Establishment and phenotype verification of mouse oviductal epithelial organoids

Objective·To establish a culture system of oviductal epithelial organoids from wild type (WT) mice and miR-34b/c-/- and miR-449-/- double knockout (dKO) mice, and verify the phenotypes.Methods·The oviduct epithelial cells of WT mice and dKO mice were isolated and purified by enzyme digestion and differential adhesion method, and the purity of the isolated oviduct epithelial cells was identified by immunofluorescence staining. The numbers, growth rates and sizes of oviductal epithelial organoids between WT mice and dKO mice were compared by counting and diameter measurement. Hematoxylin-eosin (H-E) staining and transmission electron microscope (TEM) were used to observe the morphology and structure of the oviductal epithelial organoids. The proportions of ciliated cells and secretory cells in the oviductal epithelial organoids from WT mice and dKO mice were observed and counted by immunofluorescence staining. Immunohistochemistry (IHC), real-time quantitative PCR (RT-qPCR) and Western blotting were used to observe the expression levels of marker genes of ciliated cells and secretory cells in the oviductal epithelial organoids.Results·The purity of the isolated and purified oviduct epithelial cells was high. Compared with the organoids from WT mice, the oviductal epithelial organoids from dKO mice grew faster and larger, and were more in number. But they developed more slowly than those from WT mice, as the invaginations of the dKO mice organoids appeared on the 28th day of culture, while the WT mice organoids exhibited the same structures on the 16th day. The oviductal epithelial organoids showed similar structures as those of the oviduct in vivo under hematoxylin-eosin (H-E) staining and TEM. Immunofluorescence staining showed that the ciliated cells of oviductal epithelial organoids from dKO mice were significantly reduced and the secretory cells were significantly increased (both P<0.05). IHC showed that the molecular expression patterns of the oviductal epithelial organoids were consistent with those of the oviducts in vivo, i.e. the expression levels of ciliated cell markers acetylated α-tubulin (Ac-α-tubulin) and forkhead box J1 (FOXJ1) decreased, and the expression level of the secretory cell marker paired box 8 (PAX8) increased. RT-qPCR showed that the mRNA levels of Foxj1 and tubulin β class Ⅳa (Tubb4a) decreased (both P<0.05), while Pax8 increased in the oviductal epithelial organoids of dKO mice (P<0.05). Western blotting results showed that the protein expression level of FOXJ1 in the organoids of dKO mice significantly decreased, while the expression of PAX8 significantly increased (both P<0.05).Conclusion·The culture system of oviductal epithelial organoids of WT mice and dKO mice are successfully constructed, which can simulate the phenotypes of mouse oviduct in vivo

Global characteristics and drivers of sodium and aluminum concentrations in freshly fallen plant litter

Plant litter is not only the major component of terrestrial ecosystem net productivity, the decomposition of which is also an important process for the returns of elements, including sodium (Na) and aluminum (Al), which can be beneficial or toxic for plant growth. However, to date, the global characteristics and driving factors of Na and Al concentrations in freshly fallen litter still remain elusive. Here, we evaluated the concentrations and drivers of litter Na and Al with 491 observations extracted from 116 publications across the globe. Results showed that (1) the average concentrations of Na in leaf, branch, root, stem, bark, and reproductive tissue (flowers and fruits) litter were 0.989, 0.891, 1.820, 0.500, 1.390, and 0.500 g/kg, respectively, and the concentrations of Al in leaf, branch, and root were 0.424, 0.200 and 1.540 g/kg, respectively. (2) mycorrhizal association significantly affected litter Na and Al concentration. The highest concentration of Na was found in litter from trees associated with both arbuscular mycorrhizal fungi (AM) and ectomycorrhizal fungi (ECM), followed by litter from trees with AM and ECM. Lifeform, taxonomic, and leaf form had significant impacts on the concentration of Na and Al in plant litter of different tissues. (3) leaf litter Na concentration was mainly driven by mycorrhizal association, leaf form and soil phosphorus concentration, while leaf litter Al concentration was mainly controlled by mycorrhizal association, leaf form, and precipitation in the wettest month. Overall, our study clearly assessed the global patterns and influencing factors of litter Na and Al concentrations, which may help us to better understand their roles in the associated biogeochemical cycles in forest ecosystem

Foliar litter nitrogen dynamics as affected by forest gap in the alpine forest of eastern Tibet Plateau.

There is increasing attention on the effects of seasonal snowpack on wintertime litter decomposition, as well as the processes following it, in cold biomes. However, little information is available on how litter nitrogen (N) dynamics vary with snowpack variations created by tree crown canopies in alpine forests. Therefore, to understand the effects of seasonal snowpack on litter N dynamics during different critical stages, litterbags with fir (Abies faxoniana), birch (Betula albo-sinensis), larch (Larix mastersiana) and cypress (Sabina saltuaria) foliar litter were placed on the forest floor beneath snowpack created by forest gaps in the eastern Tibet Plateau. The litterbags were sampled at the onset of freezing, deep freezing, thawing and growing stages from October 2010 to October 2012. Mass loss and N concentrations in litter were measured. Over two years of decomposition, N release occurred mainly during the first year, especially during the first winter. Litter N release rates (both in the first year and during the entire two-year decomposition study period) were higher in the center of canopy gaps than under closed canopy, regardless of species. Litter N release rates in winter were also highest in the center of canopy gaps and lowest under closed canopy, regardless of species, however the reverse was found during the growing season. Compared with broadleaf litter, needle litter N release comparisons of gap center to closed canopy showed much stronger responses to the changes in snow cover in winter and availability of sunshine during the growing season. As the decomposition proceeded, decomposing litter quality, microbial biomass and environmental temperature were important factors related to litter N release rate. This suggests that if winter warm with climate change, reduced snow cover in winter might slow down litter N release in alpine forest

