Morphological reproductive characteristics of testes and fertilization capacity of cryopreserved sperm after the Fukushima accident in raccoon (Procyon lotor).

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, we have established an archive system of livestock and wild animals from the surrounding ex-evacuation zone. Wildlife within the alert zone have been exposed to low-dose-rate (LDR) radiation for a long continuous time. In this study, we analysed the morphological characteristics of the testes and in vitro fertilization (IVF) capacity of cryopreserved sperm of racoons from the ex-evacuation zone of the FDNPP accident. The radioactivity of caesium-137 (137 Cs) was measured by gamma-ray spectrometry, and the measured radioactivity concentration was 300-6,630 Bq/kg in the Fukushima raccoons. Notably, normal spermatogenesis was observed in the seminiferous tubules of the testes, with the germinal epithelium composed of a spermatogenic cell lineage with no evident ultrastructural alterations; freeze-thawing sperm penetration ability was confirmed using the interspecific zona pellucida-free mouse oocytes IVF assays. This study revealed that the chronic and LDR radiation exposure associated with the FDNPP accident had no adverse effect on the reproductive characteristics and functions of male raccoons.

Induction of superovulation using inhibin antiserum and competence of embryo development in wild large Japanese field mice (Apodemus speciosus).

Seasonally, bred wild mice provide a unique bioresource, with high genetic diversity that differs from wild-derived mice and laboratory mice. This study aimed to establish an alternative superovulation method using wild large Japanese field mice (Apodemus speciosus) as the model species. Specifically, we investigated how the application of inhibin antiserum and equine chorionic gonadotropin (IASe) during both the reproductive and non-reproductive seasons impact the ovulation rate and competence of embryo development after in vitro fertilization (IVF) with fresh and cryopreserved sperm. When the wild mice were superovulated by injecting eCG followed by human chorionic gonadotropin (hCG), few oocytes were collected during the reproductive and non-reproductive seasons. In comparison, the number of ovulated oocytes was dramatically enhanced by the administration of IASe, followed by isolation of ovulated oocytes 24 hr after 30 IU hCG administration. The IVF oocytes that were in vitro cultured (IVC) with medium containing serum further developed to the 2- and/or 4-cell stage using both fresh and frozen-thawed sperm. In conclusion, we successfully established an alternative protocol for collecting ovulated oocytes from wild large Japanese field mice by administering IASe and hCG during both the reproductive and non-reproductive seasons. This study is the first to develop IVF-IVC wild large Japanese field mice beyond the 2- and/or 4-cell stage in vitro using fresh and cryopreserved sperm. This approach could be used in other species of wild or endangered mice to reduce the number of animals used for experiments, or in maintaining stocks of germ cells or embryos.

Effects on Glycemic Control in Impaired Wound Healing in Spontaneously Diabetic Torii (SDT) Fatty Rats.

INTRODUCTION: Impaired diabetic wound healing is an important issue in diabetic complications. The present study aims to evaluate the protective effect on glycemic control against impaired diabetic wound healing using a diabetic rat model. We investigated the wound healing process and effect on the impaired wound repair by glycemic control in the Spontaneously Diabetic Torii (SDT) fatty rat, which is a new animal model of obese type 2 diabetes and may be a good model for study impaired wound healing. MATERIAL AND METHODS: Male SDT fatty rats at 15 weeks of age were administered orally with sodium glucose co-transporter (SGLT) 2 inhibitor for 3 weeks. Wounds were induced at 2 weeks after SGLT 2 inhibitor treatment, and the wound areas were periodically examined in morphological and histological analyses. RESULTS: The SDT fatty rats showed a delayed wound healing as compared with the normal rats, but a glycemic control improved the impaired wound healing. In histological analysis in the skin of SDT fatty rats showed severe infiltration of inflammatory cell, hemorrhage and many bacterial masses in the remaining and slight fibrosis of crust on skin tissue. Thought that this results skin performance to be a delay of crust formation and regeneration of epithelium; however, these findings were ameliorated in the SGLT 2 inhibitor treated group. CONCLUSION: Glycemic control is effective for treatment in diabetic wounds and the SDT fatty rat may be useful to investigate pathophysiological changes in impaired diabetic wound healing.

Combination of spindle and first polar body chromosome images for the enhanced prediction of developmental potency of mouse metaphase II oocytes.

