Effective cryopreservation protocol for preservation of male primate (Macaca fascicularis) prepubertal fertility.

RESEARCH QUESTION: Can specimen types (cells versus tissues) and additive cryoprotectant agents contribute to efficient cryopreservation of primate spermatogonial stem cells (SSC)? DESIGN: Testicular tissues or cells from four prepubertal monkeys were used in this study. The freezing efficacy of testicular tissue was compared with cell suspensions using conventional freezing media (1.4 mol/l dimethyl sulfoxide [DMSO]) and the efficacy of cryoprotectant additives (1.4 mol/l DMSO combined with trehalose 200 mmol/l, hypotaurine 14 mmol/l, necrostatin-1 50 µmol/l or melatonin 100 µmol/l) was evaluated in testicular tissue freezing. RESULTS: The survival rate (46.0 ± 4.8% versus 33.7 ± 6.0%; P = 0.0286) and number of recovered cells (5.0 ± 1.5 × 106 cells/g versus 0.7 ± 0.8 × 106 cells/g; P = 0.0286) were significantly higher in frozen tissues than in frozen cell suspensions. After tissue freezing, a higher number of recovered PGP9.5+ cells were observed with 200 mmol/l trehalose treatment than in DMSO controls (2.4 ± 0.6 × 106 cells/g versus 1.1 ± 0.3 × 106 cells/g; P = 0.0164). Normal establishment of donor-derived colony was observed in SSC after tissue freezing with 200 mmol/l trehalose. CONCLUSIONS: Testicular tissue freezing is more effective than single cell suspension freezing for higher recovery of undifferentiated spermatogonia. Moreover, it was verified that slow freezing using 200 mmol/l trehalose, 1.4 mol/l DMSO and 10% KnockOut™ Serum Replacement in Dulbecco's phosphate-buffered saline is an effective cryopreservation protocol for primate testicular tissue.

Gestational Exposure to Bisphenol A Affects Testicular Morphology, Germ Cell Associations, and Functions of Spermatogonial Stem Cells in Male Offspring.

Exposure to bisphenol A (BPA) in the gestational period damages the reproductive health of offspring; detailed evidence regarding BPA-induced damage in testicular germ cells of offspring is still limited. In this study, pregnant mice (F0) were gavaged with three BPA doses (50 µg, 5 mg, and 50 mg/kg body weight (bw)/day; tolerable daily intake (TDI), no-observed-adverse-effect-level (NOAEL), and lowest-observed-adverse-effect level (LOAEL), respectively) on embryonic days 7 to 14, followed by investigation of the transgenerational effects of such exposure in male offspring. We observed that the NOAEL- and LOAEL-exposed F1 offspring had abnormalities in anogenital distance, nipple retention, and pubertal onset (days), together with differences in seminiferous epithelial stages and testis morphology. These effects were eradicated in the next F2 and F3 generations. Moreover, there was an alteration in the ratio of germ cell population and the apoptosis rate in germ cells increased in F1 offspring at the LOAEL dose. However, the total number of spermatogonia remained unchanged. Finally, a reduction in the stemness properties of spermatogonial stem cells in F1 offspring was observed upon LOAEL exposure. Therefore, we provide evidence of BPA-induced disruption of physiology and functions in male germ cells during the gestational period. This may lead to several reproductive health issues and infertility in offspring.

Paternal Exposure to Bisphenol-A Transgenerationally Impairs Testis Morphology, Germ Cell Associations, and Stemness Properties of Mouse Spermatogonial Stem Cells.

Bisphenol-A (BPA) exposure in an adult male can affect the reproductive system, which may also adversely affect the next generation. However, there is a lack of comprehensive data on the BPA-induced disruption of the association and functional characteristics of the testicular germ cells, which the present study sought to investigate. Adult male mice were administered BPA doses by gavage for six consecutive weeks and allowed to breed, producing generations F1-F4. Testis samples from each generation were evaluated for several parameters, including abnormal structure, alterations in germ cell proportions, apoptosis, and loss of functional properties of spermatogonial stem cells (SSCs). We observed that at the lowest-observed-adverse-effect level (LOAEL) dose, the testicular abnormalities and alterations in seminiferous epithelium staging persisted in F0-F2 generations, although a reduced total spermatogonia count was found only in F0. However, abnormalities in the proportions of germ cells were observed until F2. Exposure of the male mice (F0) to BPA alters the morphology of the testis along with the association of germ cells and stemness properties of SSCs, with the effects persisting up to F2. Therefore, we conclude that BPA induces physiological and functional disruption in male germ cells, which may lead to reproductive health issues in the next generation.

