Effect of low ethanol concentration in maturation medium on developmental ability, mitochondria, and gene expression profile in mouse oocytes.

Ethanol affects pre-conceptional oocyte quality in women. In this study, we examined the effect of low ethanol concentrations on mouse oocytes. Oocytes were collected from the ovaries of 9-10 week old mice and allowed to mature in vitro in the presence of low concentrations of ethanol (0.1% and 0.2% v/v) for 24 h. Treatment of oocytes with ethanol (0.2%) during maturation decreased the mitochondrial DNA content and membrane potential compared to that in untreated ones, whereas the ATP content did not differ between the groups. Both 0.1% and 0.2% ethanol reduced the lipid content in the oocytes. In addition, immunostaining revealed that oocytes cultured in maturation medium containing ethanol (0.2%) had reduced levels of global DNA methylation and DNMT3A compared with untreated oocytes, and decreased rate of blastocyst development with low mitochondrial protein levels (TOMM40) in embryo. RNA-sequencing of the ethanol-treated (0.2%) and untreated oocytes revealed that mitochondria were a major target of ethanol. In conclusion, treatment of oocytes with low concentration of ethanol reduces the developmental rate to the blastocyst stage, with a lower total cell number and global DNA methylation. In addition, ethanol affected mitochondrial function and mitochondria-related gene expression.

Large-scale animal model study uncovers altered brain pH and lactate levels as a transdiagnostic endophenotype of neuropsychiatric disorders involving cognitive impairment.

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer's disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

A novel alternative method for long-term evaluation of male reproductive toxicity and its recovery using a pre-pubertal mouse testis organ culture system.

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.

Improvements in in vitro spermatogenesis: oxygen concentration, antioxidants, tissue-form design, and space control.

Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10-15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.

Massive horizontal gene transfer and the evolution of nematomorph-driven behavioral manipulation of mantids.

To complete their life cycle, a wide range of parasites must manipulate the behavior of their hosts.1 This manipulation is a well-known example of the "extended phenotype,2" where genes in one organism have phenotypic effects on another organism. Recent studies have explored the parasite genes responsible for such manipulation of host behavior, including the potential molecular mechanisms.3,4 However, little is known about how parasites have acquired the genes involved in manipulating phylogenetically distinct hosts.4 In a fascinating example of the extended phenotype, nematomorph parasites have evolved the ability to induce their terrestrial insect hosts to enter bodies of water, where the parasite then reproduces. Here, we comprehensively analyzed nematomorphs and their mantid hosts, focusing on the transcriptomic changes associated with host manipulations and sequence similarity between host and parasite genes to test molecular mimicry. The nematomorph's transcriptome changed during host manipulation, whereas no distinct changes were found in mantids. We then discovered numerous possible host-derived genes in nematomorphs, and these genes were frequently up-regulated during host manipulation. Our findings suggest a possible general role of horizontal gene transfer (HGT) in the molecular mechanisms of host manipulation, as well as in the genome evolution of manipulative parasites. The evidence of HGT between multicellular eukaryotes remains scarce but is increasing and, therefore, elucidating its mechanisms will advance our understanding of the enduring influence of HGT on the evolution of the web of life.

Short-Term Dendritic Dynamics of Neonatal Cortical Neurons Revealed by In Vivo Imaging with Improved Spatiotemporal Resolution.

