Tumours and malformations in the adult offspring of cyclophosphamide-treated and control male rats--preliminary communication

Adult offspring aged 52-104 weeks, from male Sprague-Dawley rats treated chronically with cyclophosphamide (CP) were examined for tumours and gross abnormalities. Litter size at birth and at weaning was found to be greatly reduced as a result of paternal CP treatment. No unusual abnormalities were found at post-mortem examination but there was an increase in the incidence of hydronephrosis in offspring from CP-treated males compared with offspring from control males. This increase could have been indirectly caused by CP-treatment through reduced litter size. Histological examination of 26 tumours showed a variety of tumour types in the offspring of CP-treated and control males. Two of the four uterine tumours in offspring from CP-treated males were examined histologically; one was a sarcoma and the other an adenocarcinoma. Although no uterine tumours were found in offspring from control males, it is not clear whether this difference in frequency was treatment-related. The most common tumour site in female offspring from both CP-treated and control males was the mammary gland, and all six of these tumours which were examined histologically were adenofibromas. Abnormal karyotypes were observed in 2 out of 21 offspring showing abnormalities from CP-treated males and none out of 2 offspring with abnormalities from control males. These were not associated with tumours. It was concluded from this limited study that there was no clear evidence of increased tumour incidence in the offspring from CP-treated males. There was an indication that abnormal karyotypes may have been caused by the paternal CP treatment and these abnormalities persisted into adulthood.     

Malformed foetuses and karyotype abnormalities in the offspring of cyclophosphamide and allyl alcohol-treated male rats

Litters sired by male rats chronically treated with cyclophosphamide (CP) or allyl alcohol (AA), were evaluated for the incidence of foetal malformations and karyotype abnormalities. The male rats were also examined for the induction of deleterious effects on various parameters including those involved in reproductive performance. A highly significant and consistent increase in the numbers of malformed foetuses was seen in the litters from CP-treated male rats. Chromosome preparations revealed that a large proportion of the malformed foetuses carried karyotypic abnormalities. These effects were paralleled by a large increase in the number of post-implantation losses without significant differences in several sperm and semen characteristics including sperm abnormalities. No adverse reproductive effects were observed with allyl alcohol treatment.     

Congenital defects in the offspring of male mice treated with ethylnitrosourea

Daily doses of ENU (25-100 mg/kg) were injected intraperitoneally into ICR strain male mice for 5 days. The males were mated to untreated virgin females of the same strain on days 1-16 and 64-80 after the last dose. Copulations during these periods involve, respectively, treated postmeiotic cells and spermatogonial stem cells. The uterine contents were examined on day 18 of pregnancy for evidence of dominant lethal effects. The fetuses were examined for external and skeletal abnormalities. ENU treatment of either postmeiotic cells or spermatogonial stem cells caused dose-dependent significant increases in the incidence of abnormal fetuses over the control level. The induction rate per live fetus per unit dose in mg/kg by treating spermatogonial stem cells was estimated to be 1.0 X 10(-4), which is 3-fold lower than the rate previously estimated for the same endpoint at the same germ cell stage with MNU. Cleft palate was the most frequent external abnormality in the ENU-treated and the control series. Malformed vertebrae was the most frequent skeletal abnormality in the treated series. Rib fusion was the only skeletal malformation seen in the control series. Dominant lethals were clearly induced when germ cells were treated as postmeiotic cells.     

Factors predicting increased incidence of abnormal behavior in male pigtailed macaques

To identify factors predicting abnormal behavior in laboratory monkeys, we observed all available singly housed 4- to 11-year-old male pigtailed macaques (Macaca nemestrina), the species/age/sex group most likely to be referred to the Washington National Primate Research Center's Psychological Well-Being Program for behavioral assessment. Of the 87 subjects, 29 had been referred to the program whereas 58 had not. Abnormal behavior was unrelated to the subject's housing location (biocontainment vs. other facility) or invasiveness of research. Nursery-reared subjects displayed more abnormal behavior than mother-reared subjects. Across and within rearing categories, the proportion of the first 48 months of life spent singly housed was positively related to the amount of abnormal behavior at maturity. This effect was stronger for subjects separated from the mother for clinical rather than experimental reasons, and least for mother-reared subjects. Locomotor stereotypy, by far the most frequent form of abnormal behavior, was positively related to time in single housing but was unrelated to rearing. These results reinforce the importance of tactile social contact during juvenility for the prevention of abnormal behavior in social primates. They also suggest that self-directed abnormal behaviors and locomotor stereotypies have different etiologies.     

