Supplementation with autologous adipose stem cell-derived mitochondria can be a safe and promising strategy for improving oocyte quality.

PURPOSE: In our previous study, we confirmed that the supplementation of vitrified-warmed murine oocytes with autologous adipose stem cell (ASC)-derived mitochondria during intracytoplasmic sperm injection enhances post-fertilization developmental competence in mice. To ensure the safety of this technology, we conducted a thorough study in mice to investigate the potential presence of specific malformations in offspring developed from this approach. METHODS: A transgenerational comparative analysis was conducted on founder mice from embryos that developed after mitochondrial supplementation, and two subsequent generations. Reproductive performance, body growth rate, histopathological parameters, hematological parameters, daily activity patterns, and daily body temperature changes in male and female mice across these three generations were assessed in comparison to wild-type mice of the same age. RESULTS: Both male and female animals in all three generations showed comparable reproductive performance to the control group. Additionally, body growth rate by the age of 8 weeks were found to be comparable to controls across all three generations. Notably, no significant histopathological abnormalities were detected in vital organs, including the brain, heart, liver, kidneys, lungs, ovaries, and testes, in any individuals from the studied cohorts. The blood parameters were consistent with the control data. The continuous monitoring of activity and body temperature changes (both day and night) over a 1-week period revealed a pattern closely resembling that observed in the control animals. CONCLUSION: Injection of ASC-mitochondria into oocytes may be a promising technique to support developmental potential without causing adverse epigenetic events in the offspring in mice. However, before considering clinical application, additional safety screening using larger animals or non-human primates is essential.

CFP/Yit: An Inbred Mouse Strain with Slow Gastrointestinal Transit.

BACKGROUND: Gastrointestinal transit (GIT) is influenced by factors including diet, medications, genetics, and gut microbiota, with slow GIT potentially indicating a functional disorder linked to conditions, such as constipation. Although GIT studies have utilized various animal models, few effectively model spontaneous slow GIT. AIMS: We aimed to characterize the GIT phenotype of CFP/Yit (CFP), an inbred mouse strain with suggested slow GIT. METHODS: Female and male CFP mice were compared to Crl:CD1 (ICR) mice in GIT and assessed based on oral gavage of fluorescent-labeled 70-kDa dextran, feed intake, fecal amount, and fecal water content. Histopathological analysis of the colon and analysis of gut microbiota were conducted. RESULTS: CFP mice exhibited a shorter small intestine and a 1.4-fold longer colon compared to ICR mice. The median whole-GIT time was 6.0-fold longer in CFP mice than in ICR mice. CFP mice demonstrated slower gastric and cecal transits than ICR mice, with a median colonic transit time of 4.1 h (2.9-fold longer). CFP mice exhibited lower daily feed intakes and fecal amounts. Fecal water content was lower in CFP mice, apparently attributed to the longer colon. Histopathological analysis showed no changes in CFP mice, including tumors or inflammation. Moreover, CFP mice had a higher Firmicutes/Bacteroidota ratio and a relative abundance of Erysipelotrichaceae in cecal and fecal contents. CONCLUSIONS: This study indicates that CFP mice exhibit slow transit in the stomach, cecum, and colon. As a novel mouse model, CFP mice can contribute to the study of gastrointestinal physiology and disease.

Using the DNA Integrity Number to Analyze DNA Quality in Specimens Collected from Liquid-Based Cytology after Fine-Needle Aspiration of Breast Tumors and Lesions.

INTRODUCTION: Cancer genome analysis using next-generation sequencing requires adequate and high-quality DNA samples. Genomic analyses were conventionally performed using formalin-fixed paraffin-embedded sections rather than cytology samples such as cell block or smear specimens. Specimens collected from liquid-based cytology (LBC) have the potential to be sources of high-quality DNA suitable for genetic analysis even after long-term storage. METHODS: We collected breast tumor/lesion fractions from 92 residual LBC specimens using fine-needle aspiration (FNA) biopsy, including breast carcinoma (1 invasive carcinoma and 4 ductal carcinomas in situ), papillomatous lesion (5 intraductal papillomas), and fibroepithelial lesion (19 phyllodes tumors and 53 fibroadenomas) samples, and others (1 ductal adenoma, 1 hamartoma, 1 fibrocystic disease, and 7 unknown). DNA was extracted from all samples and subjected to DNA integrity number (DIN) score analysis. RESULTS: Average DIN score collected from 92 LBC specimens was significantly higher score. In addition, high-quality DNA with high DIN values (7.39 ± 0.80) was successfully extracted more than 12 months after storage of residual LBC specimens. CONCLUSION: Residual LBC specimens collected from FNA of the breast were verified to carry high-quality DNA and could serve as an alternate source for genetic analysis.

