Thirteen Ovary-Enriched Genes Are Individually Not Essential for Female Fertility in Mice.

Infertility is considered a global health issue as it currently affects one in every six couples, with female factors reckoned to contribute to partly or solely 50% of all infertility cases. Over a thousand genes are predicted to be highly expressed in the female reproductive system and around 150 genes in the ovary. However, some of their functions in fertility remain to be elucidated. In this study, 13 ovary and/or oocyte-enriched genes (Ccdc58, D930020B18Rik, Elobl, Fbxw15, Oas1h, Nlrp2, Pramel34, Pramel47, Pkd1l2, Sting1, Tspan4, Tubal3, Zar1l) were individually knocked out by the CRISPR/Cas9 system. Mating tests showed that these 13 mutant mouse lines were capable of producing offspring. In addition, we observed the histology section of ovaries and performed in vitro fertilization in five mutant mouse lines. We found no significant anomalies in terms of ovarian development and fertilization ability. In this study, 13 different mutant mouse lines generated by CRISPR/Cas9 genome editing technology revealed that these 13 genes are individually not essential for female fertility in mice.

Multiple ageing effects on testicular/epididymal germ cells lead to decreased male fertility in mice.

In mammals, females undergo reproductive cessation with age, whereas male fertility gradually declines but persists almost throughout life. However, the detailed effects of ageing on germ cells during and after spermatogenesis, in the testis and epididymis, respectively, remain unclear. Here we comprehensively examined the in vivo male fertility and the overall organization of the testis and epididymis with age, focusing on spermatogenesis, and sperm function and fertility, in mice. We first found that in vivo male fertility decreased with age, which is independent of mating behaviors and testosterone levels. Second, overall sperm production in aged testes was decreased; about 20% of seminiferous tubules showed abnormalities such as germ cell depletion, sperm release failure, and perturbed germ cell associations, and the remaining 80% of tubules contained lower number of germ cells because of decreased proliferation of spermatogonia. Further, the spermatozoa in aged epididymides exhibited decreased total cell numbers, abnormal morphology/structure, decreased motility, and DNA damage, resulting in low fertilizing and developmental rates. We conclude that these multiple ageing effects on germ cells lead to decreased in vivo male fertility. Our present findings are useful to better understand the basic mechanism behind the ageing effect on male fertility in mammals including humans.

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.

Age-related decline in spermatogenic activity accompanied with endothelial cell senescence in male mice.

Male fertility decreases with aging, with spermatogenic decline being one of its causes. Altered testis environment is suggested as a cause of the phenotype; however, the associated mechanisms remain unclear. Herein, we investigated the age-related changes in testicular somatic cells on spermatogenic activity. The number and proliferation of spermatogonia significantly reduced with aging in mice. Interestingly, senescence-associated ß-galactosidase-positive cells appeared in testicular endothelial cell (EC) populations, but not in germ cell populations, with aging. Transcriptome analysis of ECs indicated that senescence occurred in the ECs of aged mice. Furthermore, the support capacity of ECs for spermatogonial proliferation significantly decreased with aging; however, the senolytic-induced removal of senescent cells from aged ECs restored their supporting capacity to a comparable level as that of young ECs. Our results suggest that the accumulation of senescent ECs in the testis is a potential factor contributing to the age-related decline in spermatogenic activity.

Posture Classification with a Bed-Monitoring System Using Radio Frequency Identification.

Aging of the population and the declining birthrate in Japan have produced severe human resource shortages in the medical and long-term care industries. Reportedly, falls account for more than 50% of all accidents in nursing homes. Recently, various bed-release sensors have become commercially available. In fact, clip sensors, mat sensors, and infrared sensors are used widely in hospitals and nursing care facilities. We propose a simple and inexpensive monitoring system for elderly people as a technology capable of detecting bed activity, aimed particularly at preventing accidents involving falls. Based on findings obtained using that system, we aim at realizing a simple and inexpensive bed-monitoring system that improves quality of life. For this study, we developed a bed-monitoring system for detecting bed activity. It can predict bed release using RFID, which can achieve contactless measurements. The proposed bed-monitoring system incorporates an RFID antenna and tags, with a method for classifying postures based on the RFID communication status. Experimentation confirmed that three postures can be classified with two tags, seven postures with four tags, and nine postures with six tags. The detection rates were 90% for two tags, 75% for four tags, and more than 50% for six tags.