Taurine supplementation in high-fat diet fed male mice attenuates endocrine pancreatic dysfunction in their male offspring

Amino Acids - Obesity in fathers leads to DNA damage and epigenetic changes in sperm that may carry potential risk factors for metabolic diseases to the next generation. Taurine (TAU)...     

Hyperactivity and alteration of the midbrain dopaminergic system in maternally stressed male mice offspring

We recently demonstrated that prolonged maternal stress produces profound and long-lasting deficits in brain functions by programming a subset of target genes. We have now examined the possible effects of prenatal stress on the motility of adult offspring and dopamine (DA)-related gene expression in their midbrains, one of the target brain regions of stress hormones. Maternally stressed adult male mice showed impaired response habituation to novelty, and increased wheel-running activity associated with altered responses to DA receptor and DA transporter (DAT) blockers. Along with the behavioral changes, the expression profiles of several genes of the midbrain DAergic system appeared to be altered. Expression of DAT was reduced and expression of DA receptors and striatal DA-regulated neuropeptide genes was also affected. Taken together, the present findings indicate that maternal stress can cause hyperactivity in adult offspring associated with alterations in the midbrain DAergic system suggestive of a functional hyperdopaminergic state.     

Crowding pregnant mice affects attack and threat behavior of male offspring

Attack and threat behavior of adult male offspring of female mice crowded during the final third of pregnancy was investigated. In 5-min test pairings with an anosmic "standard opponent" which had 50 microliter of male mouse urine applied to its fur, the prenatally stressed group of males showed significantly less attack behavior; attack latency was longer and number of attacks, bites, amount of time spent attacking, and composite aggression scores were all lower, compared with the control group. Similarly, less threat behavior was observed in offspring from crowded dams; there were lower frequencies of tail rattles, rough grooms, and upright threats. Additionally, proportionally fewer males in the prenatally stressed group attacked or displayed threats. A second experiment was designed to investigate the effects of exogenous androgen on the aggressiveness of males from crowded mice: testosterone propionate administration (500 micrograms/animal/day, for 5 days prior to testing) abolished differences both in the proportion of males from crowded mice that fought and also apparently abolished differences in intensities of attack and threat behavior between groups. However, trends toward reduced aggression in prenatally crowded males remained. More detailed analysis of these responses, based only on animals that displayed aggression, revealed significantly reduced intensity of aggression in offspring from females crowded during pregnancy, indicating that testosterone propionate therapy did not completely restore this behavior. In order to reduce postnatal effects due to possible differences in mothering, all offspring were fostered to untreated mothers at birth. The results are discussed in terms of in utero exposure of male fetuses of crowded dams to stress-liberated adrenal steroids of maternal origin, and the possible consequences for the endocrine integrity of these offspring.     

Increased resistin expression in the adipose tissue of male prolactin transgenic mice and in male mice with elevated androgen levels.

The aim of this study was to investigate the regulation of resistin, a recently identified adipocyte-secreted peptide, in the adipose tissue of prolactin (PRL)-transgenic (tg) mice using ribonuclease protection assay. The level of resistin mRNA increased 3.5-fold in the adipose tissue of untreated male PRL-tg mice compared to controls. However, there was no difference in resistin expression in the adipose tissue of female PRL-tg mice compared to control mice. PRL-tg male mice have elevated serum testosterone levels and we therefore analyzed the effects of testosterone alone on resistin mRNA expression. Furthermore, the effects of elevated androgen levels on PRL receptor (PRLR) mRNA expression in the adipose tissue were investigated. Resistin mRNA increased 2.6-fold in the adipose tissue of control male mice with elevated serum androgen levels. In addition, PRLR mRNA expression was increased in the adipose tissue of male mice with elevated testosterone. These results suggest testosterone to be a regulator of resistin and PRLR mRNA expression in the adipose tissue of male mice.     