Mature mRNA processing that deletes 3' end sequences directs translational activation and embryonic development.

Eggs accumulate thousands of translationally repressed mRNAs that are translated into proteins after fertilization to direct diverse developmental processes. However, molecular mechanisms underlying the translation of stored mRNAs after fertilization remain unclear. Here, we report a previously unknown RNA processing of 3' end sequences of mature mRNAs that activates the translation of stored mRNAs. Specifically, 9 to 72 nucleotides at the 3' ends of zebrafish pou5f3 and mouse Pou5f1 mRNAs were deleted in the early stages of development. Reporter assays illustrated the effective translation of the truncated forms of mRNAs. Moreover, promotion and inhibition of the shortening of 3' ends accelerated and attenuated Pou5f3 accumulation, respectively, resulting in defective development. Identification of proteins binding to unprocessed and/or processed mRNAs revealed that mRNA shortening acts as molecular switches. Comprehensive analysis revealed that >250 mRNAs underwent this processing. Therefore, our results provide a molecular principle that triggers the translational activation and directs development.

Relationship Between Amplicon Size and Heat Conditions in Polymerase Chain Reaction Detection of DNA Degraded by Autoclaving.

This study examined the influence of heat exposure on DNA samples during polymerase chain reaction (PCR) detection. In this study, λDNA samples, as model DNA, were exposed to 105°C for 3-90 minutes or to 105°C-115°C for 15 minutes by autoclaving. The exposed samples were subjected to real-time PCR using nine primer sets with amplicon sizes of 45-504 bp. Regarding DNA samples exposed to 105°C by autoclaving, the data showed negative correlations between the logarithm of λDNA concentration (log λDNA) calculated using real-time PCR and exposure duration and a good relationship between the slope of the regression line and amplicon size. Regarding λDNA samples exposed to heat for 15 minutes, the data showed negative correlations between the log λDNA and exposure temperature and a good relationship between the slope of the regression line and amplicon size. These results showed that the equations used in this study could predict the degree of degradation in λDNA samples by autoclaving, and the PCR detection levels of the DNA at each amplicon size.

Human gut microbiota influences drug-metabolizing enzyme hepatic Cyp3a: A human flora-associated mice study.

Several studies revealed that gut microbiota affects the hepatic drug-metabolizing enzyme cytochrome P450 (Cyp). We hypothesized that individual gut microbiota variations could contribute to CYP activity. Human flora-associated (HFA) mice are established from germ-free mice using human feces and are often used to determine the effect of the human gut microbiota on the host. This study generated two groups of HFA mice using feces from two healthy individuals. Then, the composition of gut microbiota and hepatic Cyp activity was compared to analyze the effects of gut microbiota in healthy individuals on hepatic Cyp activity. A principal coordinate analysis based on the UniFrac distance for the composition of the cecal and fecal microbiota revealed apparent differences between the recipient groups. Hepatic Cyp, which is a marked difference in Cyp3a activity and Cyp3a11 gene expression, was observed between the recipient groups. Cyp2c and Cyp1a activities did not differ between recipient groups, with significantly lower enzymatic activities in recipients than in germ-free mice. These results indicate that the human gut microbiota affects hepatic Cyp activity. Especially, human gut microbiota composition differences have a pronounced effect on Cyp3a activity via Cyp3a11 gene expression regulation. Therefore, human gut microbiota variations among individuals may affect numerous drug metabolism, leading to drug efficacy and toxicity.

Identification of embryonic RNA granules that act as sites of mRNA translation after changing their physical properties.

Fertilized eggs begin to translate mRNAs at appropriate times and placements to control development, but how the translation is regulated remains unclear. Here, we found that pou5f3 mRNA encoding a transcriptional factor essential for development formed granules in a dormant state in zebrafish oocytes. Although the number of pou5f3 granules remained constant, Pou5f3 protein accumulated after fertilization. Intriguingly, signals of newly synthesized peptides and a ribosomal protein became colocalized with pou5f3 granules after fertilization and, moreover, nascent Pou5f3 was shown to be synthesized in the granules. This functional change was accompanied by changes in the state and internal structure of granules. Dissolution of the granules reduced the rate of protein synthesis. Similarly, nanog and sox19b mRNAs in zebrafish and Pou5f1/Oct4 mRNA in mouse assembled into granules. Our results reveal that subcellular compartments, termed embryonic RNA granules, function as activation sites of translation after changing physical properties for directing vertebrate development.