Testicular endothelial cells promote self-renewal of spermatogonial stem cells in rats†.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis in male due to their capability to multiply in numbers by self-renewal and subsequent meiotic processes. However, as SSCs are present in a very small proportion in the testis, in vitro proliferation of undifferentiated SSCs will facilitate the study of germ cell biology. In this study, we investigated the effectiveness of various cell lines as a feeder layer for rat SSCs. Germ cells enriched for SSCs were cultured on feeder layers including SIM mouse embryo-derived thioguanine and ouabain-resistant cells, C166 cells, and mouse and rat testicular endothelial cells (TECs) and their stem cell potential for generating donor-derived colonies and offspring was assessed by transplantation into recipient testes. Rat germ cells cultured on TECs showed increased mRNA and protein levels of undifferentiated spermatogonial markers. Rat SSCs derived from these germ cells underwent spermatogenesis and generated offspring when transplanted into recipients. Collectively, TECs can serve as an effective feeder layer that enhances the proliferative and self-renewal capacity of cultured rat SSCs while preserving their stemness properties.

A collaborative exercise on DNA methylation based body fluid typing.

A collaborative exercise on DNA methylation based body fluid identification was conducted by seven laboratories. For this project, a multiplex methylation SNaPshot reaction composed of seven CpG markers was used for the identification of four body fluids, including blood, saliva, semen, and vaginal fluid. A total of 30 specimens were prepared and distributed to participating laboratories after thorough testing. The required experiments included four increasingly complex tasks: (1) CE of a purified single-base extension reaction product, (2) multiplex PCR and multiplex single-base extension reaction of bisulfite-modified DNA, (3) bisulfite conversion of genomic DNA, and (4) extraction of genomic DNA from body fluid samples. In tasks 2, 3 and 4, one or more mixtures were analyzed, and specimens containing both known and unknown body fluid sources were used. Six of the laboratories generated consistent body fluid typing results for specimens of bisulfite-converted DNA and genomic DNA. One laboratory failed to set up appropriate conditions for capillary analysis of reference single-base extension products. In general, variation in the values obtained for DNA methylation analysis between laboratories increased with the complexity of the required experiments. However, all laboratories concurred on the interpretation of the DNA methylation profiles produced. Although the establishment of interpretational guidelines on DNA methylation based body fluid identification has yet to be performed, this study supports the addition of DNA methylation profiling to forensic body fluid typing.

Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing.

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200 mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs.

Bisphenol Analogs Downregulate the Self-Renewal Potential of Spermatogonial Stem Cells.

PURPOSE: In this study, we investigated the effect of bisphenol-A (BPA) and its major analogs, bisphenol-F (BPF), and bisphenol-S (BPS), on spermatogonial stem cells (SSCs) populations using in vitro SSC culture and in vivo transplantation models. MATERIALS AND METHODS: SSCs enriched from 6- to 8-day-old C57BL/6-eGFP+ male mice testes were treated with varying concentrations of bisphenols for 7 days to examine bisphenol-derived cytotoxicity and changes in SSC characteristics. We utilized flow cytometry, immunocytochemistry, real-time quantitative reverse transcription-PCR, and western blot analysis. The functional alteration of SSCs was further investigated by examining donor SSC-derived spermatogenesis evaluation through in vivo transplantation and subsequent testis analysis. RESULTS: BPF exhibited a similar inhibitory effect on SSCs as BPA, demonstrating a significant decrease in SSC survival, inhibition of proliferation, and induction of apoptosis. On the other hand, while BPS was comparatively weaker than BPA and BPF, it still showed significant SSC cytotoxicity. Importantly, SSCs exposed to BPA, BPF, and BPS exhibited a significant reduction in donor SSC-derived germ cell colonies per total number of cultured cells, indicating that, like BPA, BPF, and BPS can induce a comparable reduction in functional SSCs in the recipient animals. However, the progress of spermatogenesis, as evidenced by histochemistry and the expressions of PCNA and SSC specific markers, collectively indicates that BPA, BPF, and BPS may not adversely affect the spermatogenesis. CONCLUSIONS: Our findings indicate that the major BPA substitutes, BPF and BPS, have significant cytotoxic effects on SSCs, similar to BPA. These effects may lead to a reduction in the functional self-renewal stem cell population and potential impacts on male fertility.

