Increased postimplantation loss and malformations among the F2 progeny of male rats chronically treated with cyclophosphamide.

Cyclophosphamide, administered to the male rat, produces increased pre- and postimplantation loss in the progeny as well as an increase in the numbers of malformed and growth retarded fetuses. The purpose of this study was to determine whether the adverse effects of chronic paternal cyclophosphamide exposure are transmissible to the next generation, the F2 progeny. Adult male rats were treated by gavage daily with saline or with cyclophosphamide (3.4 or 5.1 mg/kg) for 4 or 18 weeks and mated. The male and female offspring in each treatment group (F1 generation) were randomly mated. The resulting pregnant females were killed on day 20 of gestation to evaluate progeny outcome in the F2 generation. There was a significant increase in postimplantation loss among the offspring of the group whose fathers had been treated with cyclophosphamide at a dose of 5.1 mg/kg/day. Exposure to a dose of 5.1 mg/kg/day of cyclophosphamide also resulted in an F2 generation with a significantly decreased mean fetal weight per litter and a significant increase in the number of malformed fetuses. The malformations observed among the F2 progeny included open eyes, omphalocele, generalized edema, syndactyly, gigantism, and dwarfism. Thus, exposure of the father to cyclophosphamide does result in a specific and heritable alteration in the fertility of the surviving "apparently normal" F1 progeny. Interestingly, the adverse consequences of exposure of male rats to cyclophosphamide are similar in the F2 generation to those previously reported for the F1 progeny.     

Effects of prenatal administration of testosterone and cortisone on the reproductive system of the female rat.

Testosterone propionate, cortisone, or sesame oil vehicle were given to rats during the last week of pregnancy so that effects of the hormones on anogenital distance, breeding capacity and vaginal opening of the female progeny could be contrasted. Testosterone significantly increased anogenital distance and delayed vaginal opening of progeny. When females that had been exposed to testosterone in utero were tested for breeding capacity, a significantly smaller number mated than in the control group. Female rats that had been exposed to cortisone in utero exhibited premature vaginal opening but did not differ from controls in anogenital distance, and, unlike the testosterone-exposed rats, mated. Cortisone-exposed rats carried litters to term and the litters did not differ from those of controls in numbers of pups or numbers of living pups at birth. The pups born to cortisone-exposed rats had greater birth weights and a higher survival rate to 20 days of age than pups of controls. Results indicate that testosterone administration to rats during pregnancy is far more detrimental to the development and subsequent function of the reproductive system of female progeny than cortisone and suggest that similar changes which occur in response to maternal stress or to administration of ACTH during pregnancy are more likely to result from increases in testosterone than from increases in glucocorticoid secretion.     

The embryotoxicity of gossypol

In utero development was analyzed in pregnancies that resulted from matings between gossypol-treated male rats and untreated female rats and in pregnancies in which gossypol was administered to the pregnant rat only. Gossypol treatment of males had no effect on the outcome of pregnancy. There was no significant effect on resorption, fetal growth, or malformation rate. Similarly, gossypol administered to pregnant dams at stages during organogenesis had no observable effect on pregnancy. Under the conditions of this experiment, gossypol administered to either the breeding male rat or the pregnant female rat had no demonstrable adverse effect on development in utero.     

Studies on feminine sexual behavior in the male rat: Influence of olfactory stimuli

The aim of this study was to determine if the display of lordosis behavior in the male rat could be influenced by the olfactory environment. Unexperienced adult male rats were orchidectomized (ORCH). They were primed with 75 μg estradiol benzoate and 1 mg progesterone was injected at an interval of 39 hr following long-term (LT = 3 weeks) or short-term (SHT = 8 hr 30 min) exposure to the odor of male or female urine. For 10 min they were placed in the presence of a “stimulus” male of proven sexual vigor 9 hr 30 min ± 1 hr after progesterone injection. Both LT and SHT exposure to the odor of male urine caused a significant increase in the number of ORCH rats which showed lordosis response to male mounts compared to either the ORCH rats exposed to the odor of female urine or to the controls. Following complete olfactory bulb removal (COBR), no difference was observed in the occurrence of lordosis behavior between the ORCH rats whether or not exposed to the odor of urine. For the ORCH-COBR rats exposed to male urine the proportion of animals responding to mounts did not differ from that of their nonbulbectomized counterparts. In comparing the effects of COBR vs anterior olfactory bulb removal (AOBR) lordosis behavior occurred more frequently in COBR than in AOBR-ORCH rats. The lordosis quotient (LQ) was not affected by exposure to the odor of male urine in the nonbulbectomized ORCH rats. In contrast, it appeared to be higher in both COBR and AOBR animals than in their nonbulbectomized counterparts. The olfactory bulbs were then concluded to inhibit the display of lordosis behavior in the male rat. It was also thought that the olfactory stimuli originating from male urine were capable of releasing the hypothalamic structures involved in the control of lordosis behavior of the male rat from an olfactory inhibitory influence.     