Development of incident severity classification for laboratory animals.

An incident reporting system (IRS) prevents possible adverse events by collecting and analyzing incidents that occur. However, few studies are available regarding IRSs in the laboratory animal field. This study aimed to develop an incident severity classification for laboratory animals (ISCLA) to evaluate the usefulness of the IRS in laboratory animal facilities. Twenty-three incidents reported from March 2019 to February 2020 on our IRS were retrospectively reviewed. Three of the 23 incidents failed to obtain some experimental data. Two of these incidents were harmless to animals, but the other caused the animals moderate distress. In addition, two of the three incidents made animals unsuitable for experiments. Since the inconsistent impact of incidents on animals and experiments prevented the comparison of the severity of individual incidents, we developed the ISCLA. According to the ISCLA, the above three incidents were classified into Category 3b and 4a. The others were classified into Category 0 (n=5), 1 (n=6), 2 (n=3), and 3a (n=6) in ascending order of severity. No incident was classified into Category 4b and 5. Furthermore, incidents occurring in the animal housing area were more severe than those occurring in the supporting area (P=0.002). This study showed that incident occurrences had characteristics that were not visible from individual incidents alone. Moreover, the ISCLA was considered useful when conducting the IRS and taking improvement measures in laboratory animal facilities.

Meiosis-specific ZFP541 repressor complex promotes developmental progression of meiotic prophase towards completion during mouse spermatogenesis.

During spermatogenesis, meiosis is accompanied by a robust alteration in gene expression and chromatin status. However, it remains elusive how the meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identify a protein complex that consists of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulators in mouse meiotic prophase progression. Our genetic study shows that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of the meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses identify that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrates that a germ-cell specific complex that contains ZFP541 and KCTD19 promotes the progression of meiotic prophase towards completion in male mice, and triggers the reconstruction of the transcriptional network and chromatin organization leading to post-meiotic development.

Maintenance DNA methylation in pre-meiotic germ cells regulates meiotic prophase by facilitating homologous chromosome pairing.

Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 - two essential proteins for maintenance DNA methylation - are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95- or Dnmt1-deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants. Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.

KCTD19 and its associated protein ZFP541 are independently essential for meiosis in male mice.

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.

Heat-retention effects of hydrogen-rich water bath assessed by thermography for humans.

Hydrogen-rich water bath devices are commercially available, but have been scarcely clarified for heat-retention effects. In this study, heat-retention effects of hydrogen-rich water bath were assessed by thermographic clinical trials, which employed twenty-four healthy subjects. The thermograms indicated that, under the same conditions (41 °C, 10-min bathing), hydrogen-rich water bath (hydrogen concentrations: 185-548 µg/L; oxidation-reduction potentials: -167 to -91 mV, versus 0.8 µg/L and +479 mV for normal bath, respectively) brought about the heat-retention being more marked than those of normal water bath for several body-parts in the order as follows: abdomen > upper legs > arms > hands > feet, for 30- and 60-min post-bathing, being in contrast to scarce heat-retention for head, armpits and lower legs. Then, as reflection to promotive effects on blood stream, we also examined the thickness of fingertip-capillary in hands. The thickness was expanded in the hydrogen-rich water bath more markedly than that in the normal water bath, suggesting that the hydrogen-rich water bath may have the hydrogen-based promotive effect, exceeding over mere heat retention-based effects, on blood circulation of the whole body. Meanwhile, the heat-retention in hydrogen-rich water bath weakly or moderately correlated with contents of the subcutaneous fat, whole body fat and body mass index, and inversely correlated with skeletal muscle rates, although their correlation degrees did not obviously exceed over normal water bath, with a poor relation with the basal metabolism rate. Thus, the hydrogen-rich water bath was suggested to exert heat-retention effects exceeding over normal water bath, in diverse body-parts such as abdomen, upper legs, arms and hands, via promotion to blood flow which was reflected by expanding the thickness of capillary. The heat-retention after bathing can be noted as effects of the hydrogen-rich water bath, which is applicable for most of people widespread regardless of their body composition index.

Changes in subcellular structures and states of pumilio 1 regulate the translation of target Mad2 and cyclin B1 mRNAs.

