Effects of Mating and Social Exposure on Cell Proliferation in the Adult Male Prairie Vole (Microtus ochrogaster).

Microtus ochrogaster is a rodent with a monogamous reproductive strategy characterized by strong pair bond formation after 6 h of mating. Here, we determine whether mating-induced pair bonding increases cell proliferation in the subventricular zone (SVZ), rostral migratory stream (RMS), and dentate gyrus (DG) of the hippocampus in male voles. Males were assigned to one of the four groups: (1) control: males were placed alone in a clean cage; (2) social exposure to a female (SE m/f): males that could see, hear, and smell a sexually receptive female but where physical contact was not possible, because the animals were separated by an acrylic screen with small holes; (3) social exposure to a male (SE m/m): same as group 2 but males were exposed to another male without physical contact; and (4) social cohabitation with mating (SCM): males that mated freely with a receptive female for 6 h. This procedure leads to pair bond formation. Groups 2 and 3 were controls for social interaction. Male prairie voles were injected with 5-bromo-2'-deoxyuridine (BrdU) during the behavioral tests and were sacrificed 48 h later. Brains were processed to identify the new cells (BrdU-positive) and neuron precursor cells (neuroblasts). Our principal findings are that in the dorsal region of the SVZ, SCM and SE m/f and m/m increase the percentage of neuron precursor cells. In the anterior region of the RMS, SE m/f decreases the percentage of neuron precursor cells, and in the medial region SE m/f and m/m decrease the number of new cells and neuron precursor cells. In the infrapyramidal blade of the subgranular zone of the DG, SE m/m and SCM increase the number of new neuron precursor cells and SE m/m increases the percentage of these neurons. Our data suggests that social interaction, as well as sexual stimulation, leads to pair bonding in male voles modulating cell proliferation and differentiation to neuronal precursor cells at the SVZ, RMS, and DG.

Mating and social exposure induces an opioid-dependent conditioned place preference in male but not in female prairie voles (Microtus ochrogaster).

In rodents, sexual stimulation induces a positive affective state that is evaluated by the conditioned place preference (CPP) test. Opioids are released during sexual behavior and modulate the rewarding properties of this behavior. Prairie voles (Microtus ochrogaster) are a socially monogamous species, in which copulation with cohabitation for 6h induces a pair bond. However, the mating-induced reward state that could contribute to the establishment of the long-term pair bond has not been evaluated in this species. The present study aimed to determine whether one ejaculation or cohabitation with mating for 6h is rewarding for voles. We also evaluated whether this state is opioid dependent. Our results demonstrate that mating with one ejaculation and social cohabitation with mating for 6h induce a CPP in males, while exposure to a sexually receptive female without mating did not induce CPP. In the female vole, mating until one ejaculation, social cohabitation with mating, or exposure to a male without physical interaction for 6h did not induce CPP. To evaluate whether the rewarding state in males is opioid dependent, the antagonist naloxone was injected i.p. The administration of naloxone blocked the rewarding state induced by one ejaculation and by social cohabitation with mating. Our results demonstrate that in the prairie vole, on the basis of the CPP in the testing conditions used here, the stimulation received with one ejaculation and the mating conditions that lead to pair bonding formation may be rewarding for males, and this reward state is opioid dependent.

The common gamma chain cytokine interleukin-21 is expressed by activated lymphocytes from two macropod marsupials, Macropus eugenii and Onychogalea fraenata.

In mammals, interleukin-21 is a member of the common gamma chain cytokine family that also includes IL-2, IL-4, IL-7, IL-9 and IL-15. IL-21 has pleiotropic effects on both myeloid and lymphoid immune cells and as a consequence, the biological actions of IL-21 are broad: regulating both innate and adaptive immune responses and playing a pivotal role in antiviral, inflammatory and antitumour cellular responses. While IL-21 genes have been characterized in mammals, birds, fish and amphibians, there are no reports for any marsupial species to date. We characterized the expressed IL-21 gene from immune tissues of two macropod species, the tammar wallaby (Macropus eugenii), a model macropod, and the closely related endangered bridled nailtail wallaby (Onychogalea fraenata). The open reading frame of macropod IL-21 is 462 nucleotides in length and encodes a 153-mer putative protein that has 46% identity with human IL-21. Despite the somewhat low amino acid conservation with other mammals, structural elements and residues essential for IL-21 conformation and receptor association were conserved in the macropod IL-21 predicted peptides. The detection of IL-21 gene expression in T-cell-enriched tissues, combined with analysis of the promotor region of the tammar wallaby gene, suggests that macropod IL-21 is expressed in stimulated T cells but is not readily detected in other cells and tissues. The similarity of gene expression profile and functionally important amino acid residues to eutherian IL-21 makes it unlikely that the differences in B- and T-cell responses that are reported for some marsupial species are due to a lack of important functional residues or IL-21 gene expression in this group of mammals.