Inheritance of steroid-independent male sexual behavior in male offspring of B6D2F1 mice

The importance of gonadal steroids in modulating male sexual behavior is well established. Individual differences in male sexual behavior, independent of gonadal steroids, are prevalent across a wide range of species, including man. However, the genetic mechanisms underlying steroid-independent male sexual behavior are poorly understood. A high proportion of B6D2F1 hybrid male mice demonstrates steroid-independent male sexual behavior (identified as “maters”), providing a mouse model that opens up avenues of investigation into the mechanisms regulating male sexual behavior in the absence of gonadal hormones. Recent studies have revealed several proteins that play a significant factor in regulating steroid-independent male sexual behavior in B6D2F1 male mice, including amyloid precursor protein (APP), tau, and synaptophysin. The specific goals of our study were to determine whether steroid-independent male sexual behavior was a heritable trait by determining if it was dependent upon the behavioral phenotype of the B6D2F1 sire, and whether the differential expression of APP, tau, and synaptophysin in the medial preoptic area found in the B6D2F1 sires that did and did not mate after gonadectomy was similar to those found in their male offspring. After adult B6D2F1 male mice were bred with C57BL/6J female mice, they and their male offspring (BXB₁) were orchidectomized and identified as either maters or “non-maters”. A significant proportion of the BXB₁ maters was sired only from B6D2F1 maters, indicating that the steroid-independent male sexual behavior behavioral phenotype of the B6D2F1 hybrid males, when crossed with C57BL/6J female mice, is inherited by their male offspring. Additionally, APP, tau, and synaptophysin were elevated in in the medial preoptic area in both the B6D2F1 and BXB₁ maters relative to the B6D2F1 and BXB₁ non-maters, respectively, suggesting a potential genetic mechanism for the inheritance of steroid-independent male sexual behavior.     

A male with two different familial autosomal fragile sites and a cytogenetically abnormal offspring

During the course of routine prenatal cytogenetics, a male with 2 different autosomal fragile sites (FS) was detected. The FS were at 9p21 and 12q13 and his sister also had both fragile sites, inherited from an obligate carrier father. He was the father of a foetus with an abnormal chromosome complement 46,XY/47,XY,+frag. The origin of the fragment could not be determined.     

Increased vasopressin expression in the BNST accompanies paternally induced territoriality in male and female California mouse offspring

While developmental consequences of parental investment on species-typical social behaviors has been extensively characterized in same-sex parent-offspring interactions, the impact of opposite-sex relationships is less clear. In the bi-parental California mouse (Peromyscus californicus), paternal retrieval behavior induces territorial aggression and the expression of arginine vasopressin (AVP) in adult male offspring. Although similar patterns of territorially emerge among females, the sexually dimorphic AVP system has not been considered since it is generally thought to regulate male-typical behavior. However, we recently demonstrated that male and female P. californicus offspring experience increases in plasma testosterone following paternal retrieval. Since AVP expression is androgen-dependent during development, we postulate that increases in AVP expression may accompany territoriality in female, as well as male offspring. To explore this aim, adult P. californicus offspring that received either high or low levels of paternal care (retrievals) during early development were tested for territoriality and immunohistochemical analysis of AVP within the bed nucleus of the stria terminalis (BNST), paraventricular nucleus (PVN), and supraoptic nucleus (SON). Consistent with previous studies, high care offspring were more aggressive than low care offspring. Moreover, high care offspring had significantly more AVP immunoreactive (AVP-ir) cells within the BNST than low care offspring. This pattern was observed within female as well as male offspring, suggesting an equally salient role for paternal care on female offspring physiology. Regardless of early social experience, sex differences in AVP persisted in the BNST, with males having greater expression than females.     