Temporal and spatial control of mRNA translation has emerged as a major mechanism for promoting diverse biological processes. However, the molecular nature of temporal and spatial control of translation remains unclear. In oocytes, many mRNAs are deposited as a translationally repressed form and are translated at appropriate times to promote the progression of meiosis and development. Here, we show that changes in subcellular structures and states of the RNA-binding protein pumilio 1 (Pum1) regulate the translation of target mRNAs and progression of oocyte maturation. Pum1 was shown to bind to Mad2 (also known as Mad2l1) and cyclin B1 mRNAs, assemble highly clustered aggregates, and surround Mad2 and cyclin B1 RNA granules in mouse oocytes. These Pum1 aggregates were dissolved prior to the translational activation of target mRNAs, possibly through phosphorylation. Stabilization of Pum1 aggregates prevented the translational activation of target mRNAs and progression of oocyte maturation. Together, our results provide an aggregation-dissolution model for the temporal and spatial control of translation.

Meiotic cohesins mediate initial loading of HORMAD1 to the chromosomes and coordinate SC formation during meiotic prophase.

During meiotic prophase, sister chromatids are organized into axial element (AE), which underlies the structural framework for the meiotic events such as meiotic recombination and homolog synapsis. HORMA domain-containing proteins (HORMADs) localize along AE and play critical roles in the regulation of those meiotic events. Organization of AE is attributed to two groups of proteins: meiotic cohesins REC8 and RAD21L; and AE components SYCP2 and SYCP3. It has been elusive how these chromosome structural proteins contribute to the chromatin loading of HORMADs prior to AE formation. Here we newly generated Sycp2 null mice and showed that initial chromatin loading of HORMAD1 was mediated by meiotic cohesins prior to AE formation. HORMAD1 interacted not only with the AE components SYCP2 and SYCP3 but also with meiotic cohesins. Notably, HORMAD1 interacted with meiotic cohesins even in Sycp2-KO, and localized along cohesin axial cores independently of the AE components SYCP2 and SYCP3. Hormad1/Rad21L-double knockout (dKO) showed more severe defects in the formation of synaptonemal complex (SC) compared to Hormad1-KO or Rad21L-KO. Intriguingly, Hormad1/Rec8-dKO but not Hormad1/Rad21L-dKO showed precocious separation of sister chromatid axis. These findings suggest that meiotic cohesins REC8 and RAD21L mediate chromatin loading and the mode of action of HORMAD1 for synapsis during early meiotic prophase.

Posttranscriptional regulation of maternal Pou5f1/Oct4 during mouse oogenesis and early embryogenesis.

Protein syntheses at appropriate timings are important for promoting diverse biological processes and are controlled at the levels of transcription and translation. Pou5f1/Oct4 is a transcription factor that is essential for vertebrate embryonic development. However, the precise timings when the mRNA and protein of Pou5f1/Oct4 are expressed during oogenesis and early stages of embryogenesis remain unclear. We analyzed the expression patterns of mRNA and protein of Pou5f1/Oct4 in mouse oocytes and embryos by using a highly sensitive in situ hybridization method and a monoclonal antibody specific to Pou5f1/Oct4, respectively. Pou5f1/Oct4 mRNA was detected in growing oocytes from the primary follicle stage to the fully grown GV stage during oogenesis. In contrast, Pou5f1/Oct4 protein was undetectable during oogenesis, oocyte maturation and the first cleavage stage but subsequently became detectable in the nuclei of early 2-cell-stage embryos. Pou5f1/Oct4 protein at this stage was synthesized from maternal mRNAs stored in oocytes. The amount of Pou5f1/Oct4 mRNA in the polysomal fraction was small in GV-stage oocytes but was significantly increased in fertilized eggs. Taken together, our results indicate that the synthesis of Pou5f1/Oct4 protein during oogenesis and early stages of embryogenesis is controlled at the level of translation and suggest that precise control of the amount of this protein by translational regulation is important for oocyte development and early embryonic development.

Effects of gut microbiota on in vivo metabolism and tissue accumulation of cytochrome P450 3A metabolized drug: Midazolam.