Soil fauna effects on litter decomposition are better predicted by fauna communities within litterbags than by ambient soil fauna communities

Aims:soil fauna is one of the major drivers of plant litter decomposition. This study aims to assess how soil fauna density and diversity may affect litter decomposition. Also, we assessed whether faunal communities inside the litterbags that are used to control the access of faunal groups or communities in ambient soils are better for predicting their effects on litter decomposition, given that soil fauna frequently move into and out of such litterbags. - Methods: to answer this question, we synthesized 5336 observations extracted from 46 publications to assess the effects of soil fauna communities, their density and diversity on the rate of litter decomposition (k) and litter mass loss. - Results: results showed that (1) the presence of soil fauna significantly increased k by an average of 33.0% and that the effects were mainly controlled by initial litter concentrations of phosphorus and magnesium, (2) the density and diversity of soil fauna in litterbags significantly affected k and/or mass loss, but ambient communities had limited effects, and (3) the effects of soil fauna in litterbags on k were most significant during the early stages of decomposition (0 − 30% mass loss). - Conclusions: our study clearly showed that litterbag communities were better for predicting the effects of soil fauna on litter decomposition, and that their effects were most significant during the early stages of decomposition. These results improve our ability to estimate the contribution of soil fauna in liter decomposition and the associated carbon and nutrient cycling

Litter quality and stream physicochemical properties drive global invertebrate effects on instream litter decomposition

Plant litter is the major source of energy and nutrients in stream ecosystems and its decomposition is vital for ecosystem nutrient cycling and functioning. Invertebrates are key contributors to instream litter decomposition, yet quantification of their effects and drivers at the global scale remains lacking. Here, we systematically synthesized data comprising 2707 observations from 141 studies of stream litter decomposition to assess the contribution and drivers of invertebrates to the decomposition process across the globe. We found that (1) the presence of invertebrates enhanced instream litter decomposition globally by an average of 74%; (2) initial litter quality and stream water physicochemical properties were equal drivers of invertebrate effects on litter decomposition, while invertebrate effects on litter decomposition were not affected by climatic region, mesh size of coarse-mesh bags or mycorrhizal association of plants providing leaf litter; and (3) the contribution of invertebrates to litter decomposition was greatest during the early stages of litter mass loss (0-20%). Our results, besides quantitatively synthesizing the global pattern of invertebrate contribution to instream litter decomposition, highlight the most significant effects of invertebrates on litter decomposition at early rather than middle or late decomposition stages, providing support for the inclusion of invertebrates in global dynamic models of litter decomposition in streams to explore mechanisms and impacts of terrestrial, aquatic, and atmospheric carbon fluxes

Relative proportion (%) at each decomposition stage to 2 years N release in different gaps.

<p>Abbreviations: OF = onset of freezing stage; DF = deep freezing stage; TS = thawing stage; EGS = early growing season; LGS = late growing season; G1 = gap 1, gap center south; G2 = gap 2, gap center north; G3 = gap 3, canopy edge; G4 = gap 4, expanded edge; G5 = gap 5, closed canopy.</p

MBN during litter decomposition from gap center to closed canopy at different decomposition stages over 2 years (mean ± <i>SD</i>, <i>n</i> = 5).

<p>The symbols “*” indicate the significant difference among treatments (LSD's multiple range test; <i>P</i><0.05). Abbreviations: MBN  = microbial biomass nitrogen; OF = onset of freezing stage; DF = deep freezing stage; TS = thawing stage; EGS = early growing season; LGS = late growing season; G1 = gap 1, gap center south; G2 = gap 2, gap center north; G3 = gap 3, canopy edge; G4 = gap 4, expanded edge; G5 = gap 5, closed canopy.</p

Effects of species (S) and winter snowpack (C) on N release rate in the alpine forest.

<p>OF, onset of freezing stage; DF, deep freezing stage; TS, thawing stage; EGS, early growing season; LGS, late growing season.</p><p><i>p</i>_S,effect of species; <i>p</i>_C,effect of winter snowpack.</p><p>*indicates significant difference at <i>p</i><0.05, ** indicates significant difference at <i>P</i><0.01.</p

Experimental layout in all experimental gaps by gap position.

<p>G1 to G5 were located from gap center to closed canopy. Abbreviations: G1 = gap 1, gap center south; G2 = gap 2, gap center north; G3 = gap 3, canopy edge; G4 = gap 4, expanded edge; G5 = gap 5, closed canopy.</p