Distributions of oxytocin and vasopressin 1a receptors in the Taiwan vole and their role in social monogamy.

Social monogamy is a mating strategy rarely employed by mammalian species. Laboratory studies in socially monogamous prairie voles (Microtus ochrogaster) demonstrate that oxytocin and vasopressin act within the mesolimbic dopamine pathway to facilitate pair-bond formation. Species differences in oxytocin receptor (OTR) and vasopressin 1a receptor (V1aR) distribution in this pathway are associated with species differences in mating strategy. Here we characterize the neuroanatomical distribution of OTR and V1aR binding sites in naturally occurring populations of Taiwan voles (M. kikuchii), which purportedly display social monogamy. Live trapping was conducted at two sites in 2009-2010 and receptor autoradiography for OTR and V1aR was performed on brains from 24 animals. OTR binding in two brain regions where OTR signaling regulates pair-bonding were directly compared with that of prairie voles. Our results show that like prairie voles, Taiwan voles exhibit OTR in the prefrontal cortex, insular cortex, claustrum, nucleus accumbens, caudate-putamen, dorsal lateral septal nucleus, central amygdala, and ventromedial hypothalamus. Unlike prairie voles, Taiwan voles exhibit OTR binding in the CA3 pathway of the hippocampus, as well as the indusium griseum, which has only previously been documented in tuco-tucos (Ctenomys haigi, C. sociabilis), Syrian hamsters (Mesocricetus auratus) and naked mole-rats (Heterocephalus glaber). V1aR binding was present in the ventral pallidum, lateral septum, nucleus basalis, bed nucleus of the stria terminalis, hippocampus, medial amygdala, and anterior, ventromedial and dorsomedial hypothalamus. Marked individual differences in V1aR binding were noted in the cingulate cortex and several thalamic nuclei, remarkably similar to prairie voles. While pharmacological studies are needed to determine whether oxytocin and vasopressin are involved in pair-bond formation in this species, our results lay a foundation for future investigations into the role of these neuropeptides in Taiwan vole social behavior.

Social amnesia in mice lacking the oxytocin gene.

The development of social familiarity in rodents depends predominantly on olfactory cues and can critically influence reproductive success. Researchers have operationally defined this memory by a reliable decrease in olfactory investigation in repeated or prolonged encounters with a conspecific. Brain oxytocin (OT) and vasopressin (AVP) seem to modulate a range of social behaviour from parental care to mate guarding. Pharmacological studies indicate that AVP administration may enhance social memory, whereas OT administration may either inhibit or facilitate social memory depending on dose, route or paradigm. We found that male mice mutant for the oxytocin gene (Oxt-/-) failed to develop social memory, whereas wild-type (Oxt+/+) mice showed intact social memory. Measurement of both olfactory foraging and olfactory habituation tasks indicated that olfactory detection of non-social stimuli is intact in Oxt-/- mice. Spatial memory and behavioural inhibition measured in a Morris water-maze, Y-maze, or habituation of an acoustic startle also seemed intact. Treatment with OT but not AVP rescued social memory in Oxt-/- mice, and treatment with an OT antagonist produced a social amnesia-like effect in Oxt+/+ mice. Our data indicate that OT is necessary for the normal development of social memory in mice and support the hypothesis that social memory has a neural basis distinct from other forms of memory.

Effect of cooling heat-stressed dairy cows during the dry period on insulin response.

