do not self-renew in the normal situation. Potential stem cells rapidly turn over in normal testes, suggesting that they belong to the transit-amplifying, rather than the dormant, population. During the long natural course, actual stem cells are occasionally lost and compensated for by progeny of their neighbors. In this process, potential stem cells are postulated to shift their modes from transit amplification to self-renewal, thus playing an essential role to ensure spermatogenesis integrity.

Physiological and Psychological Effects of Volatile Organic Compounds from Dried Common Rush (Juncus effusus L. var. decipiens Buchen.) on Humans.

This study compared the participants' physiological responses and subjective evaluations of air scented with different concentrations of common rush (Juncus effusus L. var. decipiens Buchen.) (30 g and 15 g, with fresh air as a control). We asked 20 participants to complete a series of visual discrimination tasks while inhaling two different air samples. We evaluated (1) brain activity, (2) autonomic nervous activity, and (3) blood pressure and pulse rate, (4) in combination with self-evaluation. In addition, we quantified the concentrations of volatile organic compounds. The participants reported the scent to be sour, pungent, and smelly; this impression was likely caused by hexanal and acetic acid. Although the self-evaluations showed that participants did not enjoy the scent, their alpha amplitudes of electroencephalogram and parasympathetic nervous activity were increased, suggesting that participants were relaxed in this atmosphere. Moreover, a lower concentration resulted in a greater induction of relaxation. While the air was not pleasant-smelling, the volatile organic compounds present had a positive psychophysiological impact.

Chemical composition and in vitro ruminal digestibility of Ludwigia grandiflora.

Ludwigia grandiflora is an amphibious plant that has been designated as a specific alien plant in Japan. Due to the risk of regeneration on land, plants are burned after eradication. Since L. grandiflora is fertile and rich in biomass, this study investigated the use of L. grandiflora as a feed for ruminants. We measured general components, detergent fiber components, and in vitro dry matter digestibility (IVDMD) using rumen microbes for three forms (terrestrial form, emergent form, and floating-leaved form) of L. grandiflora. In addition, concentrations of hazardous metallic elements were also measured from the viewpoint of feed safety. Crude protein, neutral detergent fiber, acid detergent fiber, and acid detergent lignin were contained 10.31-14.62, 27.83-42.28, 20.54-36.05, and 6.94-18.90 (%DM), respectively. IVDMD was 50.75%-68.24%. Toxic elements such as Pb, Cd, and Hg were not detected in all forms. These results suggest that L. grandiflora could be partially used as alternative roughage for ruminants.

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye.

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

A multistate stem cell dynamics maintains homeostasis in mouse spermatogenesis.

In mouse testis, a heterogeneous population of undifferentiated spermatogonia (Aundiff) harbors spermatogenic stem cell (SSC) potential. Although GFRα1+ Aundiff maintains the self-renewing pool in homeostasis, the functional basis of heterogeneity and the implications for their dynamics remain unresolved. Here, through quantitative lineage tracing of SSC subpopulations, we show that an ensemble of heterogeneous states of SSCs supports homeostatic, persistent spermatogenesis. Such heterogeneity is maintained robustly through stochastic interconversion of SSCs between a renewal-biased Plvap+/GFRα1+ state and a differentiation-primed Sox3+/GFRα1+ state. In this framework, stem cell commitment occurs not directly but gradually through entry into licensed but uncommitted states. Further, Plvap+/GFRα1+ cells divide slowly, in synchrony with the seminiferous epithelial cycle, while Sox3+/GFRα1+ cells divide much faster. Such differential cell-cycle dynamics reduces mitotic load, and thereby the potential to acquire harmful de novo mutations of the self-renewing pool, while keeping the SSC density high over the testicular open niche.

EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice.

The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1, a member of the Exocyst complex, is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. EXOC1 contributes to the pseudopod formation of spermatogonia by inactivating the Rho family small GTPase Rac1 and also functions in the spermatocyte syncytia with the SNARE proteins STX2 and SNAP23. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

The Effect of Eucalyptol on Nursing Home Residents.