The objective of this study was to classify spindle and first polar body (PB1) chromosome images in ovulated mouse oocytes over time to predict the developmental competence of metaphase II (MII) oocytes. Oocytes were collected at 12, 15, 20, and 25 h after human chorionic gonadotropin (hCG) injection, and stained for spindle tubulin, chromosomes, and PB1 chromosomes. MII spindle morphology was classified as tapered type or barrel type and PB1 chromosomes were categorized as aggregated, separated, dot, or collapsed. To determine whether differences in spindle and PB1 images in MII oocytes are associated with fertilization success, we performed in vitro fertilization (IVF) at various times after hCG injection. Barrel-type spindles and aggregate-type PB1 were dominant at 12 h after hCG injection. Oocyte spindles collected 1 h after injection were tapered, and PB1 chromosomes were separated. At 20 and 25 h after treatment, spindle and PB1 images were classified as collapsed. The rate of development to 2-cell embryos after IVF did not differ between the 12 h and 15 h treatments; however, it was significantly lower for the 25 h treatment than for other treatments. The rates of development to blastocysts at 12, 15, 20, and 25 h after hCG injection were 61, 46, 42, and 9%, respectively. MII oocytes with barrel-type spindles and aggregate-type PB1 had high rates of fertilization and blastocyst development, and spindle and PB1 characteristics were correlated with the outcomes of IVF and embryo culture. These results suggested that images of spindles combined with those of PB1 chromosomes enable the prediction of oocytic and/or embryonic quality.

Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis.

We have developed a unique method for mouse transgenesis. The transposase-enhanced pronuclear microinjection (PNI) technique described herein uses the hyperactive piggyBac transposase to insert a large transgene into the mouse genome. This procedure increased transgene integration efficiency by fivefold compared with conventional PNI or intracytoplasmic sperm injection-mediated transgenesis. Our data indicate that the transposase-enhanced PNI technique additionally requires fewer embryos to be microinjected than traditional methods to obtain transgenic animals. This transposase-mediated approach is also very efficient for single-cell embryo cytoplasmic injections, offering an easy-to-implement transgenesis method to the scientific community.

Ultrastructural Analysis of Large Japanese Field Mouse (Apodemus speciosus) Testes Exposed to Low-Dose-Rate (LDR) Radiation after the Fukushima Nuclear Power Plant Accident.

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, great attention has been paid to the impact of chronic low-dose-rate (LDR) radiation exposure on biological systems. The reproductive system is sensitive to radiation, with implications connected to infertility. We investigated the testis ultrastructure of the wild large Japanese field mouse (Apodemus speciosus) from three areas contaminated after the FDNPP accident, with different levels of LDR radiation (0.29 µSv/h, 5.11 µSv/h, and 11.80 µSv/h). Results showed good preservation of the seminiferous tubules, comparable to the unexposed animals (controls), except for some ultrastructural modifications. Increases in the numerical density of lipid droplet clusters in spermatogenic cells were found at high levels of LDR radiation, indicating an antioxidant activity rising due to radiation recovery. In all groups, wide intercellular spaces were found between spermatogenic cells, and cytoplasmic vacuolization increased at intermediate and high levels and vacuolated mitochondria at the high-level. However, these findings were also related to the physiological dynamics of spermatogenesis. In conclusion, the testes of A. speciosus exposed to LDR radiation associated with the FDNPP accident showed a normal spermatogenesis, with some ultrastructural changes. These outcomes may add information on the reproductive potential of mammals chronically exposed to LDR radiation.

Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects.

Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

Environmental radiation on large Japanese field mice in Fukushima reduced colony forming potential in hematopoietic progenitor cells without inducing genomic instability.

PURPOSE: To study the environmental radiation effects of wild animals after the Fukushima Dai-ichi nuclear power plant accident, we assessed effects on hematopoietic progenitor cells (HPCs) in large Japanese field mice (Apodemus speciosus). MATERIALS AND METHODS: A. speciosus were collected from three contaminated sites and control area. The air dose-rates at the control and contaminated areas were 0.96 ± 0.05 µGy/d (Hirosaki), 14.4 ± 2.4 µGy/d (Tanashio), 208.8 ± 31.2 µGy/d (Ide), 470.4 ± 93.6 µGy/d (Omaru), respectively. We investigated possible DNA damage and pro-inflammatory markers in the bone marrow (BM) cells. The colony-forming potential of BM cells was estimated by the number of HPC colony-forming cells. Radiation-induced genomic instability (RIGI) in HPCs was also analyzed by quantifying delayed DNA damage in CFU-GM clones. RESULTS: Although no significant differences in DNA damage and inflammation markers in BM cells from control and contaminated areas, the number of HPC colonies exhibited an inverse correlation with air dose-rate. With regard to RIGI, no significant differences in DNA damage of CFU-GM clones between the mice from the control and the three contaminated areas. CONCLUSIONS: Our study suggests that low dose-rate radiation of more than 200 Gy/d reduced HPCs, possibly eliminating genomically unstable HPCs.

Changes in ovarian morphology and hormone concentrations associated with reproductive seasonality in wild large Japanese field mice (Apodemus speciosus).

Wild large Japanese field mice (Apodemus speciosus) responses to cyclic seasonal changes are associated with physiological and behavioral changes. However, the detailed regulation of oogenesis in the ovary during the seasonal reproductive cycle in wild large Japanese field mice has not been studied. We assessed the dynamics and changes in ovarian morphology and hormone concentrations associated with reproductive seasonality throughout the year. The stages of the ovarian morphological breeding cycle of wild large Japanese field mice were classified as breeding, transition, and non-breeding periods during the annual reproductive cycle. Measurement of blood estradiol concentrations throughout the year showed that the levels in September and October were higher than those in other months. It is presumed that follicle development starts from a blood estradiol concentration of 38.4 ± 27.1 pg/mL, which marks a shift from the transitional season to the breeding season, followed by the transition to the non-breeding season at 26.1 ± 11.6 pg/mL. These results suggest that seasonal follicle development in wild rodents is correlated with estradiol regulation. We consider this species to be an alternative animal model for studying seasonal reproductive changes and the effects of environmental changes.

Evaluation of Global DNA Methylation and Gene Expression of Izumo1 and Izumo1r in Gonads after High- and Low-Dose Radiation in Neonatal Mice.

The intergenerational effects from chronic low-dose exposure are matters of concern. It is thus important to elucidate the radiation-induced effects of germ cell maturation, fertilization and embryonic development. It is well known that DNA methylation levels in CpG sites in gametes are reprogrammed in stages during their maturity. Furthermore, the binding of Izumo on the surface of sperm and Juno on the surface of oocytes is essential for fertilization. Thus, there is a possibility that these genes are useful indicators to evaluate fertility in mice after irradiation exposure. Therefore, in this study, we analyzed global DNA methylation patterns in the testes and gene expression of Izumo1 and Izumo1r (Juno) in the gonads of mice after neonatal acute high-dose ionizing radiation (HDR) and chronic low-dose ionizing radiation (LDR). One-week-old male and female mice were irradiated with a total dose of 4 Gy, with acute HDR at 7 days at a dose rate of 30 Gy/h and LDR continuously at a dose rate of 6 mGy/h from 7 to 35 days. Their gonads were subsequently analyzed. The results of global DNA methylation patterns in the testes showed that methylation level increased with age in the control group, the LDR group maintained its DNA methylation level, and the HDR group showed decreased DNA methylation levels with age. In the control group, the gene expression level of Izumo1 in the testis did not show age-related changes, although there was high expression at 100 days of age. However, in the LDR group, the expression level recovered after the end of irradiation, while it remained low regardless of age in the HDR group. Conversely, gene expression of Izumo1r (Izumo1 receptor) in the ovary decreased with age in the control group. Although the gene expression of Izumo1r decreased with age in the LDR group, it remained low in the HDR group. Our results indicate that LDR can induce different DNA methylation patterns, and both high- and low-dose radiation before sexual maturity might affect gametogenesis and fertility.

Effects of rice feeding and carnitine addition on growth performance and mRNA expression of protein metabolism-related genes in broiler grower chicks.

This study was carried out to evaluate the nutritional effects of rice feeding and carnitine addition to a diet for broiler chicks. Thirty-six male 10-day-old broiler chicks were assigned to one of the following four treatment groups: corn-based diet (corn group), rice-based diet (rice group), and each diet with added carnitine (100 ppm). The experimental period was 2 weeks. Rice feeding resulted in significantly higher growth performance (body weight gain and feed efficiency) compared to corn feeding. Carnitine addition also resulted in higher growth performance. Breast muscle and thigh muscle weight (g) were significantly higher in broiler chicks fed rice and those fed diets with added carnitine. Liver mRNA expression of IGF-I was significantly higher in broiler chicks fed rice compared to those fed corn. There was no significant difference in mRNA expression of muscle atrogin-1 or liver CPT-I between broiler chicks fed rice and those fed corn, not between broilers chicks fed diets containing carnitine or not. Overall, these results show that rice feeding and carnitine addition improve the growth performance of broiler chicks by increasing mRNA expression of liver IGF-I. In addition, carnitine action is not affected by different cereals (corn and rice).