New strategies for germ cell cryopreservation: Cryoinjury modulation.

Cryopreservation is an option for the preservation of pre- or post-pubertal female or male fertility. This technique not only is beneficial for human clinical applications, but also plays a crucial role in the breeding of livestock and endangered species. Unfortunately, frozen germ cells, including oocytes, sperm, embryos, and spermatogonial stem cells, are subject to cryoinjury. As a result, various cryoprotective agents and freezing techniques have been developed to mitigate this damage. Despite extensive research aimed at reducing apoptotic cell death during freezing, a low survival rate and impaired cell function are still observed after freeze-thawing. In recent decades, several cell death pathways other than apoptosis have been identified. However, the relationship between these pathways and cryoinjury is not yet fully understood, although necroptosis and autophagy appear to be linked to cryoinjury. Therefore, gaining a deeper understanding of the molecular mechanisms of cryoinjury could aid in the development of new strategies to enhance the effectiveness of the freezing of reproductive tissues. In this review, we focus on the pathways through which cryoinjury leads to cell death and propose novel approaches to enhance freezing efficacy based on signaling molecules.

Transcriptome alterations in spermatogonial stem cells exposed to bisphenol A.

Owing to their self-renewal and differentiation abilities, spermatogonial stem cells (SSCs) are essential for maintaining male fertility and species preservation through spermatogenesis. With an increase in exposure to plasticizers, the risk of endocrine-disrupting chemicals exerting mimetic effects on estrogen receptors, such as bisphenol A (BPA), has also increased. This has led to concerns regarding the preservation of male fertility. BPA impairs spermatogenesis and the maintenance of SSCs; however, the transcriptome differences caused by BPA in SSCs are poorly understood. Thus, this study aimed to investigate the transcriptome differences in SSCs exposed to BPA, using RNA sequencing (RNA-Seq) analysis. We found that cell proliferation and survival were suppressed by SSC exposure to BPA. Therefore, we investigated transcriptome differences through RNA-Seq, functional annotation, and gene set enrichment analysis. Our results showed repetitive and abundant terms related to two genes of lysosomal acidification and five genes of glycosaminoglycan degradation. Furthermore, we validated the transcriptome analyses by detecting mRNA and protein expression levels. The findings confirmed the discovery of differentially expressed genes (DEGs) and the mechanism of SSCs following exposure to BPA. Taken together, we expect that the identified DEGs and lysosomal mechanisms could provide new insights into the preservation of male fertility and related research.

Autophagy modulation alleviates cryoinjury in murine spermatogonial stem cell cryopreservation.

BACKGROUND: Cryopreservation can expand the usefulness of spermatogonial stem cells (SSCs) in various fields. However, previous investigations that have attempted to modulate cryoinjury-induced mechanisms to increase cryoprotective efficiency have mainly focused on apoptosis and necrosis. OBJECTIVES: This study aimed to establish an effective molecular-based cryoprotectant for SSC cryopreservation via autophagy modulation. MATERIALS AND METHODS: To determine the efficacy of autophagy modulation, we assessed the recovery rate and relative proliferation rate and performed western blotting for the determination of autophagy flux, immunocytochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) for SSC characterization, and spermatogonial transplantation for in vivo SSC functional activity. RESULTS: The results showed that a basal level of autophagy caused a higher relative proliferation rate (pifithrin-µ 0.01 µM, 184.2 ± 11.2%; 3-methyladenine 0.01 µM, 175.3 ± 10.3%; pifithrin-µ 0.01 µM + 3-methyladenine 0.01 µM, P3, 224.6 ± 22.3%) than the DMSO control (100 ± 6.2%). All treatment groups exhibited normal characteristics, suggesting that these modulators could be used as effective cryoprotectants without changing the properties of the undifferentiated germ cells. According to the results of the in vivo spermatogonial transplantation assay, the colonies per total number of cultured SSCs was significantly higher in the pifithrin-µ 0.01 µM (1596.7 ± 172.5 colonies), 3-methyladenine 0.01 µM (1522.1 ± 179.2 colonies), and P3 (1727.5 ± 196.5 colonies) treatment groups than in the DMSO control (842.8 ± 110.08 colonies), which was comparable to that of the fresh control (1882.1 ± 132.1 colonies). DISCUSSION: A basal level of autophagy is more essential for resilience in frozen SSCs after thawing, rather than the excessive activation or inhibition of autophagy. CONCLUSION: A basal level of autophagy plays a critical role in the pro-survival response of frozen SSCs after thawing; herein, a new approach by which SSC cryoprotective efficiency can be improved was identified.