The link between drug-metabolizing enzymes and gut microbiota is well established. In particular, hepatic cytochrome P450 (CYP) 3A activities are presumed to be affected by gut microbiota. However, there is no direct evidence that the gut microbiota affects CYP3A metabolism or the clearance of clinically relevant drugs in vivo. Our purpose was to evaluate the effects of gut microbiota on in vitro and in vivo drug metabolism and on the clearance of midazolam, which is a standard CYP3A metabolized drug. Hepatic Cyp3a activity and in vitro midazolam hydroxylase activity were compared using specific pathogen-free (SPF) and germ-free (GF) mice. In a pharmacokinetics (PK) study, SPF and GF mice were intraperitoneally injected with 60 mg/kg of midazolam, and plasma and tissue concentrations were measured. Hepatic Cyp3a activity and midazolam hydroxylase activity were significantly lower in GF mice than in SPF mice. Notably, in the PK study, the area under the plasma concentration-time curve from time zero to infinity and the elimination half-life were approximately four-fold higher in GF mice compared with SPF mice. Furthermore, the concentration of midazolam in the brain 180 min after administration was about 14-fold higher in GF mice compared with SPF mice. Together, our results demonstrated that the gut microbiota altered the metabolic ability of Cyp3a and the tissue accumulation of midazolam.

Gene disruption of medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2) causes movement retardation.

Transglutaminases are an enzyme family that catalyses protein cross-linking essential for several biological functions. In the previous studies, we characterized the orthologues of the mammalian transglutaminase family in medaka (Oryzias latipes), an established fish model. Among the human isozymes, tissue-type transglutaminase (TG2) has multiple functions that are involved in several biological phenomena. In this study, we established medaka mutants deficient for the orthologue of human TG2 using the CRISPR/Cas9 and transcription activator-like effector nucleases systems. Although apparent morphological changes in the phenotype were not observed, movement retardation was found in the mutant fish when evaluated by a tank-diving test. Furthermore, comparative immunohistochemistry analysis using in this fish model revealed that orthologue of human TG2 was expressed at the periventricular layer of the optic tectum. Our findings provide novel insight for the relationship between tissue-type transglutaminase and the nervous system and the associated behaviour.

Germ cell-intrinsic effects of sex chromosomes on early oocyte differentiation in mice.

A set of sex chromosomes is required for gametogenesis in both males and females, as represented by sex chromosome disorders causing agametic phenotypes. Although studies using model animals have investigated the functional requirement of sex chromosomes, involvement of these chromosomes in gametogenesis remains elusive. Here, we elicit a germ cell-intrinsic effect of sex chromosomes on oogenesis, using a novel culture system in which oocytes were induced from embryonic stem cells (ESCs) harboring XX, XO or XY. In the culture system, oogenesis using XO and XY ESCs was severely disturbed, with XY ESCs being more strongly affected. The culture system revealed multiple defects in the oogenesis of XO and XY ESCs, such as delayed meiotic entry and progression, and mispairing of the homologous chromosomes. Interestingly, Eif2s3y, a Y-linked gene that promotes proliferation of spermatogonia, had an inhibitory effect on oogenesis. This led us to the concept that male and female gametogenesis appear to be in mutual conflict at an early stage. This study provides a deeper understanding of oogenesis under a sex-reversal condition.

Pumilio1 phosphorylation precedes translational activation of its target mRNA in zebrafish oocytes.

SummaryTranslational regulation of mRNAs is crucial for promoting various cellular and developmental processes. Pumilio1 (Pum1) has been shown to play key roles in translational regulation of target mRNAs in many systems of diverse organisms. In zebrafish immature oocytes, Pum1 was shown to bind to cyclin B1 mRNA and promote the formation of cyclin B1 RNA granules. This Pum1-mediated RNA granule formation seemed critical to determine the timing of translational activation of cyclin B1 mRNA during oocyte maturation, leading to activation of maturation/M-phase-promoting factor (MPF) at the appropriate timing. Despite its fundamental importance, the mechanisms of translational regulation by Pum1 remain elusive. In this study, we examined the phosphorylation of Pum1 as a first step to understand the mechanisms of Pum1-mediated translation. SDS-PAGE analyses and phosphatase treatments showed that Pum1 was phosphorylated at multiple sites during oocyte maturation. This phosphorylation began in an early period after induction of oocyte maturation, which preceded the polyadenylation of cyclin B1 mRNA. Interestingly, depolymerization of actin filaments in immature oocytes caused phosphorylation of Pum1, disassembly of cyclin B1 RNA granules, and polyadenylation of cyclin B1 mRNA but not translational activation of the mRNA. Overexpression of the Pum1 N-terminus prevented the phosphorylation of Pum1, disassembly of cyclin B1 RNA granules, and translational activation of the mRNA even after induction of oocyte maturation. These results suggest that Pum1 phosphorylation in the early period of oocyte maturation is one of the key processes for promoting the disassembly of cyclin B1 RNA granules and translational activation of target mRNA.

High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes.

BACKGROUND: Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method. RESULTS: The mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs. CONCLUSIONS: This high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization.