Pcgf1 gene disruption reveals primary involvement of epigenetic mechanism in neuronal subtype specification in the enteric nervous system.

The enteric nervous system (ENS) regulates gut functions independently from the central nervous system (CNS) by its highly autonomic neural circuit that integrates diverse neuronal subtypes. Although several transcription factors are shown to be necessary for the generation of some enteric neuron subtypes, the mechanisms underlying neuronal subtype specification in the ENS remain elusive. In this study, we examined the biological function of Polycomb group RING finger protein 1 (PCGF1), one of the epigenetic modifiers, in the development and differentiation of the ENS by disrupting the Pcgf1 gene selectively in the autonomic-lineage cells. Although ENS precursor migration and enteric neurogenesis were largely unaffected, neuronal differentiation was impaired in the Pcgf1-deficient mice, with the numbers of neurons expressing somatostatin (Sst+ ) decreased in multiple gut regions. Notably, the decrease in Sst+ neurons was associated with the corresponding increase in calbindin+ neurons in the proximal colon. These findings suggest that neuronal subtype conversion may occur in the absence of PCGF1, and that epigenetic mechanism is primarily involved in specification of some enteric neuron subtypes.

Uts2b is a microbiota-regulated gene expressed in vagal afferent neurons connected to enteroendocrine cells producing cholecystokinin.

Enteroendocrine cells (EECs) are the primary sensory cells that sense the gut luminal environment and secret hormones to regulate organ function. Recent studies revealed that vagal afferent neurons are connected to EECs and relay sensory information from EECs to the brain stem. To date, however, the identity of vagal afferent neurons connected to a given EEC subtype and the mode of their gene responses to its intestinal hormone have remained unknown. Hypothesizing that EEC-associated vagal afferent neurons change their gene expression in response to the microbiota-related extracellular stimuli, we conducted comparative gene expression analyses of the nodose-petrosal ganglion complex (NPG) using specific pathogen-free (SPF) and germ-free (GF) mice. We report here that the Uts2b gene, which encodes a functionally unknown neuropeptide, urotensin 2B (UTS2B), is expressed in a microbiota-dependent manner in NPG neurons. In cultured NPG neurons, expression of Uts2b was induced by AR420626, the selective agonist for FFAR3. Moreover, distinct gastrointestinal hormones exerted differential effects on Uts2b expression in NPG neurons, where cholecystokinin (CCK) significantly increased its expression. The majority of Uts2b-expressing NPG neurons expressed CCK-A, the receptor for CCK, which comprised approximately 25% of all CCK-A-expressing NPG neurons. Selective fluorescent labeling of Uts2b-expressing NPG neurons revealed a direct contact of their nerve fibers to CCK-expressing EECs. This study identifies the Uts2b as a microbiota-regulated gene, demonstrates that Uts2b-expressing vagal afferent neurons transduce sensory information from CCK-expressing EECs to the brain, and suggests potential involvement of UTS2B in a modality of CCK actions.

Enhanced enteric neurogenesis by Schwann cell precursors in mouse models of Hirschsprung disease.

Hirschsprung disease (HSCR) is characterized by congenital absence of enteric neurons in distal portions of the gut. Although recent studies identified Schwann cell precursors (SCPs) as a novel cellular source of enteric neurons, it is unknown how SCPs contribute to the disease phenotype of HSCR. Using Schwann cell-specific genetic labeling, we investigated SCP-derived neurogenesis in two mouse models of HSCR; Sox10 haploinsufficient mice exhibiting distal colonic aganglionosis and Ednrb knockout mice showing small intestinal aganglionosis. We also examined Ret dependency in SCP-derived neurogenesis using mice displaying intestinal aganglionosis in which Ret expression was conditionally removed in the Schwann cell lineage. SCP-derived neurons were abundant in the transition zone lying between the ganglionated and aganglionic segments, although SCP-derived neurogenesis was scarce in the aganglionic region. In the transition zone, SCPs mainly gave rise to nitrergic neurons that are rarely observed in the SCP-derived neurons under the normal condition. Enhanced SCP-derived neurogenesis was also detected in the transition zone of mice lacking RET expression in the Schwann cell lineage. Increased SCP-derived neurogenesis in the transition zone suggests that reduction in the vagal neural crest-derived enteric neurons promotes SCP-derived neurogenesis. SCPs may adopt a neuronal subtype by responding to changes in the gut environment. Robust SCP-derived neurogenesis can occur in a Ret-independent manner, which suggests that SCPs are a cellular source to compensate for missing enteric neurons in HSCR.