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.

Development of a remotely controllable 4 m long aerial-hose-type firefighting robot.

In a fire outbreak, firefighters are expected to rapidly extinguish fires to stop the spread of damage and prevent secondary disasters. We proposed the concept of a dragon firefighter (DFF), which is a flying-hose-type firefighting robot. We developed a 3.6 m long DFF equipped with two nozzle units and achieved stable flight. However, the system was not yet completed because the root of the robot, which should have been operated remotely, was operated manually. In addition, the system's reliability was insufficient to successfully repeat the demonstration several times. The development of a robot demonstration system is crucial for the practical application of such a firefighting robot. In this study, we developed a demonstration system for a remotely controllable 4 m flying firehose robot for demonstration at the World Robot Summit 2020 (WRS 2020) opening ceremony in Fukushima as a milestone. This paper focuses on the following issues: 1): installation of the remotely controllable mobile base, 2): redesign of the water channels (the sizes of nozzle outlets) to get enough thrusts to fly with a fire engine, 3): development of nozzle units with a larger movable range (1.5 times larger than the conventional nozzle) in addition to waterproofing technique to improve system reliability, and 4): redesign of a passive damping mechanism to ensure better stability. Thus, a firefighting demonstration was successfully conducted at the opening ceremony of the World Robot Summit 2020 in Fukushima, Japan, and we discuss the lessons learned through the demonstration. We found that the developed DFF system incorporating a mobile base could achieve remote fire extinguishing.

Efficient simultaneous double DNA knock-in in murine embryonic stem cells by CRISPR/Cas9 ribonucleoprotein-mediated circular plasmid targeting for generating gene-manipulated mice.

Gene targeting of embryonic stem (ES) cells followed by chimera production has been conventionally used for developing gene-manipulated mice. Although direct knock-in (KI) using murine zygote via CRISPR/Cas9-mediated genome editing has been reported, ES cell targeting still has merits, e.g., high throughput work can be performed in vitro. In this study, we first compared the KI efficiency of mouse ES cells with CRISPR/Cas9 expression vector and ribonucleoprotein (RNP), and confirmed that KI efficiency was significantly increased by using RNP. Using CRISPR/Cas9 RNP and circular plasmid with homologous arms as a targeting vector, knock-in within ES cell clones could be obtained efficiently without drug selection, thus potentially shortening the vector construction or cell culture period. Moreover, by incorporating a drug-resistant cassette into the targeting vectors, double DNA KI can be simultaneously achieved at high efficiency by a single electroporation. This technique will help to facilitate the production of genetically modified mouse models that are fundamental for exploring topics related to human and mammalian biology.

Osmotic Pressure and Diffusion of Ions in Charged Nanopores.

The transport of ions and water in nanopores is of interest for a number of natural and technological processes. Due to their practically identical long straight cylindrical pores, nanoporous track-etched membranes are suitable materials for investigation of its mechanisms. This communication reports on simultaneous measurements of osmotic pressure and salt diffusion with a 24 nm pore track-etched membrane. Due to the use of dilute electrolyte solutions (1-4 mM KCl and LiCl), this pore size was commensurate with the Debye screening length. Advanced interpretation of experimental results using a full version of the space-charge model has revealed that osmotic pressure and salt diffusion can be quantitatively correlated with electrostatic interactions of ions with charged nanopore walls. The surface-charge density is shown to increase with electrolyte concentration in agreement with the mechanism of deprotonation of weakly acidic surface groups. Moreover, a lack of significant surface-charge dependence on the kind of cation (K+ or Li+) demonstrates that binding of salt counterions does not play a major role in this system.