Eucalyptol is one of the most popular volatile components. It is used in many essential oils for relieving sinus and lung congestion caused by a variety of conditions. This pilot study sought to analyze clinical evidence for the effect of the scent of eucalyptol on the cognitive function of elderly people. Seventy nursing-home residents with cognitive impairment were recruited. Three one-week experiments were performed: eucalyptol scent was diffused in bedrooms with a diffuser only at wake-up time in the first experiment, and at wake-up time and bedtime in the second and third experiments. Results showed that although an improvement was not seen when using Mini Mental State Examination (MMSE) and Cohen-Mansfild Agitation Inventory (CMAI) measures, Dementia Behavior Disturbance Scale (DBD) scores improved significantly, even though no subject reported perceiving the scent. The significant improvements of the behaviour were found not only among the subjects whose room had a diffuser but also among the subjects who were exposed to an unperceivable level of eucalyptol drifted in the living room.

A new aliphatic ester of hydroxysalicylic acid from fermented Carica papaya L. preparation with a potential hair growth stimulating activity.

An aliphatic ester of hydroxysalicylic acid (6), reported for the first time from a natural source in addition to five known compounds were isolated from the fermented Carica papaya L. preparation, a commercialized functional food. The known compounds were identified as 5-hydroxymethylfurfuraldehyde (1), trans-caffeic acid (2), butyl 4-hydroxybenzoate (butylparaben) (3), lycopene (4), benzyl isothiocyanate (5). Compounds 1 and 3 were reported for the first time from Papaya fruits through this study. The new compound showed a moderate antioxidant activity and a potent hair growth stimulating activity in vitro.

Competition for Mitogens Regulates Spermatogenic Stem Cell Homeostasis in an Open Niche.

In many tissues, homeostasis is maintained by physical contact between stem cells and an anatomically defined niche. However, how stem cell homeostasis is achieved in environments where cells are motile and dispersed among their progeny remains unknown. Using murine spermatogenesis as a model, we find that spermatogenic stem cell density is tightly regulated by the supply of fibroblast growth factors (FGFs) from lymphatic endothelial cells. We propose that stem cell homeostasis is achieved through competition for a limited supply of FGFs. We show that the quantitative dependence of stem cell density on FGF dosage, the biased localization of stem cells toward FGF sources, and stem cell dynamics during regeneration following injury can all be predicted and explained within the framework of a minimal theoretical model based on "mitogen competition." We propose that this model provides a generic and robust mechanism to support stem cell homeostasis in open, or facultative, niche environments.

Effect of polyphenols from Vicia faba L on lipase activity and melanogenesis.

Two new flavonoid glycosides, kaempferol 3-O-α-L-rhamnopyranosyl (1→6) (3''-acetyl)-ß-D-galactopyranoside 1 and kaempferol 3-O-α-L-arabinopyranosyl-5-O-α-L-rhamnopyranoside 2, along with six known ones 3-8 were isolated from the flowers of Vicia faba L. (Fabaceae). Methanol extract and the isolated compounds were tested against lipase and melanogenesis inhibition activities and resulted in that compound 2 showed 53 and 77% lipase inhibition activity in concentrations of 400 and 800 µg/mL, respectively. For melanogenesis, compounds 2, 3 and 4 exhibited potent melanogenesis inhibition activity where the melanin content in melanoma cells was decreased to be about 57.5, 56 and 61%, respectively, with no obvious melanocytotoxicity. The rest of compounds showed weak to moderate activity. The results of melanogenesis inhibition activity of this study suggested the potential use of Vicia faba flowers as a skin-whitening agent and reveal the flowers to be a rich source of important phytochemicals with antilipase and melanogenesis inhibitory activity.

Changes in content of triterpenoids and polysaccharides in Ganoderma lingzhi at different growth stages.