Antioxidant or Apoptosis Inhibitor Supplementation in Culture Media Improves Post-Thaw Recovery of Murine Spermatogonial Stem Cells.

We postulated that supplementation of antioxidant or apoptosis inhibitor in post-thaw culture media of spermatogonial stem cells (SSCs) alleviates reactive oxygen species (ROS) generation and apoptosis. Our aim was to develop an effective culture media for improving post-thaw recovery of SSCs. To determine the efficacy of supplementation with hypotaurine (HTU), α-tocopherol (α-TCP), and Z-DEVD-FMK (ZDF), we assessed the relative proliferation rate and SSC functional activity and performed a ROS generation assay, apoptosis assay, and western blotting for determination of the Bax/Bcl-xL ratio, as well as immunocytochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) for SSC characterization. The relative proliferation rates with HTU 400 µM (133.7 ± 3.2%), α-TCP 400 µM (158.9 ± 3.6%), and ZDF 200 µM (133.1 ± 7.6%) supplementation were higher than that in the DMSO control (100 ± 3.6%). ROS generation was reduced with α-TCP 400 µM (0.8-fold) supplementation in comparison with the control (1.0-fold). Early apoptosis and Bax/Bcl-xL were lower with α-TCP 400 µM (2.4 ± 0.4% and 0.5-fold) and ZDF 200 µM (1.8 ± 0.4% and 0.3-fold) supplementation in comparison with the control (5.3 ± 1.4% and 1.0-fold) with normal characterization and functional activity. Supplementation of post-thaw culture media with α-TCP 400 µM and ZDF 200 µM improved post-thaw recovery of frozen SSCs via protection from ROS generation and apoptosis after cryo-thawing.

Inhibition of Caspase-8 Activity Improves Freezing Efficiency of Male Germline Stem Cells in Mice.

Cryopreservation of male germline stem cells (GSCs) is an essential technique for their long-term preservation and utilization in various fields. However, the specific apoptosis pathways involved in cryoinjury during freezing remain unclear. Therefore, our study sought to identify the pathways involved in cryoinjury-induced apoptosis and thereby to improve freezing efficiency during GSC cryopreservation through the creation of a specific molecular-based cryoprotectant. The activities of caspase-8, caspase-9, caspase-3, and caspase-7 were assessed by Western blot analyses to determine the role of specific apoptosis pathways in GSC cryoinjury. Specifically, the role of a specific caspase was identified by recovery rate, relative proliferation rate, Annexin V/propidium iodide co-staining, and caspase activity using its inhibitor and activator. Moreover, the safety of the cryoprotectant was assessed by immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, the efficacy of the molecular-based cryoprotectant was assessed using frozen cells in the presence of dimethyl sulfoxide (DMSO) (control), trehalose, a caspase-8 inhibitor Z-IETD-FMK [ZIF], or a mixture of the aforementioned compounds, after which the changes in Src signaling were measured. Our results demonstrated that caspase-8 plays a major role in cryoinjury-induced apoptosis and therefore its inhibition improves freezing efficiency. Specifically, a significantly higher relative proliferation rate was observed in the Z-IETD-FMK 0.01 µM-treated cells than in the DMSO control (100% ± 6.2% vs. 189.8% ± 9.5%), with decreases in both early apoptosis (16.6% ± 2.2% vs. 7.5% ± 1.0%) and caspase-8 activity (1.0-fold vs. 0.4-fold). The relative proliferation rate was significantly higher in the cryoprotectant mixture (246.0% ± 12.2%) than other individual treatment groups (trehalose 200 mM, 189.8% ± 9.5%; Z-IETD-FMK 0.01 µM, 189.7% ± 2.2%) with no significant differences in Src signaling. Therefore, our findings provide novel insights into the development of freezing protocols to enhance GSC freezing efficiency, thereby facilitating the wider adoption of GSCs in the livestock industry and/or clinical trials.