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene.

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (RetM919T , RetN396K and RetY792F ) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. RetM919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in RetM919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.

Mice conditionally expressing RET(C618F) mutation display C cell hyperplasia and hyperganglionosis of the enteric nervous system.

Medullary thyroid carcinoma (MTC) develops from hyperplasia of thyroid C cells and represents one of the major causes of thyroid cancer mortality. Mutations in the cysteine-rich domain (CRD) of the RET gene are the most prevalent genetic cause of MTC. The current consensus holds that such cysteine mutations cause ligand-independent dimerization and constitutive activation of RET. However, given the number of the CRD mutations left uncharacterized, our understanding of the pathogenetic mechanisms by which CRD mutations lead to MTC remains incomplete. We report here that RET(C618F), a mutation identified in MTC patients, displays moderately high basal activity and requires the ligand for its full activation. To assess the biological significance of RET(C618F) in organogenesis, we generated a knock-in mouse line conditionally expressing RET(C618F) cDNA by the Ret promoter. The RET(C618F) allele can be made to be Ret-null and express mCherry by Cre-loxP recombination, which allows the assessment of the biological influence of RET(C618F) in vivo. Mice expressing RET(C618F) display mild C cell hyperplasia and increased numbers of enteric neurons, indicating that RET(C618F) confers gain-of-function phenotypes. This mouse line serves as a novel biological platform for investigating pathogenetic mechanisms involved in MTC and enteric hyperganglionosis.

Development of the intrinsic and extrinsic innervation of the gut.

The gastrointestinal (GI) tract is innervated by intrinsic enteric neurons and by extrinsic efferent and afferent nerves. The enteric (intrinsic) nervous system (ENS) in most regions of the gut consists of two main ganglionated layers; myenteric and submucosal ganglia, containing numerous types of enteric neurons and glial cells. Axons arising from the ENS and from extrinsic neurons innervate most layers of the gut wall and regulate many gut functions. The majority of ENS cells are derived from vagal neural crest cells (NCCs), which proliferate, colonize the entire gut, and first populate the myenteric region. After gut colonization by vagal NCCs, the extrinsic nerve fibers reach the GI tract, and Schwann cell precursors (SCPs) enter the gut along the extrinsic nerves. Furthermore, a subpopulation of cells in myenteric ganglia undergoes a radial (inward) migration to form the submucosal plexus, and the intrinsic and extrinsic innervation to the mucosal region develops. Here, we focus on recent progress in understanding the developmental processes that occur after the gut is colonized by vagal ENS precursors, and provide an up-to-date overview of molecular mechanisms regulating the development of the intrinsic and extrinsic innervation of the GI tract.

Neuronal Differentiation in Schwann Cell Lineage Underlies Postnatal Neurogenesis in the Enteric Nervous System.

Elucidation of the cellular identity of neuronal precursors provides mechanistic insights into the development and pathophysiology of the nervous system. In the enteric nervous system (ENS), neurogenesis persists from midgestation to the postnatal period. Cellular mechanism underlying the long-term neurogenesis in the ENS has remained unclear. Using genetic fate mapping in mice, we show here that a subset of Schwann cell precursors (SCPs), which invades the gut alongside the extrinsic nerves, adopts a neuronal fate in the postnatal period and contributes to the ENS. We found SCP-derived neurogenesis in the submucosal region of the small intestine in the absence of vagal neural crest-derived ENS precursors. Under physiological conditions, SCPs comprised up to 20% of enteric neurons in the large intestine and gave rise mainly to restricted neuronal subtypes, calretinin-expressing neurons. Genetic ablation of Ret, the signaling receptor for glial cell line-derived neurotrophic factor, in SCPs caused colonic oligoganglionosis, indicating that SCP-derived neurogenesis is essential to ENS integrity. Identification of Schwann cells as a physiological neurogenic source provides novel insight into the development and disorders of neural crest-derived tissues. SIGNIFICANCE STATEMENT: Elucidating the cellular identity of neuronal precursors provides novel insights into development and function of the nervous system. The enteric nervous system (ENS) is innervated richly by extrinsic nerve fibers, but little is known about the significance of extrinsic innervation to the structural integrity of the ENS. This report reveals that a subset of Schwann cell precursors (SCPs), which invades the gut alongside the extrinsic nerves, adopts a neuronal fate and differentiates into specific neuronal subtypes. SCP-specific ablation of the Ret gene leads to colonic oligoganglionosis, demonstrating a crucial role of SCP-derived neurogenesis in ENS development. Cross-lineage differentiation capacity in SCPs suggests their potential involvement in the development and pathology of a wide variety of neural crest-derived cell types.