Heat stress (HT) during the dry period affects hepatic gene expression and adipose tissue mobilization during the transition period. In addition, it is postulated that HT may alter insulin action on peripheral tissues. Our objective was to evaluate the effect of cooling heat-stressed cows during the dry period on insulin effects on peripheral tissues during the transition period. Cows were dried off 46 d before expected calving and assigned to 1 of 2 treatments: HT (n = 16) or cooling (CL, n = 16). During the dry period, the average temperature-humidity index was 78, but CL cows were cooled with sprinklers and fans, whereas HT cows were not. After calving, all cows were housed and managed under the same conditions. Rectal temperatures were measured twice daily (0730 and 1430 h) and respiration rate recorded 3 times weekly during the dry period. Dry matter intake was recorded daily from dry-off to 42 d relative to calving (DRC). Body weight and body condition score were measured weekly from dry-off to 42 DRC. Milk yield and composition were recorded daily to 42 wk postpartum. Glucose tolerance tests (GTT) and insulin challenges (IC) were performed at dry-off, -14, 7, and 28 DRC in a subset of cows (HT, n = 8; CL, n = 8). Relative to HT, CL cows had lower rectal temperatures (39.3 vs. 39.0°C) in the afternoon and respiration rate (69 vs. 48 breath/min). Cows from the cooling treatment tended to consume more feed than HT cows prepartum and postpartum. Compared with HT, CL cows gained more weight before calving but lost more weight and body condition in early lactation. Cows from the cooling treatment produced more milk than HT cows (34.0 vs. 27.7 kg/d), but treatments did not affect milk composition. Treatments did not affect circulating insulin and metabolites prepartum, but CL cows had decreased glucose, increased nonesterified fatty acid, and tended to have lower insulin concentrations in plasma postpartum compared with HT cows. Cooling prepartum HT cows did not affect the insulin responses to GTT and IC during the transition period and glucose responses to GTT and IC at -14 and 28 DRC were not affected by treatments. At 7 DRC, CL cows tended to have slower glucose clearance to GTT and weaker glucose response to IC relative to HT cows. Cows from the cooling treatment had stronger nonesterified fatty acid responses to IC postpartum but not prepartum compared with HT. In conclusion, cooling heat-stressed dairy cows in the dry period reduced insulin effects on peripheral tissues in early lactation but not in the dry period.

The Philippine cynomolgus monkey (Macaca fasicularis) provides a new nonhuman primate model of tuberculosis that resembles human disease.

A nonhuman primate model of tuberculosis that closely resembles human disease is urgently needed. We have evaluated the Philippine cynomolgus monkey, Macaca fasicularis, as a model of TB. Cynomolgus monkeys challenged intratracheally with extremely high doses of Mycobacterium tuberculosis (10(5) or 10(4) CFU) developed an acute, rapidly progressive, highly fatal multilobar pneumonia. However, monkeys challenged with moderate or low doses of M. tuberculosis (

Lower CSF oxytocin concentrations in women with a history of childhood abuse.

Early-life disruption of the parent-child relationship, for example, in the form of abuse, neglect or loss, dramatically increases risk for psychiatric, as well as certain medical, disorders in adulthood. The neuropeptide oxytocin (OT) plays a seminal role in mediating social affiliation, attachment, social support, maternal behavior and trust, as well as protection against stress and anxiety. We therefore examined central nervous system OT activity after early-life adversity in adult women. We measured OT concentrations in cerebrospinal fluid (CSF) collected from 22 medically healthy women, aged 18-45 years, categorized into those with none-mild versus those with moderate-severe exposure to various forms of childhood abuse or neglect. Exposure to maltreatment was associated with decreased CSF OT concentrations. A particularly strong effect was identified for emotional abuse. There were inverse associations between CSF OT concentrations and the number of exposure categories, the severity and duration of the abuse and current anxiety ratings. If replicated, the association of lower adult CSF OT levels with childhood trauma might indicate that alterations in central OT function may be involved in the adverse outcomes of childhood adversity.

Human retinoblastoma susceptibility gene: cloning, identification, and sequence.

Recent evidence indicates the existence of a genetic locus in chromosome region 13q14 that confers susceptibility to retinoblastoma, a cancer of the eye in children. A gene encoding a messenger RNA (mRNA) of 4.6 kilobases (kb), located in the proximity of esterase D, was identified as the retinoblastoma susceptibility (RB) gene on the basis of chromosomal location, homozygous deletion, and tumor-specific alterations in expression. Transcription of this gene was abnormal in six of six retinoblastomas examined: in two tumors, RB mRNA was not detectable, while four others expressed variable quantities of RB mRNA with decreased molecular size of about 4.0 kb. In contrast, full-length RB mRNA was present in human fetal retina and placenta, and in other tumors such as neuroblastoma and medulloblastoma. DNA from retinoblastoma cells had a homozygous gene deletion in one case and hemizygous deletion in another case, while the remainder were not grossly different from normal human control DNA. The gene contains at least 12 exons distributed in a region of over 100 kb. Sequence analysis of complementary DNA clones yielded a single long open reading frame that could encode a hypothetical protein of 816 amino acids. A computer-assisted search of a protein sequence database revealed no closely related proteins. Features of the predicted amino acid sequence include potential metal-binding domains similar to those found in nucleic acid-binding proteins. These results provide a framework for further study of recessive genetic mechanisms in human cancers.