Individual neurons in sensory cortices exhibit specific receptive fields based on their dendritic patterns. These dendritic morphologies are established and refined during the neonatal period through activity-dependent plasticity. This process can be visualized using two-photon in vivo time-lapse imaging, but sufficient spatiotemporal resolution is essential. We previously examined dendritic patterning from spiny stellate (SS) neurons, the major type of layer 4 (L4) neurons, in the mouse primary somatosensory cortex (barrel cortex), where mature dendrites display a strong orientation bias toward the barrel center. Longitudinal imaging at 8 h intervals revealed the long-term dynamics by which SS neurons acquire this unique dendritic pattern. However, the spatiotemporal resolution was insufficient to detect the more rapid changes in SS neuron dendrite morphology during the critical neonatal period. In the current study, we imaged neonatal L4 neurons hourly for 8 h and improved the spatial resolution by uniform cell surface labeling. The improved spatiotemporal resolution allowed detection of precise changes in dendrite morphology and revealed aspects of short-term dendritic dynamics unique to the neonatal period. Basal dendrites of barrel cortex L4 neurons were highly dynamic. In particular, both barrel-inner and barrel-outer dendrites (trees and branches) emerged/elongated and disappeared/retracted at similarly high frequencies, suggesting that SS neurons acquire biased dendrite patterns through rapid trial-and-error emergence, elongation, elimination, and retraction of dendritic trees and branches. We also found correlations between morphology and behavior (elongation/retraction) of dendritic tips. Thus, the current study revealed short-term dynamics and related features of cortical neuron dendrites during refinement.

Culture-space control is effective in promoting haploid cell formation and spermiogenesis in vitro in neonatal mice.

The classical organ culture method, in which tissue is placed at the gas‒liquid interphase, is effective at inducing mouse spermatogenesis. However, due to reginal variations in the supply of oxygen and nutrients within a tissue, the progress of spermatogenesis was observed only in limited areas of a tissue. In addition, haploid cell formation and its differentiation to spermatozoon, i.e. spermiogenesis, were infrequent and inefficient. Here, we show that the polydimethylsiloxane (PDMS)-chip ceiling (PC) method, which ensures a uniform supply of nutrients and oxygen throughout the tissue by pressing it into a thin, flat shape, can provide control over the culture space. We used this method to culture testis tissue from neonatal mice, aged 1 to 4 days, and found that modulating the culture space during the experiment by replacing one chip with another that had a higher ceiling effectively increased tissue growth. This adjustment also induced more efficient spermatogenesis, with the process of spermiogenesis being particularly promoted. Meiotic cells were observed from culture day 14 onward, and haploid cells were confirmed at the end of each experiment. This technique was also shown to be a sensitive assay for testicular toxicity. Culture-space control will be a critical regulation parameter for sophisticated tissue culture experiments.

Generation of rat offspring using spermatids produced through in vitro spermatogenesis.

An in vitro spermatogenesis method using mouse testicular tissue to produce fertile sperm was established more than a decade ago. Although this culture method has generally not been effective in other animal species, we recently succeeded in improving the culture condition to induce spermatogenesis of rats up to the round spermatid stage. In the present study, we introduced acrosin-EGFP transgenic rats in order to clearly monitor the production of haploid cells during spermatogenesis in vitro. In addition, a metabolomic analysis of the culture media during cultivation revealed the metabolic dynamics of the testis tissue. By modifying the culture media based on these results, we were able to induce rat spermatogenesis repeatedly up to haploid cell production, including the formation of elongating spermatids, which was confirmed histologically and immunohistochemically. Finally, we performed a microinsemination experiment with in vitro produced spermatids, which resulted in the production of healthy and fertile offspring. This is the first demonstration of the in vitro production of functional haploid cells that yielded offspring in animals other than mice. These results are expected to provide a basis for the development of an in vitro spermatogenesis system applicable to many other mammals.

Effect of a low ethanol concentration during in vitro maturation of bovine oocytes and subsequent embryo development.