GDNF signaling levels control migration and neuronal differentiation of enteric ganglion precursors.

Pleiotropic growth factors play a number of critical roles in continuous processes of embryonic development; however, the mechanisms by which a single regulatory factor is able to orchestrate diverse developmental events remain imperfectly understood. In the development of the enteric nervous system (ENS), myenteric ganglia (MGs) form initially, after which the submucosal ganglia (SMGs) develop by radial inward migration of immature ENS precursors from the myenteric layer. Here, we demonstrate that glial cell line-derived neurotrophic factor (GDNF) is essential for the formation not only of the MGs, but the SMGs as well, establishing GDNF as a long-term acting neurotrophic factor for ENS development in a mouse model. GDNF promotes radial migration of SMG precursors. Interestingly, premigratory SMG precursors in the myenteric layer were distinguished from the surrounding neuronally differentiating cells by their lower activation of the GDNF-mediated MAPK pathway, suggesting that low activation of GDNF downstream pathways is required for the maintenance of the immature state. ENS precursors devoid of GDNF signaling during midgestation halt their migration, survive, and remain in an undifferentiated state over the long-term in vivo. Reactivation of GDNF signaling in these dormant precursors restores their migration and neuronal differentiation in gut organ culture. These findings suggest that pleiotropic function of GDNF is at least in part governed by modulating levels of intracellular activation of GDNF downstream pathways; high activation triggers neuronal differentiation, whereas low activation is crucial for the maintenance of progenitor state.

A Single RET Mutation in Hirschsprung Disease Induces Intestinal Aganglionosis Via a Dominant-Negative Mechanism.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of the enteric nervous system (ENS). HSCR potentially involves multiple gene aberrations and displays complex patterns of inheritance. Mutations of the RET gene, encoding the RET receptor tyrosine kinase, play a central role in the pathogenesis of HSCR. Although a wide variety of coding RET mutations have been identified, their pathogenetic significance in vivo has remained largely unclear. METHODS: We introduced a HSCR-associated RET missense mutation, RET(S811F), into the corresponding region (S812) of the mouse Ret gene. Pathogenetic impact of Ret(S812F) was assessed by histologic and functional analyses of the ENS and by biochemical analyses. Interactions of the Ret(S812F) allele with HSCR susceptibility genes, the RET9 allele and the Ednrb gene, were examined by genetic crossing in mice. RESULTS: RetS812F/+ mice displayed intestinal aganglionosis (incidence, 50%) or hypoganglionosis (50%), impaired differentiation of enteric neurons, defecation deficits, and increased lethality. Biochemical analyses revealed that Ret(S811F) protein was not only kinase-deficient but also abrogated function of wild-type RET in trans. Moreover, the Ret(S812F) allele interacted with other HSCR susceptibility genes and caused intestinal aganglionosis with full penetrance. CONCLUSIONS: This study demonstrates that a single RET missense mutation alone induces intestinal aganglionosis via a dominant-negative mechanism. The RetS812F/+ mice model HSCR displays dominant inheritance with incomplete penetrance and serves as a valuable platform for better understanding of the pathogenetic mechanism of HSCR caused by coding RET mutations.

Neural precursor death is central to the pathogenesis of intestinal aganglionosis in Ret hypomorphic mice.