Seasonal abundance of anopheline mosquitoes and their association with rainfall and malaria along the Matapí River, Amapá, [corrected] Brazil.

Three communities separated by 1.5-7.0 km, along the Matapí River, Amapá State, Brazil, were sampled monthly from April 2003 to November 2005 to determine relationships between seasonal abundance of host-seeking anophelines, rainfall and malaria cases. Out of the 759 821 adult female anophelines collected, Anopheles darlingi Root (Diptera: Culicidae) was the most abundant (56.2%) followed by An. marajoara Galvão & Damasceno (24.6%), An. nuneztovari Gabaldón (12.4%), An. intermedius (Chagas) (4.4%) and An. triannulatus (Neiva and Pinto) (2.3%). Vector abundance, as measured by human landing catches, fluctuated during the course of the study and varied in species-specific ways with seasonal patterns of rainfall. Anopheles darlingi and An. triannulatus were more abundant during the wet-dry transition period in June to August, whereas An. marajoara began to increase in abundance in February in two villages, and during the wet-dry transition in the other village. Anopheles nuneztovari and An. intermedius increased in abundance shortly after the rains began in January to February. A generalized linear mixed model (GLMM) analysis of 32 consecutive months of collections showed significant differences in abundance for each species by village and date (P < 0.0001). Correlations between lagged rainfall and abundances also differed among species. A strong positive correlation of An. darlingi abundance with rainfall lagged by 4 and 5 months (Pearson's r = 0.472-0.676) was consistent among villages and suggests that rainfall may predict vector abundance. Significant correlations were detected between numbers of malaria cases and abundances of suspected vector species. The present study shows how long-term field research may connect entomological and climatological correlates with malaria incidence.

Effects of sociosexual environment on serum testosterone in captive male African rhinoceros.

The relationships between testosterone concentrations in male African rhinoceros and the presence of conspecific males and females were investigated. Serum testosterone concentrations were measured using enzyme-linked immunoassay (EIA) in 37 male black rhinoceros (Diceros bicornis) and 21 male white rhinoceros (Ceratotherium simum) housed at 37 institutions in the USA. Testosterone concentrations in both black (n=37) and white (n=21) rhinoceros males rose with increasing numbers of females present (P<0.05). Average testosterone concentrations also rose with an increased number of conspecific males (n=34) in black rhinoceros (P<0.05). However, no specific pattern was found among male white rhinoceros housed with other males. We inferred that introduction of females to a male may play an important role in stimulating libido and spermatogenesis. The similar response of black rhinoceros and white rhinoceros to increased numbers of females suggested that, at least historically, herd structure for blacks may have been more similar to whites than previously realized, and should be investigated further.

Limitations in the isolation and stimulation of splenic mononuclear cells in a dasyurid marsupial, Phascogale calura.

OBJECTIVE: Marsupials suffer from an increasing number of stressors in this changing world. Functional studies are thus needed to broaden our understanding of the marsupial immune system. The red-tailed phascogale (Phascogale calura) is a small Australian marsupial previously used in descriptive immunological studies. Here, we aimed to develop functional assays by isolating and stimulating blood and spleen mononuclear cells in vitro. RESULTS: While peripheral blood mononuclear cell (PBMC) were relatively easy to isolate, only 105 mononuclear cells (> 90% purity and > 75% viability) could be recovered from the spleen, independently of the sex and age of the animal or the centrifugation time and speed tested. The pores of the mesh sieve used for tissue homogenization might have been too big to yield a single cell suspension. Nevertheless, in spite of the overall low number of cells recovered, PBMC and splenic mononuclear cells were successfully activated in preliminary trials with phytohemaglutinin. This activation state was evidenced by a change in shape and the presence of small cell aggregations in the mitogen-stimulated cultures. A non-radioactive colorimetric assay was also performed to confirm cell proliferation in these wells. This work highlights the importance of developing and reporting detailed methodological protocols in non-traditional research species.