Seasonal changes in the spermatogenesis of the large Japanese field mice (Apodemus speciosus) controlled by proliferation and apoptosis of germ cells.

The aim of this study was to investigate the proliferation and apoptosis of male germ cells during the seasonal reproductive cycle of the large Japanese field mice (Apodemus speciosus). Male mice residing in their natural habitat were captured in Niigata, Japan. Testis sections were stained with haematoxylin and eosin, and mitotic male germ cells were identified using immunofluorescence staining for proliferating cell nuclear antigen (PCNA). Apoptosis was analysed using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay. The phases of spermatogenesis during the seasonal reproductive cycle were classified as active, transitional, and inactive based on the diameter of the seminiferous tubules. The number of PCNA-positive germ cells was less during the inactive than other phases. The percentage of TUNEL-positive germ cells per seminiferous tubule was greater during the inactive than active and transitional phases. Spermatogenesis during the seasonal reproductive cycle is controlled by proliferation and apoptosis in male germ cells. This species of undomesticated mice could be used as an animal model to study spermatogenesis as a valuable indicator of the effects of ecological and anthropogenic factors on animal reproduction.

Intestinal Bacteria as Powerful Trapping Lifeforms for the Elimination of Radioactive Cesium.

In March 2011, an accident at the Fukushima Daiichi Nuclear Power Plant led to major problems, including the release of radionuclides such as Cesium (Cs)-137 into the environment. Ever since this accident, Cs-137 in foods has become a serious problem. In this study, we determined the concentration of Cs-137 in the feces, urine, and ruminal contents of cattle and demonstrated the possibility of its elimination from the body by intestinal bacteria. The results revealed a high Cs-137 concentration in the feces; in fact, this concentration was higher than that in skeletal muscles and other samples from several animals. Furthermore, intestinal bacteria were able to trap Cs-137, showing an uptake ratio within the range of 38-81% in vitro. This uptake appeared to be mediated through the sodium-potassium (Na+-K+) ion pump in the bacterial cell membrane. This inference was drawn based on the fact that the uptake ratio of Cs-137 was decreased in media with high potassium concentration. In addition, it was demonstrated that intestinal bacteria hindered the trapping of Cs-137 by the animal. Cattle feces showed high concentration of Cs-137 and intestinal bacteria trapped Cs-137. This study is the first report showing that intestinal bacteria contribute to the elimination of Cs-137 from the body.

Haematological analysis of Japanese macaques (Macaca fuscata) in the area affected by the Fukushima Daiichi Nuclear Power Plant accident.

Several populations of wild Japanese macaques (Macaca fuscata) inhabit the area around Fukushima Daiichi Nuclear Power Plant (FNPP). To measure and control the size of these populations, macaques are captured annually. Between May 2013 and December 2014, we performed a haematological analysis of Japanese macaques captured within a 40-km radius of FNPP, the location of a nuclear disaster two years post-accident. The dose-rate of radiocaesium was estimated using the ERICA Tool. The median internal dose-rate was 7.6 µGy/day (ranging from 1.8 to 219 µGy/day) and the external dose-rate was 13.9 µGy/day (ranging from 6.7 to 35.1 µGy/day). We performed multiple regression analyses to estimate the dose-rate effects on haematological values in peripheral blood and bone marrow. The white blood cell and platelet counts showed an inverse correlation with the internal dose-rate in mature macaques. Furthermore, the myeloid cell, megakaryocyte, and haematopoietic cell counts were inversely correlated and the occupancy of adipose tissue was positively correlated with internal dose-rate in femoral bone marrow of mature macaques. These relationships suggest that persistent whole body exposure to low-dose-rate radiation affects haematopoiesis in Japanese macaques.

90Sr specific activity of teeth of abandoned cattle after the Fukushima accident - teeth as an indicator of environmental pollution.

90Sr specific activity in the teeth of young cattle that were abandoned in Kawauchi village and Okuma town located in the former evacuation areas of the Fukushima-Daiichi Nuclear Power Plant (FNPP) accident were measured. Additionally, specific activity in contaminated surface soils sampled from the same area was measured. (1) All cattle teeth examined were contaminated with 90Sr. The specific activity, however, varied depending on the developmental stage of the teeth during the FNPP accident; teeth that had started development before the accident exhibited comparatively lower values, while teeth developed mainly after the accident showed higher values. (2) Values of 90Sr-specific activity in teeth formed after the FNPP accident were higher than those of the bulk soil but similar to those in the exchangeable fraction (water and CH3COONH4 soluble fractions) of the soil. The findings suggest that 90Sr was incorporated into the teeth during the process of development, and that 90Sr in the soluble and/or leachable fractions of the soil might migrate into teeth and contribute to the amount of 90Sr in the teeth. Thus, the concentration of 90Sr in teeth formed after the FNPP accident might reflect the extent of 90Sr pollution in the environment.

Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution.

The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs-Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm.

Trehalose enhanced the freezability of Poodle dog sperm collected by an artificial vagina (AV).

In an attempt to develop a suitable freezing method for Poodle dog sperm, an experiment was conducted to investigate semen collection methods of digital stimulation and an artificial vagina (AV), using Tris and trehalose-egg yolk extender, on the characteristics and cryopreservation of sperm. Two dogs (dogs A and B) were subjected to semen collection by digital stimulation and AV. The volume, sperm concentration, sperm motility index (SMI) and acrosome status of ejaculates were determined immediately after collection. The remainder was frozen as pellets in Tris and trehalose-egg yolk extender. Sperm motility index was evaluated after thawing and during a thermal resistance test, and acrosome integrity was also assessed. No significant differences regarding sperm concentrations, SMI and acrosome integrity were observed between semen collected by AV and digital stimulation. However, when dog sperm were collected by an AV and frozen in trehalose-egg yolk extender, the motility index of frozen-thawed sperm was significantly improved compared to sperm frozen in Tris-egg yolk extender which were collected by digital stimulation. In conclusion, semen collected by an AV and frozen in trehalose-egg yolk extender was effective in enhancing the freezability of Poodle dog sperm.

Location and expression of Juno in mice oocytes during maturation.

OBJECTIVE: Oocyte-sperm interaction is the essential step in fertilization. Juno, which has been known as Folate receptor 4, is the Izumo1 receptor expressed on the oocyte membrane. This study aims to investigate the location and expression of Juno in mice oocytes during maturation. METHODS: To confirm the stage at which Juno expression begins in the mice oocytes and its location pattern, we performed immunostaining methods. Next, we evaluated Juno mRNA expression by a half quantitative RT-PCR. Juno knockdown oocytes were generated by microinjecting siRNA into the germinal vesicle (GV) stage oocytes, and analyzed the maturation rate. RESULTS: Our results showed that Juno was expressed on the surface of the oocyte cytoplasmic membrane at the GV stage and it continues to be expressed at similar levels in the metaphase II (MII) stages of oocytes maturation. Interestingly, Juno is also expressed on the first polar body membrane at the MII stage. Fluorescence showing Juno expression was decreased in the oolemma of siRNA injected oocytes, but it was not completely disappearing in knock down oocytes. MII stage-rates of siRNA injected oocytes were not significantly different from sham controls. CONCLUSION: Juno was expressed in oocytes at the GV stage and it continues to be expressed at similar levels in later stages of oocytes maturation. Juno accumulation in oolemma during oocyte maturation is essential for fertilization, such as membrane recognition of both gametes.

Analysis of the Effect of Chronic and Low-Dose Radiation Exposure on Spermatogenic Cells of Male Large Japanese Field Mice ( Apodemus speciosus ) after the Fukushima Daiichi Nuclear Power Plant Accident.

In this study we analyzed the effect of chronic and low-dose-rate (LDR) radiation on spermatogenic cells of large Japanese field mice ( Apodemus speciosus ) after the Fukushima Daiichi Nuclear Power Plant (FNPP) accident. In March 2014, large Japanese field mice were collected from two sites located in, and one site adjacent to, the FNPP ex-evacuation zone: Tanashio, Murohara and Akogi, respectively. Testes from these animals were analyzed histologically. External dose rate from radiocesium (combined 134Cs and 137Cs) in these animals at the sampling sites exhibited 21 µGy/day in Tanashio, 304-365 µGy/day in Murohara and 407-447 µGy/day in Akogi. In the Akogi group, the numbers of spermatogenic cells and proliferating cell nuclear antigen (PCNA)-positive cells per seminiferous tubule were significantly higher compared to the Tanashio and Murohara groups, respectively. TUNEL-positive apoptotic cells tended to be detected at a lower level in the Murohara and Akogi groups compared to the Tanashio group. These results suggest that enhanced spermatogenesis occurred in large Japanese field mice living in and around the FNPP ex-evacuation zone. It remains to be elucidated whether this phenomenon, attributed to chronic exposure to LDR radiation, will benefit or adversely affect large Japanese field mice.