The RET tyrosine kinase is required for the migration, proliferation, and survival of the enteric neural crest-derived cells (ENCCs) that form the enteric nervous system (ENS). Hypomorphic RET alleles cause intestinal aganglionosis [Hirschsprung disease (HSCR)], in which delayed migration and successive nonapoptotic ENCC death are considered to be major contributory factors. The significance of ENCC death in intestinal aganglionosis, however, has remained unclear. We show that elevated expression of Bcl-xL inhibits ENCC death in both Ret-null and hypomorphic states. However, the rescued Ret-null mice showed ENS malfunction with reduced nitric oxide synthase expression in colonic neurons, revealing the requirement of RET for neuronal differentiation. In contrast, the inhibition of cell death allows morphologically and functionally normal ENS formation in Ret hypomorphic mice. These results indicate that ENCC death is a principal cause of intestinal aganglionosis in a Ret hypomorphic state, and suggest that the inhibition of cell death is a route to the prevention of HSCR.

Diminished Ret expression compromises neuronal survival in the colon and causes intestinal aganglionosis in mice.

Mutations in the RET gene are the primary cause of Hirschsprung disease (HSCR), or congenital intestinal aganglionosis. However, how RET malfunction leads to HSCR is not known. It has recently been shown that glial cell line-derived neurotrophic factor (GDNF) family receptor alpha1 (GFRalpha1), which binds to GDNF and activates RET, is essential for the survival of enteric neurons. In this study, we investigated Ret regulation of enteric neuron survival and its potential involvement in HSCR. Conditional ablation of Ret in postmigratory enteric neurons caused widespread neuronal death in the colon, which led to colonic aganglionosis. To further examine this finding, we generated a mouse model for HSCR by reducing Ret expression levels. These mice recapitulated the genetic and phenotypic features of HSCR and developed colonic aganglionosis due to impaired migration and successive death of enteric neural crest-derived cells. Death of enteric neurons was also induced in the colon, where reduction of Ret expression was induced after the period of enteric neural crest cell migration, indicating that diminished Ret expression directly affected the survival of colonic neurons. Thus, enteric neuron survival is sensitive to RET dosage, and cell death is potentially involved in the etiology of HSCR.

Increased RET Activity Coupled with a Reduction in the RET Gene Dosage Causes Intestinal Aganglionosis in Mice.

Mutations of the gene encoding the RET tyrosine kinase causes Hirschsprung's disease (HSCR) and medullary thyroid carcinoma (MTC). Current consensus holds that HSCR and MTC are induced by inactivating and activating RET mutations, respectively. However, it remains unknown whether activating mutations in the RET gene have adverse effects on ENS development in vivo We addressed this issue by examining mice engineered to express RET51(C618F), an activating mutation identified in MTC patients. Although Ret51(C618F)/51(C618F) mice displayed hyperganglionosis of the ENS, Ret51(C618F)/- mice exhibited severe intestinal aganglionosis because of premature neuronal differentiation. Reduced levels of glial cell-derived neurotrophic factor (GDNF), a RET-activating neurotrophic factor, ameliorated the ENS phenotype of Ret51(C618F)/- mice, demonstrating that GDNF-mediated activation of RET51(C618F) is responsible for severe aganglionic phenotype. The RET51(C618F) allele showed genetic interaction with Ednrb gene, one of modifier genes for HSCR. These data reveal that proliferation and differentiation of ENS precursors are exquisitely controlled by both the activation levels and total dose of RET. Increased RET activity coupled with a decreased gene dosage can cause intestinal aganglionosis, a finding that provides novel insight into HSCR pathogenesis.

Identifying target genes regulated downstream of Cdx2 by microarray analysis.

The caudal-related homeobox transcription factor (Cdx2) plays an important role in intestinal development, differentiation, and homeostasis. However, only a limited number of Cdx2-regulated target genes have been elucidated. To delineate the molecular mechanism regulated downstream of Cdx2, we aimed to define Cdx2-regulated genes. We engineered a rat intestinal epithelial cell line, IEC-6, with minimal endogenous Cdx2 expression to express exogenous Cdx2. The gene expression patterns for Cdx2-inducing cells and control cells were examined using oligonucleotide arrays. In the present study, differential expression of 23 genes was confirmed by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis using gene-specific primers. Increased expression of genes was involved in the Notch signaling pathway, xenobiotic metabolism, enzymes associated with tumor suppression, RNA binding protein, receptors, signal transduction, and transcription factors. The wide-ranging collection of such inducing genes suggests to the functions of Cdx2 in cell fate decision and maintenance of intestinal epithelia.