The present study investigated the effects of low ethanol exposure on bovine oocytes. Cumulus-oocyte complexes (COCs) were aspirated for the antral follicles of slaughterhouse-derived ovaries. These COCs were incubated in maturation medium containing 0, 0.1, and 0.2% ethanol for 21 h and subjected to fertilization and in vitro development, and then the rates of nuclear maturation, mitochondrial DNA copy number (Mt-cn) and protein (TOMM40), ATP content and lipid content in oocyte, fertilization, and blastulation were examined. Furthermore, COCs were incubated with 0 or 0.1% ethanol and then mitochondrial membrane potential (MMP) and the glucose consumption of COCs was determined. In addition, gene expression in oocytes was examined by RNA sequencing. Ethanol (0.1 and 0.2%) increased Mt-cn and Mt-protein levels whereas 0.2% ethanol increased the blastulation rate and ATP content in oocytes and decreased lipid content in oocytes. Ethanol (0.1%) increased MMP in oocytes and decreased glucose consumption of COCs. Eight stage embryos derived from 0.1% ethanol treated oocytes had higher levels of trimethyl-H3K9 compared with that of nontreated counterpart. RNA sequencing revealed that differentially expressed genes were associated with glycolysis/gluconeogenesis, carbon metabolism, sphingolipid metabolism, amino acid metabolism, and fatty acid degradation pathways. In conclusion, even 0.1% concentrations of ethanol during in vitro maturation considerably affects oocyte metabolism and histone configuration of embryos.

Monitoring the Resolution of Acute Exacerbation of Airway Bronchoconstriction in an Asthma Attack Using Capnogram Waveforms.

Patients with acute bronchospasm can show a distinct slope of the capnogram ("shark fin") as a result of asynchronous alveolar excretion. Although the slope of the upward alveolar plateau (phase III) in the capnogram waveforms of non-intubated patients is known to help monitor the therapeutic response to acute bronchospasm, little is known about the significance of its slope among intubated patients. Therefore, we quantified the phase III slope of an intubated patient with acute asthma to investigate whether capnogram waveforms could be useful for identifying the response to antibronchospasm treatment in real time. CASE SUMMARY: The patient was a 53-year-old man who had a history of asthma. He presented to the emergency department with the primary complaint of respiratory distress. He was diagnosed with severe asthma attack and required invasive mechanical ventilation for 10 days, during which we quantified the phase III slope of the capnogram. The phase III slope decreased during treatment, with a significant reduction from the third to the fourth day; however, a significant decrease in end-tidal carbon dioxide (EtCO2) was observed from the fifth to the sixth day. We found that the slope values decreased earlier than EtCO2 reduction, although the absolute EtCO2 values eventually decreased in response to antibronchospasm treatment. CONCLUSION: There were several reports that evaluated the phase III slope in non-intubated patients with asthma, but this is the first report measuring the phase III slope in an intubated patient over several days. Capnogram waveforms may serve as useful real-time indicators to monitor acute bronchospasm among mechanically ventilated patients.

In vitro spermatogenesis in isolated seminiferous tubules of immature mice.

Mouse spermatogenesis, from spermatogonial stem cell proliferation to sperm formation, can be reproduced in vitro by culturing testis tissue masses of neonatal mice. However, it remains to be determined whether this method is also applicable when testis tissues are further divided into tiny fragments, such as segments of the seminiferous tubule (ST), a minimal anatomical unit for spermatogenesis. In this study, we investigated this issue using the testis of an Acrosin-GFP/Histone H3.3-mCherry (Acr/H3) double-transgenic mouse and monitored the expression of GFP and mCherry as indicators of spermatogenic progression. Initially, we noticed that the cut and isolated stretches of ST shrunk rapidly and conglomerated. We therefore maintained the isolation of STs in two ways: segmental isolation without truncation or embedding in soft agarose. In both cases, GFP expression was observed by fluorescence microscopy. By whole-mount immunochemical staining, meiotic spermatocytes and round and elongating spermatids were identified as Sycp3-, crescent-form GFP-, and mCherry-positive cells, respectively. Although the efficiency was significantly lower than that with tissue mass culture, we clearly showed that spermatogenesis can be induced up to the elongating spermatid stage even when the STs were cut into short segments and cultured in isolation. In addition, we demonstrated that lowered oxygen tension was favorable for spermatogenesis both for meiotic progression and for producing elongating spermatids in isolated STs. Culturing isolated STs rather than tissue masses is advantageous for explicitly assessing the various environmental parameters that influence the progression of spermatogenesis.