The pivotal role of pituitary adenylate cyclase-activating polypeptide for lactate production and secretion in astrocytes during fear memory.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an essential role in the modulation of astrocyte functions. Although lactate secretion from astrocytes contributes to many forms of neuronal plasticity in the central nervous system, including fear learning and memory, the role of PACAP in lactate secretion from astrocytes is unclear. METHODS: The amygdala and hippocampus of PACAP (+ / +) and PACAP (-/-) mice were acquired 1 h after memory acquisition and recall in the passive avoidance test. The concentration of glycogen and lactate in these regions was measured. The concentration of lactate in the hippocampus's extracellular fluid was also measured by microdialysis during memory acquisition or intracerebroventricular administration of PACAP. RESULTS: We observed that memory acquisition caused a significant decrease in glycogen concentration and increased lactate concentration in the PACAP (+ / +) mice's hippocampus. However, memory acquisition did not increase in the lactate concentration in PACAP (-/-) mice's hippocampus. Further, memory retrieval evoked lactate production in the amygdala and the hippocampus of PACAP (+ / +) mice. Still, there was no significant increase in lactate concentration in the same regions of PACAP (-/-) mice. In vivo microdialysis in rats revealed that the hippocampus's extracellular lactate concentration increased after a single PACAP intracerebroventricular injection. Additionally, the hippocampus's extracellular lactate concentration increased with the memory acquisition in PACAP (+ / +) mice, but not in PACAP (-/-) mice. CONCLUSIONS: PACAP may enhance lactate production and secretion in astrocytes during the acquisition and recall of fear memories.

RNA-binding protein Ptbp1 regulates alternative splicing and transcriptome in spermatogonia and maintains spermatogenesis in concert with Nanos3.

PTBP1, a well-conserved RNA-binding protein, regulates cellular development by tuning posttranscriptional mRNA modification such as alternative splicing (AS) or mRNA stabilization. We previously revealed that the loss of Ptbp1 in spermatogonia causes the dysregulation of spermatogenesis, but the molecular mechanisms by which PTBP1 regulates spermatogonium homeostasis are unclear. In this study, changes of AS or transcriptome in Ptbp1-knockout (KO) germline stem cells (GSC), an in vitro model of proliferating spermatogonia, was determined by next generation sequencing. We identified more than 200 differentially expressed genes, as well as 85 genes with altered AS due to the loss of PTBP1. Surprisingly, no differentially expressed genes overlapped with different AS genes in Ptbp1-KO GSC. In addition, we observed that the mRNA expression of Nanos3, an essential gene for normal spermatogenesis, was significantly decreased in Ptbp1-KO spermatogonia. We also revealed that PTBP1 protein binds to Nanos3 mRNA in spermatogonia. Furthermore, Nanos3+/-;Ptbp1+/- mice exhibited abnormal spermatogenesis, which resembled the effects of germ cell-specific Ptbp1 KO, whereas no significant abnormality was observed in mice heterozygous for either gene alone. These data implied that PTBP1 regulates alternative splicing and transcriptome in spermatogonia under different molecular pathways, and contributes spermatogenesis, at least in part, in concert with NANOS3.

EFCAB2 is a novel calcium-binding protein in mouse testis and sperm.

Calcium-binding proteins regulate ion metabolism and the necessary signaling pathways for the maturational events of sperm. Our aim is to identify the novel calcium-binding proteins in testis. The gene EFCAB2 (GenBank NM_026626.3, NP_080902.1) was not previously examined, and its properties and exact mechanisms of action are unknown. In this study, we performed phylogenetic and structure prediction analyses of EFCAB2, which displays definitive structural features. Additionally, the distribution, localization, and calcium binding ability of mouse EFCAB2 were investigated. Results revealed extensive conservation of EFCAB2 among different eukaryotic orthologs. The constructed 3D model predicted that mouse EFCAB2 contains seven α-helices and two EF-hand motifs. The first EF-hand motif is located in N-terminal, while the second is located in C-terminal. By aligning the 3D structure of Ca2+-binding loops from EFCAB2 with calmodulin, we predicted six residues that might be involved in Ca2+ binding. The distribution of the Efcab2 mRNA, as determined by northern blotting, was detected only in the testis among mouse tissues. Native and recombinant EFCAB2 protein were detected by western blotting as one band at 20 kDa. In situ hybridization and immunohistochemical analyses showed its localization specifically in spermatogenic cells from primary spermatocytes to elongate spermatids within the seminiferous epithelium, but neither spermatogonia nor somatic cells were expressed. Moreover, EFCAB2 was specifically localized to the principal piece of cauda epididymal sperm flagellum. Furthermore, the analyses of purified recombinant EFCAB2 by Stains-all, ruthenium red staining, and by applying in vitro autoradiography assay showed that the physiological function of this protein is Ca2+ binding. These results suggested that EFCAB2 might be involved in the control of sperm flagellar movement. Altogether, here we describe about EFCAB2 as a novel calcium-binding protein in mouse testis and sperm.