Dominant cataract and recessive specific locus mutations in offspring of X-irradiated male mice

Male mice were X-irradiated with 3.0 + 3.0 Gy or 5.1 + 5.1 Gy (fractionation interval 24 h). The offspring were screened for dominant cataract and recessive specific locus mutations. In the 3.0 + 3.0-Gy spermatogonial treatment group, 3 dominant cataract mutations were confirmed in 15 551 offspring examined and 29 specific locus mutations were recovered in 18 139 offspring. In the post-spermatogonial treatment group, 1 dominant cataract mutation was obtained in 1120 offspring and 1 recessive specific locus mutation was recovered in 1127 offspring. The induced mutation rate per locus, per gamete, per Gy calculated for recessive specific locus mutations is 2.0 X 10(-5) in post-spermatogonial stages and 3.7 X 10(-5) in spermatogonia. For dominant cataract mutations, assuming 30 loci, the induced mutation rate is 5.0 X 10(-6) in the post-spermatogonial stages and 1.1 X 10(-6) in spermatogonia. In the 5.1 + 5.1-Gy spermatogonial treatment group, 3 dominant cataract mutations were obtained in 11 205 offspring, whereas in 13 201 offspring 27 recessive specific locus mutations were detected in the spermatogonial group. In the post-spermatogonial treatment group no dominant cataract mutation was observed in 425 offspring and 2 recessive specific locus mutations were detected in 445 offspring. The induced mutation rate per locus, gamete and Gy in spermatogonia for recessive specific locus mutations is 2.8 X 10(-5) and for dominant cataract mutations 0.9 X 10(-6). In post-spermatogonial stages, the mutation rate for recessive specific locus alleles is 6.2 X 10(-5). In the concurrent untreated control group, in 11 036 offspring no dominant cataract mutation and in 23 518 offspring no recessive specific locus mutation was observed. Litter size and the number of carriers at weaning have been determined in the confirmation crosses of the obtained dominant cataract mutants as indicators of viability and penetrance effects. Two mutants had a statistically significantly reduced litter size and one mutant had a statistically significantly reduced penetrance.     

Dominant cataract and recessive specific-locus mutations detected in offspring of procarbazine-treated male mice

The induction of dominant cataract mutations by procarbazine was studied concomitantly with the induction of specific-locus mutations in treated male mice. The most effective dose in the specific-locus test, 600 mg/kg of procarbazine, and a fractionated dose of 5 X 200 mg/kg were used. The frequencies of dominant cataract mutations were higher, but not significantly different from the historical control. The ratio between the number of recovered specific-locus and dominant cataract mutations was in accordance with that found in our experiments with gamma-rays (Ehling et al., 1982; Kratochvilova, 1981) or in experiments with ethylnitrosourea (Favor, 1986). A total of 3 dominant cataract mutations were recovered in the offspring of procarbazine-treated spermatogonial stem cells. Two mutations had complete penetrance while the third exhibited a reduced penetrance of approximately 70%. The viability and fertility of the heterozygotes of all 3 mutations were not affected. Only 1 mutation was shown to be viable as a homozygote.     

Effects of recombinant murine granulocyte-macrophage colony-stimulating factor in cyclophosphamide-treated mice

To evaluate the efficacy of recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) in attenuating the myelosuppression associated with chemotherapy, the effects of 100 and 300 ng rGM-CSF, administered twice daily by intraperitoneal injection for 6 consecutive days to mice 24 hours after a dose of 200 mg/kg cyclophosphamide, were measured. Six days after the initial injection of rGM-CSF, a significant increase occurred in the absolute myeloid count compared to that of vehicle-treated animals. The difference was most pronounced on day 7, attaining levels of 327% and 428% of the control; these increases slowly declined to that of the control level by day 19. No significant effect was produced by rGM-CSF on the packed red cell volume or on the platelet count. Furthermore, the administration of rGM-CSF did not alter bone marrow cellularity or increase the number of marrow-derived hematopoietic stem cells. In contrast, a significant splenomegaly occurred, starting on day 6 and continuing until day 17. This was characterized by a pronounced increase in splenic-derived granulocyte (CFU-G), granulocyte-macrophage (CFU-GM), macrophage (CFU-M), megakaryocyte (CFU-MK), and erythroid (BFU-E, CFU-E) stem cells. The increases occurred between days 6 and 9 following the initial administration of rGM-CSF. These findings indicated that the administration of rGM-CSF to cyclophosphamide-treated animals causes an absolute increase in circulating myeloid cells and that these increases are derived from the spleen. The use of recombinant hematopoietic growth factors may permit the administration of more intensive chemotherapy through amelioration of chemically induced leukopenia.     