Cdx2 homeodomain protein regulates the expression of MOK, a member of the mitogen-activated protein kinase superfamily, in the intestinal epithelial cells.

Regulatory protein kinases are involved in various cellular processes such as proliferation, differentiation, and apoptosis. Using cDNA differential display, we identified MOK, a member of the mitogen-activated protein kinase superfamily, as one of the genes induced by a caudal-related homeobox transcription factor, Cdx2. Analysis of the 5'-flanking region of the MOK gene led to the identification of primary Cdx2 responsive element, and an electrophoretic mobility shift assay indicated that Cdx2 binds to that element. The interaction of Cdx2 with the MOK promoter region was further confirmed in vivo by chromatin immunoprecipitation assays. The expression of MOK mRNA and protein was limited to the crypt epithelial cells of the mouse intestine. We also determined the MOK activity associated with the growth arrest and induction of differentiation by sodium butyrate or Cdx2 expression in the human colon cancer cell line HT-29. Taken together, these data indicate that MOK is a direct target gene for Cdx2, and that MOK may be involved in growth arrest and differentiation in the intestinal epithelium.

A water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia suppresses azoxymethane-induction of colon cancers in male F344 rats.

The present study was designed to investigate the protective effect of a dietary water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi or Mannentake) mycelia (designated as MAK) on the induction and development of azoxymethane (AOM)-induced colon tumors in male F344/Du Crj rats. A total of 80 animals were divided into five groups at six weeks of age, groups 2, 3 and 4 being given weekly subcutaneous injections of AOM (15 mg/kg body weight) for the initial 3 weeks to induce colon tumors. Rats in group 1 and 5 were injected with the vehicle, 0.9% (w/v) saline, following the same schedule. Rats in groups 1, 2, 3, 4 and 5 were fed MF, MF, 1.25% MAK, 2.5% MAK and 2.5% MAK diets, respectively, starting 1 week before AOM treatment and throughout the six-month experimental period. There were no significant differences in number of ACF, total AC and AC per site among groups 2 to 4, but the tumor incidence was significantly lower, and tumor size was smaller in group 4 (AOM + 2.5% MAK) than in group 2 (AOM + MF). Additionally, beta-catenin positive tumor cell nuclei were significantly decreased in the MAK-fed rats (groups 3 and 4), which also demonstrated lowering of the PCNA labeling index and a shortened germinal region in the colon. The present results thus indicate that dietary MAK could act as a potent chemopreventive agent for colon carcinogenesis.

Prevention by long-term fermented miso of induction of colonic aberrant crypt foci by azoxymethane in F344 rats.

The present study was designed to investigate the effects of fermented miso in the diet on the induction of aberrant crypt foci (ACF) by azoxymethane (AOM) in male F344 rats. A total of 50 rats, 8 weeks of age, were divided into 5 groups and given weekly subcutaneous injections of AOM (15 mg/kg body wt) for 3 weeks. Rats were fed a normal control MF solid diet, or solid diet containing 10% long-term fermented (aged), medium- or short-term fermented miso, or 2.2% NaCl for 5 weeks, starting one week before the first AOM dosing. It was found that, compared to the control (MF) diet, the long-term fermented diet significantly decreased (by 22.2%) ACF/colon, but increased (by 18.2%) the number of aberrant crypts (Acs)/focus. The latter was also increased by the medium-term fermented diet (by 25.3%). The PCNA labeling index was only affected by the short-term fermented diet (36.9% increase) and by 2.2% NaCl diet (27.2% increased). The present results indicate that aged or completely fermented miso supplemented into the diet, could act as a chemopreventive agent for colon carcinogenesis.

Inhibition by long-term fermented miso of induction of gastric tumors by N-methyl-N'-nitro-N-nitrosoguanidine in CD (SD) rats.