A case of tooth fracture occurred upon medicating bisphosphonate for an elderly person: Preservation therapy and responses for Stage 0 of bisphosphonate-related osteonecrosis of jaw.

This case report aimed to report the progress of preservation therapy and response of symptoms and signs for Stage 0 of bisphosphonate-related osteonecrosis of jaw (BRONJ). A 68-year-old female was recognized having a tooth at the left upper first molar fracture upon medicating bisphosphonate (BP) in 2007. At that time, the extraction of the tooth was an absolute contraindication. Therefore, we performed preservation therapy. We observed the symptoms and signs every month. After 5 months, swelling and redness in the entire first molar tooth were seen and fistula formed partly. Bone exposure was not seen. We administrated antibiotics immediately. As a result, symptoms disappeared. On April 10, 2009, the patient visited us as she felt a sense of incongruity in the lower left first and second molar teeth. Clinically, there were no symptoms of pain. However, we observed the radiolucent finding in about 5 mm diameter at apical position by X-ray photography; we considered a possibility of Stage 0 for BRONJ. We immediately administered medicine for 5 days and the symptoms disappeared. At present, no inflammation with signs and symptoms at the upper left first molar and lower left first, second molar parts is shown. We performed preservation therapy for tooth fracture case medicating of BP. Immediate responses for inflammation and symptoms of the Stage 0 of BRONJ have led to success. Hence, dentists should perform regular clinical observation, and enough education to the patient for BRONJ is necessary.

Occlusal Dysesthesia: A Clinical Report on the Psychosomatic Management of a Japanese Patient Cohort.

PURPOSE: A cohort of Japanese patients diagnosed with occlusal dysesthesia (OD) was clinically analyzed for psychosomatic background, management, and treatment outcome. MATERIALS AND METHODS: The study group comprised 61 patients (17 men and 44 women) who met the OD criteria. Treatment outcomes were categorized as improvement, interruption, and transfer to another department. RESULTS: The diagnosed OD was resolved in 25 patients (41%), 20 patients (33%) discontinued treatment, 13 (21%) were referred or transferred to other specialties such as psychiatry, and 3 (5%) continued to receive treatment following an engagement period of 3 months, 2 years, and 5 years, respectively. Among the 20 patients who discontinued treatment, complaints persisted for 10 and they did not comply with treatment, 1 had immodithymia characterized by adherence to symptoms, 3 had depressive states, 2 were suspected to have schizophrenia, and 2 were suspected to have so-called phantom bite syndrome. CONCLUSION: This study suggests that OD treatment should take into account the underlying psychiatric disorder manifesting as physical complaints.

Catalytic hypervalent iodine oxidation using 4-iodophenoxyacetic acid and oxone: oxidation of p-alkoxyphenols to p-benzoquinones.

A catalytic hypervalent iodine oxidation of p-alkoxyphenols using 4-iodophenoxyacetic acid (1a) and Oxone((R)) was developed. Reaction of p-alkoxyphenol (2) with a catalytic amount of 1a in the presence of Oxone((R)) as a co-oxidant in 2,2,2-trifluoroethanol-water (1 : 2) gave the corresponding p-quinone (3) in excellent yield without special operation. The substituent effect on iodobenzene ring in the oxidation was observed; p-alkoxy is the most effective, with the series following the approximate order p-RO>p-Me, o-MeO, m-MeO>H>o-CO(2)H. And remarkable solvent effects were observed.

Catalytic hypervalent iodine oxidation of p-dialkoxybenzenes to p-quinones using 4-iodophenoxyacetic acid and Oxone.

A catalytic hypervalent iodine oxidation of p-dialkoxybenzenes using 4-iodophenoxyacetic acid (1) and 2KHSO5 x KHSO4 x K2SO4 (Oxone) was developed. Reaction of p-dialkoxybenzenes (2) with a catalytic amount of 1 in the presence of Oxone as a co-oxidant in 2,2,2-trifluoroethanol-water (1 : 2) gave the corresponding p-quinones (3) in excellent yields without purification. This procedure was applied to synthesis of blattellaquinone (9), the sex pheromone of the German cockroach, Blattella germanica.