Effect of serum replacement on murine spermatogonial stem cell cryopreservation.

Cryopreservation of spermatogonial stem cells (SSCs) is a necessity to preserve the genetic information of valuable livestock herds and to produce transgenic animals. However, serum, a key component that allows efficient cryopreservation, has many limitations attributed to its undefined composition, inter-batch variations, and contamination potential. Therefore, we aimed to establish a method for serum-free cryopreservation of SSCs. To evaluate the cryopreservation efficiency of serum replacements, we assessed the recovery rate, relative proliferation potential, proliferation capacity, and apoptosis capacity. SSCs were characterized, and their functional activity was determined through immunofluorescence, RT-qPCR, and spermatogonial transplantation. The efficiency of each serum replacement was compared to that of the negative control (10% DMSO in DPBS) and positive control (10% DMSO and 40% FBS in DPBS). Our results indicated that cryopreservation with 5% human serum albumin (rHSA) exhibited a higher relative proliferation potential (274.0 ± 13.4%) than with DMSO control (100 ± 8.6%), with no significant difference from the 40% FBS (190.0 ± 20.1%). Moreover, early apoptosis also significantly decreased to a greater extent with 5% rHSA (5.1 ± 0.7%) than with DMSO control (12.9 ± 0.8%) and was at a level comparable to the 40% FBS (4.9 ± 0.8%). In addition, the SSCs cryopreserved with 5% rHSA exhibited normal self-renewal and differentiation abilities. In conclusion, 5% rHSA is a potential serum replacement for SSC cryopreservation, with properties comparable to that of serum. These results would contribute to the application of SSCs in improving livestock and in future clinical trials for human infertility treatment.

Necrostatin-1 improves the cryopreservation efficiency of murine spermatogonial stem cells via suppression of necroptosis and apoptosis.

Cryopreservation of spermatogonial stem cells (SSCs) is important to preserve the lineages of valuable livestock and produce transgenic animals. Although interest in molecular-based cryopreservation methods have been increasing to improve their efficiency, the issue of necroptosis has not yet been considered. Therefore, the purpose of this study was to understand the role of necroptosis using necrostatin-1 (Nec-1), necroptosis inhibitor, in SSC cryopreservation, and to investigate the potential application of Nec-1 as a cryoprotectant. To determine the cryopreservation efficiency of Nec-1, we assessed recovery rate, proliferation potential, cellular membrane damage, RIP1 protein expression, apoptosis, and its mechanism. Stable characterization and functional activity of SSCs was determined via immunofluorescence, RT-qPCR, and in vivo transplantation of SSCs. Our results showed a higher proliferation potential in 50 µM Nec-1 (146.5 ± 16.8%) than in DMSO controls (100.0 ± 3.4%). Furthermore, the cryoprotective effects of Nec-1 were verified by a decrease in RIP1 expression (3.1 ± 0.2-fold vs. 1.3 ± 0.3-fold) and in early apoptosis (4.3 ± 0.8% vs. 2.6 ± 0.1%) compared to DMSO controls. Normal functional activity was observed in the SSCs after cryopreservation with 50 µM Nec-1. In conclusion, necroptosis could be a cause of cryoinjury, and their inhibitor may serve as potential effective cryoprotectant. This study will contribute to establish a molecular-based cryopreservation method, and thereby expanding the use of SSCs into the domestic livestock industry as well as for clinical applications.

Effect of Equilibration Time and Temperature on Murine Spermatogonial Stem Cell Cryopreservation.