Gender-related differences in expression and function of hepatic P-glycoprotein and multidrug resistance-associated protein (Mrp2) in rats

To clarify whether gender-related differences exist in the expression and function of hepatic P-glycoprotein- and/or multidrug resistance-associated protein (Mrp2), we measured the hepatobiliary excretion of doxorubicin and their protein levels in male and female Sprague-Dawley rats. When rats received a single intravenous injection of doxorubicin (5 mg/kg), a delay in the disappearance of doxorubicin from plasma was observed in male rats. When rats received a constant-rate infusion of doxorubicin, no significant gender-related differences in the apparent biliary clearance of doxorubicin based on the steady state plasma concentrations were observed between male and female rats. However, the net biliary clearance of doxorubicin based on the liver concentration, which represents the actual function of P-glycoprotein and/or Mrp2, was higher in female rats than in male rats. These results suggest that the actual function of the hepatobiliary transport of doxorubicin is greater in female than in male rats. Western blot analysis revealed that the expression of P-glycoprotein and Mrp2 in the liver of female rats was significantly higher than in male rats, similar to results of hepatobiliary excretion experiments. The expression of hepatic cytochrome P450 (CYP) 2B1, which is involved in the metabolism of doxorubicin, was significantly higher in male than in female rats. By pretreatment with testosterone (10 mg/day for 7 days), the actual biliary clearance of doxorubicin in female rats was nearly that of male rats. The protein levels of P-glycoprotein and Mrp2 in female rats were also lowered by treatment with testosterone so as to be nearer those in male rats. These results suggest that gender-related differences exist in P-glycoprotein- and Mrp2-mediated hepatobiliary transport and that these two transporters may be regulated by sex hormones.     

Maternal hyperinsulinemia predisposes rat fetuses for hyperinsulinemia, and adult-onset obesity and maternal mild food restriction reverses this phenotype

We have previously shown that artificial rearing of newborn female rat pups on a high-carbohydrate (HC) milk formula resulted in chronic hyperinsulinemia and adult-onset obesity (HC phenotype) and that the maternal HC phenotype was transmitted to their progeny (2-HC rats) because of fetal development in the HC female rat. The aims of this study were to investigate 1) the fetal adaptations that predisposed the progeny for the expression of the HC phenotype in adulthood and 2) whether the transfer of the HC phenotype to the progeny could be reversed by maternal food restriction. Fetal parameters such as plasma insulin and glucose levels, mRNA level of preproinsulin gene, pancreatic insulin content, and islet insulin secretory response in vitro were determined. On gestational day 21, 2-HC fetuses were hyperinsulinemic, had increased insulin content and mRNA level of the preproinsulin gene in their pancreata and demonstrated an altered glucose-stimulated insulin secretory response by isolated islets. Modification of the intrauterine environment in HC female rats was achieved by pair feeding them to the amount of diet consumed by age-matched control rats from the time of their weaning. This mild dietary restriction reversed their HC phenotype and also prevented the development of the HC phenotype in their progeny. These findings show that mal-programming of the progeny of the hyperinsulinemic-obese HC female for the expression of the HC phenotype is initiated in utero and that normalization of the maternal environment in HC female rats by mild food restriction resulted in the normal phenotype in their progeny.     

Differential effects of testosterone metabolites in the neonatal period on open-field behavior and lordosis in the rat

Two experiments were done to compare the effects of neonatal exposure to testosterone and its major metabolites, dihydrotestosterone (DHT) and estradiol (E₂), on the development of sex differences in open-field behavior in the rat. In Experiment 1 female rats administered either testosterone propionate (TP), DHT, or estradiol benzoate (EB) were found as adults to have low activity scores, more typical of adult males, when compared to the high scores of oil-treated females. In Experiment 2 the adult open-field behavior of female rats treated neonatally with testosterone or the metabolites was compared to that of male rats treated from Day 1 to 10 of life with the aromatizing enzyme inhibitor, androst-1,4,6-triene-3,17-dione (ATD). These same animals were later tested for lordotic behavior after gonadectomy and priming with EB and progesterone. All male animals and female animals exposed neonatally to testosterone or to either of the metabolites had suppressed open-field activity scores compared to oil-treated females. However, the lordotic behavior of females exposed to DHT and of males exposed to ATD was not defeminized and was comparable to that of oil-treated females. These observations were discussed in terms of a role for the androgenic actions of testosterone in establishing sex differences in nonreproductive behavior in the rat.     