Neonatal oxytocin manipulations have long-lasting, sexually dimorphic effects on vasopressin receptors.

Developmental exposure to oxytocin (OT) or oxytocin antagonists (OTAs) has been shown to cause long-lasting and often sexually dimorphic effects on social behaviors in prairie voles (Microtus ochrogaster). Because regulation of social behavior in monogamous mammals involves central receptors for OT, arginine vasopressin (AVP), and dopamine, we examined the hypothesis that the long-lasting, developmental effects of exposure to neonatal OT or OTA might reflect changes in the expression of receptors for these peptides. On postnatal day 1, prairie voles were injected intraperitoneally with either OT (1 mg/kg), an OTA (0.1 mg/kg), saline vehicle, or were handled only. At approximately 60 days of age, vasopressin V1a receptors, OT receptors (OTR) and dopamine D2 receptor binding were quantified using receptor autoradiography in brain tissue taken from males and females. Significant treatment effects on V1a binding were found in the bed nucleus of the stria terminalis (BNST), cingulate cortex (CgCtx), mediodorsal thalamus (MdThal), medial preoptic area of the hypothalamus (MPOA), and lateral septum (LS). The CgCtx, MPOA, ventral pallidum, and LS also showed significant sex by treatment interactions on V1a binding. No significant treatment or sex differences were observed for D2 receptor binding. No significant treatment difference was observed for OTR receptor binding, and only a marginal sex difference. Changes in the neuropeptide receptor expression, especially the V1a receptor, may help to explain sexually dimorphic changes in behavior that follow comparable neonatal manipulations.

Specific activation of the cellular Harvey-ras oncogene in dimethylbenzanthracene-induced mouse mammary tumors.

Genomic DNAs from dimethylbenzanthracene-induced BALB/c mouse mammary tumors arising from the transplantable hyperplastic outgrowth (HPO) line designated DI/UCD transformed NIH 3T3 cells upon transfection. Transforming activity was attributed to the presence of activated Harvey ras-1 oncogenes containing an A----T transversion at the middle adenosine nucleotide in codon 61. DNAs from untreated DI/UCD HPO cells and radiation-induced and spontaneous mammary tumors from the DI/UCD HPO line failed to transform NIH 3T3 cells. The results indicated that the mutation activation of Harvey ras-1 oncogenes was specific to dimethylbenzanthracene treatment in the mouse mammary tumor system.

Substrate recognition by the 2 micron circle site-specific recombinase: effect of mutations within the symmetry elements of the minimal substrate.

The minimal substrate for the 2 microns circle site-specific recombinase FLP consists of a nearly perfect 13-base-pair dyad symmetry with an 8-base-pair core. By using a series of chemically synthesized FLP substrates in in vitro FLP recombination and FLP-binding assays, we have identified four positions within each of the symmetry elements that are important contact points for the FLP protein. Furthermore, the binding and recombination data provide evidence for cooperativity between the two symmetry elements of a substrate and between the symmetry elements of two partner substrates during FLP recombination.

Culture and stimulation of tammar wallaby lymphocytes.

We describe the culture and stimulation of lymphocytes from the model marsupial, the tammar wallaby (Macropus eugenii). We also describe the capacity of tammar wallaby lymphocytes isolated from blood, spleen and lymph nodes to produce soluble immunomodulatory factors. Culture conditions were optimized for mitogen-driven stimulation using the plant lectin phytohaemagglutinin (PHA). Products secreted by stimulated cells were harvested and crudely fractionated before they were added back to freshly isolated lymphocytes. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay, both stimulatory and inhibitory bioactive factors were detected in serum-free supernatants harvested from mitogen-treated peripheral blood mononuclear cells. This paper describes the capacity of leukocytes of the tammar wallaby to respond to mitogenic stimulation and to produce soluble, low-molecular-weight bioactive molecules that possess cytokine-like activity.

Neuroendocrine bases of monogamy.