Cryopreservation of spermatogonial stem cells (SSCs) is essential for preservation of valuable livestock and clinical applications. Although optimal equilibration of cryoprotectants has emerged as a promising approach to improve the cryopreservation efficiency, standard equilibration protocols have not yet been considered in cryopreservation of SSCs. This study aimed to establish a standard equilibration protocol to improve the cryopreservation efficiency of murine germ cells enriched for SSCs. After time- and temperature-dependent equilibration, the germ cells were cryopreserved with 10% dimethyl sulfoxide (DMSO) and 200 mM trehalose. To investigate cryopreservation efficiency at different equilibration conditions, the survival and proliferation rates were assessed after thawing, and then, cytotoxicity and intracellular trehalose quantification were analyzed. Protein (PLZF, GFRα1, VASA, and c-Kit) and gene (Bcl6b, Erm, Dazl, and Sycp1) expression was determined using immunofluorescence and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. The proliferation rate increased significantly following equilibration for 20 minutes at room temperature (RT; 163.7% ± 24.6%) or 4°C (269.0% ± 18.2%). Cytotoxicity was reduced in 10% DMSO with 200 mM trehalose compared with that of 10% DMSO alone. Also, intracellular trehalose was observed after equilibration. The immunofluorescence and RT-qPCR data revealed that the murine germ cells enriched for SSCs retained their self-renewal ability after cryopreservation following equilibration. The most effective protocol was equilibration with 10% DMSO and 200 mM trehalose for 20 minutes at RT or 4°C, which is due to synergistic effects of intracellular and extracellular trehalose. This improved methodology will contribute toward the development of a standardized freezing protocol for murine germ cells enriched for SSCs and thereby expand their application in various fields.

Epigenetic age signatures in bones.

Age prediction can help identify skeletal remains by limiting the search range for a missing person. Although age prediction methods based on odontology and anthropology are frequently used in the forensic field, DNA methylation is particularly promising age-predictive biomarker. In this study, we generated genome-wide DNA methylation profiles of bone samples from 32 identified skeletal remains with an age at death ranging from 31 to 96 years. Only 12 provided more than 800 K quality-filtered CpG methylation values using Illumina's Infinium MethylationEPIC BeadChip array. Methylation ages of the bone samples calculated using a recently developed skin & blood clock composed of 391 CpG sites were found to be very similar to their actual ages (MAD = 6.4 years). However, the low success rate in methylation profiling of bone DNA samples may prevent researchers from applying the array to this type of samples. Therefore, we selected a set of CpG sites that would enable age prediction based on only a few CpG sites in bone DNA samples. Nineteen age-associated CpG marker candidates were selected from 720 K quality-filtered CpG values of 21 male skeletal remain samples. Because age signatures for blood, such as markers on the ELOVL2, FHL2, KLF14 and TRIM59 genes, had showed strong age associations in 12 bone samples, we further tested the age association of the 5 well-known markers in a blood-based model and the 13 out of 19 CpG markers from the array of 21 bone samples with an independent set of 30 skeletal remain samples using SNaPshot multiplex based on single nucleotide primer extension. Four CpG sites on TMEM51, TRIM59, ELOVL2, and EPHA6 genes showed moderate or weak correlations between methylation and age, which suggests further investigation of these markers to predict the age of bones.

Characterization of the Chaetoceros salsugineum nuclear inclusion virus coat protein gene.

Analysis of the genome of Chaetoceros salsugineum nuclear inclusion virus (CsNIV) revealed the presence of six putative open reading frames (ORFs) in the genome. We further characterized ORF3, which encodes a putative coat protein. Polymerase chain reaction (PCR) using ORF3 gene-specific primers amplified a single DNA band nearly 1.2kb. This amplified product was gel-purified, cloned, sequenced, and expressed in Escherichia coli. Specific antiserum was raised against the recombinant protein and used for Western blotting to test whether the ORF3 protein is the CsNIV coat protein. One major CsNIV protein of approximately 46kDa reacted positively with the antiserum, suggesting that this antiserum is specific for the CsNIV coat protein. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of the 46kDa structural band revealed 14 peptide sequences that matched the ORF3 regions of CsNIV. The expression of ORF3 in host cells was examined by constructing a cDNA library of CsNIV-infected cells. Nucleotide sequences of the cDNA clones were complementary to various regions of both CsNIV ORF3 and ORF4; however, no clones containing only the ORF3 region were identified. Also, Northern blotting revealed a single 2.5-kb transcript, indicating that ORF3 could be transcribed together with ORF4.