An efficient environmental DNA detection method for rare species: a case study of a small salamander (Hynobius boulengeri).

Loss of biodiversity is a serious concern, and amphibians are particularly threatened. Most small salamanders in Japan are endangered. Distributional information is fundamental to the conservation of these rare species; however, small salamanders are generally difficult to locate or catch. Environmental DNA analysis is an effective survey method for monitoring such rare species. The conventional polymerase chain reaction (PCR) method, which combines PCR amplification with subsequent electrophoresis, and the real-time PCR method, which uses fluorescent material, are commonly used for this purpose. In this study, a comparison of these two detection methods was conducted using a rare salamander species, Hynobius boulengeri, as a model case. We compared three points: (i) detection sensitivity, (ii) influence of environmental factors related to detection, and (iii) time and financial costs of the two methods. To perform this comparison, we developed a real-time PCR detection assay, conducted field surveys, and compared the time and financial costs of conventional and real-time PCR methods. The comparison showed no statistical difference in the detection sensitivity from field samples, and the effects of environmental factors tended to be similar. In addition, the financial cost was lower for the conventional PCR method while the time cost was lower for the real-time PCR method. Therefore, selecting eDNA detection methods based on objectives, time, and financial costs will promote efficient monitoring and contribute to the conservation of rare species.

Seasonal ecosystem linkages contribute to the maintenance of migratory polymorphism in a salmonid population.

The influence of resource subsidies on animal growth, survival and reproduction is well understood, but their ultimate effects on life history have been less explored. Some wild species have a partially migratory life history, wherein migration is dictated based upon threshold traits regulated in part by the seasonal availability of resources. We conducted a large-scale field manipulation experiment where we provided a terrestrial invertebrate subsidy to red-spotted masu salmon. Individuals in stream reaches that received a subsidy had, on average, a 53% increase in growth rate relative to those in control reaches. This increased growth resulted in a greater proportion of individuals reaching the threshold body size and smolting in the autumn. Consequently, 19-55% of females in subsidized reaches became migratory, whereas 0-14% became migratory in the control reaches. Our findings highlight seasonal ecosystem linkage as a key ecosystem property for maintaining migratory polymorphism in partially migratory animals.

Floating plastic accumulation and distribution around Kuroshio Current, western North Pacific.

The distribution of floating plastic debris around the Kuroshio Current which transports plastics from the coastal waters of Asian countries to North Pacific subtropical gyre, was investigated in 2014. The mean abundance and weight of plastic debris on the sea surface were 100,376 counts/km2 and 446.16 g/km2, respectively. Intensive plastic accumulation was observed in the frontal area between the northern edge of the Kuroshio and coastal waters off Shikoku, while a relatively higher abundance in the south of Kuroshio was generally associated with anticyclonic mesoscale eddies. Such an accumulation resulted from the eddy-Kuroshio interactions which are specifically associated with the offshore non-large meandering Kuroshio path. Overall, white, fragmented, small-sized (≤1 mm) particles with polyethylene and polypropylene polymers were dominant. In the southern area of Kuroshio, the contribution of polystyrene and larger-sized plastic was higher, suggesting a rapid influx of fresh particles from western Japan to offshore by the northwest monsoon.

Paternal aging impacts mitochondrial DNA content and telomere length in mouse embryos.

Mitochondrial DNA (mtDNA) copy number and telomere length (TL) in blastocysts derived from the same male mice at young (10-19-week-old) and aged (40-49-week-old) time points and mtDNA and TL in the hearts of offspring derived from young and aged male mice were examined. Paternal aging correlated with reduced mtDNA and TL in blastocysts. mtDNA and TL were significantly correlated, which was also observed in bovine blastocysts. Moreover, mtDNA in the heart of offspring was reduced in male mice with paternal aging. In conclusion, paternal aging affects embryonic mtDNA and TL, potentially impacting their offspring.