A number of studies have implicated the neurohypophyseal peptides oxytocin and vasopressin in the central mediation of complex social behaviors, including affiliation, parental care and territorial aggression. Research on a monogamous rodent, the prairie vole (Microtus ochrogaster), suggests that these neuropeptides are also involved in the control of several behaviors associated with monogamy, including pair bonding, paternal care and mate guarding. Comparative studies using several species of vole have identified species-specific patterns of oxytocin- and vasopressin-receptor expression in the brain that appear to be associated with a monogamous versus non-monogamous social structure. Molecular studies suggest that changes in the regulation of oxytocin- and vasopressin-receptor gene expression underlie these species differences in receptor distribution and might provide a mechanism for the evolution of monogamy in voles.

Neuropeptides and the evolution of social behavior.

Comparative studies over the past year have revealed two new insights into the role of neuropeptides in the evolution of social behaviors. First, across vertebrate taxa, certain neuropeptide effects appear to be gender-specific. Second, species variations in receptor gene structure can alter neuropeptide receptor distribution and thereby contribute to species differences in social behavior.

Oxytocin receptors in the nucleus accumbens facilitate "spontaneous" maternal behavior in adult female prairie voles.

Oxytocin and the nucleus accumbens have been extensively implicated in the regulation of maternal behavior, and the processing of pup-related stimuli relevant for this behavior. Oxytocin receptor density in the nucleus accumbens is highly variable in virgin female prairie voles, as is their behavioral response to pups, ranging from neglecting and infanticidal to full maternal behavior. We hypothesized that oxytocin receptor in the nucleus accumbens facilitates the expression of "spontaneous" maternal behavior in prairie voles. Forty sexually-naive adult females were exposed to pups for the first time and tested for maternal behavior. Oxytocin receptor binding in the nucleus accumbens and other brain regions was later determined using autoradiography. Females that showed maternal behavior (lick and groom the pups and hover over them for at least 30 s, n=24) had higher oxytocin receptor density in the nucleus accumbens (shell subregion) (P<0.05) than females that did not show maternal behavior or attacked the pups (n=16). No differences were found in other brain regions (medial preoptic area, septum, prelimbic cortex). In a second experiment, we tested whether infusions of the oxytocin receptor antagonist (d(CH2)5(1),Tyr(Me)2,Orn8)-AVT into the nucleus accumbens would block "spontaneous" maternal behavior. As a control region, oxytocin receptor antagonist was also infused into the caudate putamen. Ten females were infused bilaterally into the nucleus accumbens or caudate putamen with either 2 ng/0.5 microl of oxytocin receptor antagonist or CSF (vehicle). While five of 10 nucleus accumbens CSF-infused animals showed maternal behavior, none of the nucleus accumbens oxytocin receptor antagonist-infused subjects did (0/10; chi2, P<0.01). Nucleus accumbens oxytocin receptor antagonist-infused females recovered the next day and were not different from controls. Animals infused with CSF or oxytocin receptor antagonist into the caudate putamen did not differ (four/10, four/10). This is the first study to show that the nucleus accumbens is involved in the regulation of "spontaneous" maternal behavior and that oxytocin receptor in this brain region facilitates maternal responses.

Alterations of acquired mouse mammary tumor virus DNA during mammary tumorigenesis in BALB/cfC3H mice.

The patterns of the milk-transmitted (exogenous) mouse mammary tumor virus (MuMTV) DNA restriction endonuclease fragments in the nodule and tumor stages of BALB/cfC3H mouse mammary neoplasia were compared with the use of the Southern blot analysis. Acquired MuMTV restriction fragments were detected in DNA from hyperplastic alveolar nodules (HAN), from primary hyperplastic outgrowths (HPO), from families of transplanted HPO, from tumors from HPO, and from serially transplanted tumors. The restriction fragment patterns suggested that the HAN were composed of clonal dominant populations. Transplantation of subdivisions of individual HAN resulted in HPO with DNA restriction patterns suggesting that HAN also contained two or more subpopulations. In all cases, HAN subpopulations shared MuMTV restriction fragments suggesting a common origin. Forty-seven tumors arising from HPO shared MuMTV restriction fragments with the HPO. Most but not all tumors had additional acquired MuMTV restriction fragments not detected in the progenitor HPO, indicating that they were composed of a distinct subpopulation that originated from the HPO. The restriction fragment pattern in some tumor lines was remarkably stable through many transplant generations. Some tumors had no major additional restriction fragments, suggesting that major rearrangements of MuMTV DNA are not required for tumorigenesis.