Delayed teratogenic effect of methylmercury on hepatic cytochrome P-450-dependent monooxygenase systems of rats.

Hepatic cytochrome P-450-dependent monooxygenase systems were depressed in adult male rats which had been exposed prenatally to methylmercury. The effect was not seen in immature male or female rats or in adult female rats. This latent teratogenic effect of methylmercury appeared only when exposure occurred during early fetal development. These studies demonstrate that the expression of a chemically induced teratogenic effect may be considerably delayed in the rat and that the stage of development of the fetus at the time of exposure to the chemical agent may be a determining factor.     

Environmental levels of oestrogenic and antiandrogenic compounds feminize digit ratios in male rats and their unexposed male progeny

Digit length ratios, especially the second-to-fourth digit ratio (2D : 4D), are associated with various pathological and behavioural conditions in many species including humans and are dependent upon prenatal androgen to oestrogen balance. It is unknown whether digit ratios are modified by environmental exposure to ubiquitous endocrine disruptors. We studied the effect on adult male Wistar rat digit ratios of a gestational exposure to the oestrogenic and antiandrogenic compounds bisphenol A (BPA), genistein and vinclozolin, in low doses, and in combination with investigating in parallel a possible sexual dimorphism of this trait. We also investigated the effects on the male progeny not exposed during gestation. X-rays were taken of the left and right forepaws, and 2D–5D proximal to distal phalanx distances were measured by a standardized procedure based on semi-automatic image analysis. We provide evidence that there is a sexual dimorphism of digit ratios in the Wistar rat, and we found that BPA alone or in combination with genistein and vinclozolin significantly feminized digit ratios in male rats. Intriguingly, significant feminization of digit ratios was also found in the unexposed male progeny of males that had been exposed to compound mixtures. In conclusion, prenatal environmental levels of endocrine-active substances permanently disrupt digit ratios. Digit ratio measurement in adults is thus a promising biomarker of prenatal exposure to low-dose endocrine disruptors in rodents, with potential implications for future studies in humans.     

Annual changes in the copulatory behavior of male rats maintained under constant laboratory conditions

ABSTRACT

The objective of the present study was to evaluate the sexual behavior of male rats kept under constant laboratory conditions for one entire year. A total of 213 sexually-inexperienced, male Wistar rats were maintained in controlled environmental conditions from birth. Depending the month in which they reached the age 3-month-old, the male rats were divided into 12 groups, one for each month of the year, and their sexual behavior was evaluated. Records of their sexual behavior were made from 09:00 to 11:00 hrs am. The following parameters were recorded: mount (latency and number), intromission (latency and number), ejaculation latency, and intromission rate. During the months of March, June, July and September, the rats showed lower mount and intromission latencies than in January, February, April, May and October-to-December. Similarly, in March, June, July and August they had higher copulatory efficiency than in January, February, April and December. Results suggest that male rats exposed to controlled environmental conditions could have endogenous mechanisms that regulate sexual behavior but are independent of seasonal environmental signals. The annual variability in the sexual behavior of male rats maintained under constant laboratory conditions should be considered when planning research and experiments.     

Elevated Micronucleus Frequency in Bone Marrow Erythrocytes in the Progeny of Male Mice Exposed to Chronic Low-Dose Gamma Irradiation

The micronucleus frequency in bone marrow erythrocytes from the F1 progeny of male mice exposed to chronic low-dose -irradiation was determined. Male BALB/c mice were irradiated with 10, 25 and 50 cGy at dose rates of 1, 5, and 15 cGy/day and mated with unirradiated females on day 15 after irradiation. The obtained offspring had an elevated micronucleus frequency in bone marrow erythrocytes at the age of 2 months. This suggests the transmission of genome instability from damaged germ-line cells of irradiated male parents to somatic cells of the progeny.     

Effects of paternal ethylene dibromide exposure on F1 generation behavior in the rat

The effects of ethylene dibromide (EDB) exposure to the male rat were studied through behavioral assessments of their F1 progeny. Exposed males were bred with untreated female rats at 4 or 9 weeks after 5 daily EDB treatments. Behavioral assessment of motor reflexes and motor coordination were examined up to 21 days of age. Significant differences in the development of motor coordination and motor activity were observed in the F1 progeny of EDB-exposed males. These results support the evidence of EDB genotoxicity and further demonstrates the utility of behavioral end-points of the offspring as a sensitive means of assessing paternal reproductive risk.     