The present study was designed to investigate the effects of fermented miso in the diet on the induction of gastric tumors by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in male CD (SD) rats. A total of 120 animals, 6 weeks of age, were divided into 6 groups and given MNNG (100 ppm) in the drinking water for 16 weeks. Starting 1 week before the carcinogen treatment the rats were fed a normal control MF solid diet, or the same diet containing 10% long-term fermented, medium- or short-term fermented miso, or 1% NaCl until the end of the MNNG exposure period. They were then maintained on the MF control diet and normal tap water until the autopsy time point at 52 weeks. The long-term fermented miso significantly reduced the size of the gastric tumors as compared with the other groups. The present results thus indicate that dietary supplementation with long-term fermented miso could act as a chemopreventive agent for gastric carcinogenesis.

Prevention of development of N,N'-dimethylhydrazine-induced colon tumors by a water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia in male ICR mice.

The protective effects of a dietary water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi or Mannentake) mycelia (designated as MAK) against development of colon tumors were investigated in male ICR mice. The animals were given weekly injections of N,N'-dimethylhydrazine (DMH, 10 mg/kg body weight) for the initial 10 weeks to induce colon carcinogenesis, and then fed on diet with or without 5% MAK for 10 weeks. There were no significant differences in incidence and the total number of colon tumors between the groups. However, the MAK diet group demonstrated significantly reduced sizes of tumors in comparison with the MF diet group. Moreover, this was linked to a lowered PCNA positive index and shortening of the germinal region in the colon. beta-catenin positive tumor cell nuclei were also significantly decreased in the MAK group. The present results thus indicate that dietary MAK could act as a potent chemopreventive agent for colon carcinogenesis.

Effects of weaning by surrogate mothers (ACI) on tumor development in SD rats treated with methylnitrosourea (MNU) and/or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

In this experiment, methylnitrosourea (MNU) was administered, followed by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), to assess effects of surrogate mothering on tumor. One or two day old male SD pups were treated with or without 30 mg/kg body weight of MNU and nursed by SD or ACI surrogate mothers for 5 weeks. When 6-weeks-old they were then treated with 100 ppm MNNG or tap water for 16 weeks. The tumor incidence in the MNNG alone group was significantly lower than with MNU alone or MNU+MNNG (p < 0.01). Kidney or nerve tumors mainly developed in the MNU group, gastric tumors in the MNNG group, and the two combined in the MNU+MNNG group. The incidence and mean number of tumors did not significantly differ between the two weaning groups. However, mean survival time with the ACI surrogate mothers after treatment with MNU was increased as compared with the SD mother group. Cumulative development of tumors in the ACI surrogate mother group was also delayed (p < 0.05). Similar results were obtained with MNU+MNNG and MNNG alone. The present experiment suggested that tumor induction might be effected by components of the mother's milk.

Damage of the mouse testis by tritiated water and 137Cs-gamma-rays.

Tritiated water at 23.2, 46.3 or 92.5 MBq/animal and 137Cs-gamma-rays at 9.5 Gy (equivalent 370 MBq) or lower doses were administered to 6-week old male C3H/HeNCrj and C57BL/6NCrj mice, as well as F1 Crj: B6C3F1 (C3H x C57BL) progeny. Each set of six to ten animals were autopsied 30 days after the first irradiation. Testis weights were decreased dose dependently, relative values being highest in the C3H and lowest in the C57BL case, with B6C3F1 intermediate. Vacuolization in seminiferous tubules appeared in the 23.2 MBq group and increased with the dose. Focal pyknosis and karyomegaly were found at 46.3 MBq, while primary and secondary spermatocytes and spermatids disappeared with 92.5 MBq. Only a few spermatogonia and Sertoli cells remained after exposure to 9.5 Gy 137Cs-gamma-rays. Sizes of seminiferous tubules were decreased dose dependently, with no strain differences. When male B6C3F1 mice were irradiated with Cs-gamma-rays at 0.119 (equivalent 4.63 MBq tritiated water) or 2.38 Gy (equivalent 92.5 MBq tritiated water), body weights and size of the seminiferous tubules were decreased at both doses, and the larger dose also caused reduction of testis weight and abnormal sperm. However, all changes except for the alteration in weights had disappeared 1 month after the final irradiation. It is considered that the size of seminiferous tubules may be a good parameter for radiation damage in the testis.