DNA methylation of the ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59 genes for age prediction from blood, saliva, and buccal swab samples.

Many studies have reported age-associated DNA methylation changes and age-predictive models in various tissues and body fluids. Although age-associated DNA methylation changes can be tissue-specific, a multi-tissue age predictor that is applicable to various tissues and body fluids with considerable prediction accuracy might be valuable. In this study, DNA methylation at 5 CpG sites from the ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59 genes were investigated in 448 samples from blood, saliva, and buccal swabs. A multiplex methylation SNaPshot assay was developed to measure DNA methylation simultaneously at the 5 CpG sites. Among the 5 CpG sites, 3 CpG sites in the ELOVL2, KLF14 and TRIM59 genes demonstrated strong correlation between DNA methylation and age in all 3 sample types. Age prediction models built separately for each sample type using the DNA methylation values at the 5 CpG sites showed high prediction accuracy with a Mean Absolute Deviation from the chronological age (MAD) of 3.478 years in blood, 3.552 years in saliva and 4.293 years in buccal swab samples. A tissue-combined model constructed with 300 training samples including 100 samples from each blood, saliva and buccal swab samples demonstrated a very strong correlation between predicted and chronological ages (r = 0.937) and a high prediction accuracy with a MAD of 3.844 years in the 148 independent test set samples of 50 blood, 50 saliva and 48 buccal swab samples. Although more validation might be needed, the tissue-combined model's prediction accuracies in each sample type were very much similar to those obtained from each tissue-specific model. The multiplex methylation SNaPshot assay and the age prediction models in our study would be useful in forensic analysis, which frequently involves DNA from blood, saliva, and buccal swab samples.

Platform-independent models for age prediction using DNA methylation data.

Age prediction has been in the spotlight recently because it can provide an important information about the contributors of biological evidence left at crime scenes. Specifically, many researchers have actively suggested age-prediction models using DNA methylation at several CpG sites and tested the candidates using platforms such as the HumanMethylation 450 array and pyrosequencing. With DNA methylation data obtained from each platform, age prediction models were constructed using diverse statistical methods typically with multivariate linear regression. However, because each developed model is based on single-platform data, the prediction accuracy is reduced when applying DNA methylation data obtained from other platforms. In this study, bisulfite sequencing data for 95 saliva samples were generated using massively parallel sequencing (MPS) and compared with methylation SNaPshot data from the same 95 individuals. The predicted age obtained by applying MPS data to an age-prediction model built for methylation SNaPshot data differed greatly from the chronological age due to platform differences. Therefore, novel variables were introduced to indicate the platform type, and construct platform-independent age predictive models using a neural network and multivariate linear regression. The final neural network model had a mean absolute deviation (MAD) of 3.19 years between the predicted and chronological age, and the mean absolute percentage error (MAPE) was 8.89% in the test set. Similarly, the linear regression model showed 3.69 years of MAD and 10.44% of MAPE in the same test set. The platform-independent age-prediction model was made extensible to an increasing number of platforms by introducing platform variables, and the idea of platform variables can be applied to age prediction models for other body fluids.

Direct modification of spermatogonial stem cells using lentivirus vectors in vivo leads to efficient generation of transgenic rats.

Spermatogonial stem cells (SSCs) transmit genetic information to the next progeny in males. Thus, SSCs are a potential target for germline modifications to generate transgenic animals. In this study, we report a technique for the generation of transgenic rats by in vivo manipulation of SSCs with a high success rate. SSCs in juvenile rats were transduced in vivo with high titers of lentivirus harboring enhanced green fluorescent protein and mated with wild-type females to create founder rats. These founder rats expressed the transgene and passed on the transgene with an overall success rate of 50.0%. Subsequent generations of progeny from the founder rats both expressed and passed on the transgene. Thus, direct modification of SSCs in juvenile rats is an effective means of generating transgenic rats through the male germline. This technology could be adapted to larger animals, in which existing methods for gene modification are inadequate or inapplicable, resulting in the generation of transgenic animals in a variety of species.