Can prenatal exposure to a 900 MHz electromagnetic field affect the morphology of the spleen and thymus,and alter biomarkers of oxidative damage in 21-day-old male rats?

We investigated the effects of a 900 Megahertz (MHz) electromagnetic field (EMF), applied during the prenatal period, on the spleen and thymus of 21-day-old male rat pups. Pregnant Sprague-Dawley rats were divided into control and EMF groups. We applied 900 MHz EMF for 1 h/day to the EMF group of pregnant rats. Newborn male rat pups were removed from their mothers and sacrificed on postnatal day 21. Spleen and thymus tissues were excised and examined. Compared to the control group, thymus tissue malondialdehyde levels were significantly higher in the group exposed to EMF, while glutathione levels were significantly decreased. Increased malondialdehyde and glutathione levels were observed in splenic tissue of rats exposed to EMF, while a significant decrease occurred in superoxide dismutase values compared to controls. Transmission electron microscopy showed pathological changes in cell morphology in the thymic and splenic tissues of newborn rats exposed to EMF. Exposure to 900 MHz EMF during the prenatal period can cause pathological and biochemical changes that may compromise the development of the male rat thymus and spleen.     

AP-PCR assay of DNA alterations in the progeny of male mice exposed to low-level gamma-radiation

By comparative analysis of fingerprints of arbitrarily primed polymerase chain reaction (AP-PCR) products, DNA alterations in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-doses of gamma-radiation was investigated. Male BALB/c mice exposed to 10-50 cGy were mated with unirradiated females 15 days after irradiation. DNA was isolated from biopsies taken from tail tips of 2-month-old progeny. Preliminary AP-PCRs were carried out with 17 primers representing core sequences of micro- and/or minisatellites or their flanking oligonucleotides. Best quantitatively reproduced AP-PCR fingerprints of genomic DNA were obtained with one of these primers, a 20-mer oligonucleotide flanking the micro-satellite locus Atplb2 on mouse chromosome 11. Comparative analysis of individual fingerprints of AP-PCR products obtained on DNA templates from the progeny of irradiated and intact males revealed an increased variability of micro-satellite-associated sequences and an increased frequency of "non-parental bands" in DNA-fingerprints from the progeny of males chronically exposed to gamma-radiation 15 days before mating (at the postmeiotic stage of spermatogenesis). The results show that increased micro-satellite instability can be initiated by irradiation of the male parent to subsequently arise or be transmitted to the soma of the F1 generations.     

Paternal cyclophosphamide treatment causes postimplantation loss via inner cell mass-specific cell death.

Treatment of the father with the anticancer alkylating agent cyclophosphamide has negative effects on embryonic development in the rat. Four-week treatment of male rats with a low dose of cyclophosphamide causes a dramatic, dose-dependent increase in postimplantation death of the progeny. Several recent studies have indicated that the paternal genome is required for the development of the extraembryonic tissues. Thus, the purpose of this study was to determine which tissues of the implanting embryo were affected by paternal exposure to cyclophosphamide. Male Sprague-Dawley rats were given cyclophosphamide (6 mg/kg/day) or saline by gavage and bred to untreated female rats after 4 weeks of treatment. Pregnant female rats were killed on day 7 of gestation, and implantation sites were dissected from the uterus, fixed, embedded in Epon for semithin serial sectioning, and stained for subsequent light microscopy. Strikingly, many of the implantation sites of affected embryos sired by treated males displayed an apparently normal trophectoderm enclosing a region of dying cells, containing dark-stained pyknotic nuclei. Very few or no inner cell mass-derived embryonic cells were present in these implantation sites. Therefore, there is a selective death of inner cell mass-derived cells in day 7 implantation sites obtained from the progeny of cyclophosphamide-treated males. The results of this study suggest that treatment of the male with cyclophosphamide can affect paternal genes specifically required for development of the inner cell mass cells of the embryo, without an apparent effect on those genes required for normal trophectoderm.     

遗传性大鼠脐疝模型的选育

目的近交繁殖先天性脐疝大鼠获得能稳定遗传的大鼠脐疝模型,大鼠脐疝结构观察及治疗。方法每代大鼠全同胞交配,记录产子数及脐疝情况,分析大鼠脐疝率;取F2代6月龄脐疝雌雄大鼠各6只,其中雌雄各2只进行解剖观察,雌雄大鼠各4只进行外科手术缝合。结果随近交系数增大,大鼠脐疝率升高,F12、F13代大鼠均为脐疝;F1代至F13代雌性大鼠总体脐疝率显著高于雄性大鼠（x2=11.1,P=0.001）;雌雄大鼠脐疝结构一致,手术后3~4周痊愈无复发。结论经连续13代全同胞交配获得了遗传性状稳定的大鼠脐疝模型。     