Increased bone mass in male and female mice following tamoxifen administration

Tamoxifen is capable of preserving bone mass in gonadectomized rodents as well as intact female mice; however, a detailed 3D quantitative analysis of the structural changes produced in the growing skeleton of intact mice of both genders by this agent is lacking. Employing quantitative microcomputed tomography (muCT), we assessed the effects of 4-hydroxytamoxifen (OHT) on the femora of C57BL/6J mice administered this agent either for 12 (males and females) or 2 (females) weeks. In mice of either gender, but especially in females, 12 weeks of OHT exposure led to dramatic increases in both cortical and trabecular bone. Females exposed to OHT for either 2 or 12 weeks demonstrated significantly increased cortical wall thickness, trabecular bone volume, connectivity, and number, as well as decreased trabecular separation. Significant increases in several of these parameters were also evident in males after 12 weeks of OHT administration. In view of the expanding use of OHT to induce Cre-mediated recombination events, our findings suggest that care should be exercised when interpreting the skeletal phenotypes of mice exposed this agent, particularly in situations where the effects of OHT might synergize with the phenotypic outcome of a specific genetic alteration.     

Frequency of congenital defects and dominant lethals in the offspring of male mice treated with methylnitrosourea

ICR strain male mice injected intraperitoneally with daily doses of MNU (5–25 mg/kg) for 5 days and mated to untreated virgin females of the same strain on days 1–7, 8–14, 15–21 and 64–80 after the last dose. Copulations during these periods involve, respectively, spermatozoa, late spermatids, early spermatids, and spermatogonial stem cells at the time of the last treatment. The uterine contents were examined on day 18 of pregnancy for post-implantation losses (dominant lethality). Fetuses were examined for exteranal and skeletal abnormalities. In contrast to the results reproted for specific-locus mutations, MNU treatment of either postmeiotic cells or spermatogonial stem cells caused dose-dependent significant increases in the incidence of congenital defects and of dominant lethals over the control levels. The relative sensitivity of germ cells sampled on days 1–7, 8–21 and 64–80 to MNU-induced congenital defects was 1:1.6:2. For the induction of dominant lethals, the sensitivity ratio was 1:1.8:0.5. It is proposed that congenital defects in the offspring of mice following paternal treatment with MNU may represent mostly chromasomal rather than genic changes. Cleft palate was the most frequent of the external abnormalities, which were significantly induced in every treatment series; fused ribs were the most frequent of the skeletal abnormalities, which were significatly induced in the treatment series for spermatogonial stem cells.     

Increased blood pressure in the offspring of diabetic mothers

Studies were conducted in rats to determine the effect of maternal diabetes and the consequent hyperglycemia on cardiovascular function in the offspring. Diabetes was induced in pregnant Wistar rats through streptozotocin injection (50 mg/kg). Cardiovascular parameters were measured in 2-mo-old offspring animals of diabetic (OD, n=12) and control rats (OC, n=8). Arterial pressure (AP), heart rate (HR), baroreflex sensitivity, and vascular responsiveness to phenylephrine (PH) and sodium nitroprusside (SN) were measured. Angiotensin-converting enzyme (ACE) activity in heart, kidney, and lung was determined. OD rats exhibited increases in systolic AP (138+/-8 vs. 119+/-6 mmHg, OD vs. OC), with no change in HR (342+/-21 vs. 364+/-39 beats per minute (bpm), OD vs. OC). The reflex tachycardia elicited by SN was reduced in OD rats, as indicated by the slope of the linear regression (-2.2+/-0.4 vs. -3.6+/-0.8 bpm/mmHg, OD vs. OC). Vascular responsiveness to PH was increased 63% in OD rats compared with OC. OD rats showed increases in ACE activity in heart, kidney, and lung (1.13+/-0.24, 3.04+/-0.86, 40.8+/-8.9 vs. 0.73+/-0.19, 1.7+/-0.45, 28.1+/-6 nmol His-Leu.min-1 mg protein-1, OD vs. OC). Results suggest that diabetes during pregnancy affects cardiovascular function in offspring, seen as hypertension, baroreflex dysfunction, and activation of tissue renin-angiotensin system.