Ganoderma lingzhi is a traditional medicinal mushroom, and its extract contains many bioactive compounds. Triterpenoids and polysaccharides are the primary bioactive components that contribute to its medicinal properties. In this study, we quantified 18 triterpenoids, total triterpenoid content and total polysaccharide content in the ethanol and water extracts of G. lingzhi at different growth stages. Triterpenoids were quantified by liquid chromatograph-tandem mass spectrometry in the multiple-reaction-monitoring mode. Total triterpenoid and total polysaccharide content were determined by colorimetric analysis. The results indicated that the fruit bodies at an early growth stage had a higher content of ganoderic acid A, C2, I and LM2, as well as of ganoderenic acid C and D, than those at a later growth stage. In contrast, ganoderic acid K, TN and T-Q contents were higher in mature fruit bodies (maturation stage). The highest total triterpenoid and total polysaccharide contents were found in fruit bodies before maturity (stipe elongation stage or early stage of pileus formation). Our results provide information which will contribute to the establishment of an efficient cultivation system for G. lingzhi with a higher content of triterpenoids.

Stem cell heterogeneity: actual and potential stem cell compartments in mouse spermatogenesis.

In order to assess the behavior of the spermatogonia that contribute to the stem cell system in steady-state mouse spermatogenesis, a series of pulse-labeling experiments of a subset of "undifferentiated spermatogonial" was performed using transgenic approaches. The results suggest that, in addition to the spermatogonial population that actually acts as the stem cells (actual stem cell compartment), a second set of undifferentiated spermatogonia also exists that possesses the potential to self-renew, but does not normally do so (potential stem cell compartment). The potential stem cells turn over rather rapidly, suggesting that they act as the transit amplifying cells in the normal situation. The potential stem cells seem to change their mode from transientamplification to self-renewal upon transplantation, regeneration, or loss of actual stem cells that occurs during the long reproduction period. Such a heterogeneous composition of the stem cell system may be important for the robustness of overall spermatogenesis.

Biological Activities of Extracts from Different Parts of Cryptomeria japonica.

Cryptomeria japonica is one of the most important forest tree species in Japan. To increase the demand for domestic timber, broad uses related to the functional attributes of C. japonica must be developed. Several studies have examined the usefulness of C. japonica in terms of its biological activities, but a comprehensive study subjecting all parts of the C. japonica plant to the same solvent for extraction has not been done. Here, methanol extractions from the leaves, branches, bearing branches, male flowers, female flowers, cones, bark, heartwood, sapwood, pith, rhizomes, roots, and pollen were subjected to several in vitro assays of their biological activities such as antioxidant activity, anti-lipase activity, antibacterial activity and melanin-biosynthesis-inhibition activity. Their total phenolic content was also determined. The methanol extracts from each part of C. japonica except for pollen showed strong activities in the bioactivity assays. Furthermore, the methanol extracts were analyzed by GC/MS. The phytochemical profile varied among extracts from various parts of C. japonica. Our results suggest the great potential of C. japonica for use as a functional ingredient in health-related products.

Determination of Seed Soundness in Conifers Cryptomeria japonica and Chamaecyparis obtusa Using Narrow-Multiband Spectral Imaging in the Short-Wavelength Infrared Range.

Regeneration of planted forests of Cryptomeria japonica (sugi) and Chamaecyparis obtuse (hinoki) is the pressing importance to the forest administration in Japan. Low seed germination rate of these species, however, has hampered low-cost production of their seedlings for reforestation. The primary cause of the low germinability has been attributed to highly frequent formation of anatomically unsound seeds, which are indistinguishable from sound germinable seeds by visible observation and other common criteria such as size and weight. To establish a method for sound seed selection in these species, hyperspectral imaging technique was used to identify a wavelength range where reflectance spectra differ clearly between sound and unsound seeds. In sound seeds of both species, reflectance in a narrow waveband centered at 1,730 nm, corresponding to a lipid absorption band in the short-wavelength infrared (SWIR) range, was greatly depressed relative to that in adjacent wavebands on either side. Such depression was absent or less prominent in unsound seeds. Based on these observations, a reflectance index SQI, abbreviated for seed quality index, was formulated using reflectance at three narrow SWIR wavebands so that it represents the extent of the depression. SQI calculated from seed area-averaged reflectance spectra and spatial distribution patterns of pixelwise SQI within each seed area were both proven as reliable criteria for sound seed selection. Enrichment of sound seeds was accompanied by an increase in germination rate of the seed lot. Thus, the methods described are readily applicable toward low-cost seedling production in combination with single seed sowing technology.