Social agents as catalysts: Social dynamics in the classroom with book introduction robot.

One of the possible benefits of robot-mediated education is the effect of the robot becoming a catalyst between people and facilitating learning. In this study, the authors focused on an asynchronous active learning method mediated by robots. Active learning is believed to help students continue learning and develop the ability to think independently. Therefore, the authors improved the UGA (User Generated Agent) system that we have created for long-term active learning in COVID-19 to create an environment where children introduce books to each other via robots. The authors installed the robot in an elementary school and conducted an experiment lasting more than a year. As a result, it was confirmed that the robot could continue to be used without getting bored even over a long period of time. They also analyzed how the children created the contents by analyzing the contents that had a particularly high number of views. In particular, the authors observed changes in children's behavior, such as spontaneous advertising activities, guidance from upperclassmen to lowerclassmen, collaboration with multiple people, and increased interest in technology, even under conditions where the new coronavirus was spreading and children's social interaction was inhibited.

Spontaneous activity in whisker-innervating region of neonatal mouse trigeminal ganglion.

Spontaneous activity during the early postnatal period is thought to be crucial for the establishment of mature neural circuits. It remains unclear if the peripheral structure of the developing somatosensory system exhibits spontaneous activity, similar to that observed in the retina and cochlea of developing mammals. By establishing an ex vivo calcium imaging system, here we found that neurons in the whisker-innervating region of the trigeminal ganglion (TG) of neonatal mice generate spontaneous activity. A small percentage of neurons showed some obvious correlated activity, and these neurons were mostly located close to one another. TG spontaneous activity was majorly exhibited by medium-to-large diameter neurons, a characteristic of mechanosensory neurons, and was blocked by chelation of extracellular calcium. Moreover, this activity was diminished by the adult stage. Spontaneous activity in the TG during the first postnatal week could be a source of spontaneous activity observed in the neonatal mouse barrel cortex.

Characterization of human epithelial resident memory regulatory T cells.

Human resident memory regulatory T cells (Tregs) exist in the normal, noninflamed skin. Except one, all previous studies analyzed skin Tregs using full-thickness human skin. Considering that thick dermis contains more Tregs than thin epidermis, the current understanding of skin Tregs might be biased toward dermal Tregs. Therefore, we sought to determine the phenotype and function of human epidermal and epithelial Tregs. Human epidermis and epithelium were allowed to float on a medium without adding any exogenous cytokines and stimulations for two days and then emigrants from the explants were analyzed. Foxp3 was selectively expressed in CD4+CD103- T cells in the various human epithelia, as it is highly demethylated. CD4+CD103-Foxp3+ cells suppressed proliferation of other resident memory T cells. The generation and maintenance of epithelial Tregs were independent of hair density and Langerhans cells. Collectively, immune-suppressive CD4+CD103-Foxp3+ Tregs are present in the normal, noninflamed human epidermis and mucosal epithelia.

The Relationship between Vector Species Richness and the Risk of Vector-Borne Infectious Diseases.

AbstractInfectious diseases can impact human welfare and impede wildlife management. Much recent research explores whether biodiversity increases or decreases infectious disease risk. Here, we theoretically study the relationship between vector species richness and the risk of vector-borne diseases using an epidemiological model of a single host and multiple vectors. The model considers that vectors are involved in interspecific feeding interference that causes transmission interference and in interspecific recruitment competition that mediates susceptible vector regulation. The model reveals three possible shapes of the vector richness-disease risk relationship: monotonic amplification, hump-shaped, and monotonic dilution patterns. The monotonic amplification pattern occurs across a wide parameter region. The hump-shaped and monotonic dilution patterns are found when transmission interference is strong and recruitment competition is weak. Unexpectedly, susceptible vector regulation not only promotes dilution but can strengthen amplification if coupled with strong transmission interference. Our results suggest that vector richness might be more likely to cause amplification rather than dilution, and shifts in the community mean trait values of vectors could also affect disease risk along the vector richness gradient.