Maternal high-fat diet consumption results in fetal malprogramming predisposing to the onset of metabolic syndrome-like phenotype in adulthood

Chronic consumption of a high-fat (HF) diet by female rats in their postweaning period resulted in significant increases in body weight and plasma levels of insulin, glucose, and triglycerides during pregnancy compared with female rats consuming a standard rodent laboratory chow (LC). On gestational day 21, plasma insulin levels and the insulin secretory response of islets to various secretogogues were significantly increased in HF fetuses. The HF male progeny weaned onto LC (HF/LC) demonstrated increases in body weight from postnatal day 60 onward. In adulthood, HF/LC male rats were significantly heavier than controls, had increased plasma levels of insulin, glucose, free fatty acids, and triglycerides, and demonstrated glucose intolerance. HF/LC male islets secreted increased amounts of insulin in response to low glucose concentrations, but their response to a high glucose concentration was similar to that of LC/LC islets. In another set of experiments, when the male progeny of HF female rats were weaned onto a high-sucrose diet (HF/HSu), their metabolic profile was further worsened. These results indicate that chronic consumption of a HF diet by female rats malprograms the male progeny for glucose intolerance and development of increased body weight in adulthood. The long-term high-fat feeding to female rats employed in this study bears resemblance to the dietary habits in Western societies. The results of this study implicate dietary practices of women in the etiology of the present epidemic of human obesity and related disorders.     

Postnatal fate of the ultimobranchial remnants in the rat thyroid gland

The ultimobranchial follicles (UBFs) are considered embryonic remnants from the ultimobranchial body (UBB). They are follicular structures that vary in size and appearance depending on the age of the rat. The main objective of this article was to study the progressive changes in shape, size, and frequency of the UBFs in the postnatal rat, from birth to old‐age. To accomplish that objective, a systematic morphometric and incidental study of the UBF has been carried out in 110 Wistar rats of different ages and both sexes, divided into three groups: 1) young rats (5–90‐day‐old); 2) adult rats (6–15‐month‐old), and 3) old rats (18–24‐month‐old). The glands were serially sectioned and immunostained for calcitonin at five equidistant levels. According to our results, UBFs were observed in all thyroid glands but a more exhaustive sampling was occasionally necessary in male rats. In young rats, immature UBFs predominantly appeared whereas in adult rats, mature UBFs with cystic appearance and variable luminal content prevailed. We frequently found spontaneous anomalous UBFs in old rats, which we have termed as “ultimobranchial cystadenomata.” Additionally, in young rats, UBF areas significantly increased with age and they were larger when compared to that of normal thyroid follicles. Likewise, in adult rats, UBFs were significantly larger than normal thyroid follicles but only in female rats. In general, UBFs in females were also significantly larger than those found in male rats. Finally, all these differences related to UBFs together with a higher incidence in females of UB cystadenomata suggest a sexual dimorphism in regard to the destiny of these embryonic remnants during postnatal thyroid development. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Intra- and interspecific competition between biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) from Spain

Biotypes B and Q of Bemisia tabaci (Gennadius), collected from the islands of Tenerife and Majorca respectively, were exposed to competition conditions on tomato cv. Marmande in the laboratory. Both biotypes were established in single and mixed cultures at different densities. Increased mortality of biotype Q females and immature instars was observed together with a lower rate of fecundity and progeny size compared to biotype B, when reared in single or mixed cultures. The female:male sex ratio of F1 individuals of biotype Q was higher in single than in mixed cultures. However, the sex ratio of F1 individuals of biotype B was the same in single and mixed cultures, suggesting reproductive interference. Whitefly density did not affect interspecific interactions. It had a moderate effect on developmental rate of both biotypes, and on mortality of immature instars and progeny size of biotype B only. The results indicate that under laboratory conditions the studied biotype B should displace biotype Q.     

The decline of atrial natriuretic peptide (ANP) gene expression in older rats and the effects of ginsenoside on ANP gene expression.

1. The levels of atrial natriuretic peptide(ANP) gene expression in rat atria at 2-3 and 14-18 months of age and the effects of ginsenosides on r-ANP gene expression by determining the concentration of ANP-mRNA were investigated. The male and female rats were abdominally (i.p.) injected with aqueous solution of ginsenosides prepared from ginseng stems and leaves (G-PSL) and ginseng roots (G-PR), 50 mg/kg body wt, once a day for 7 days. Atria total RNA was extracted by the cold phenol method. The ANP-mRNA contents were determined using the Northern blot and dot blot hybridization technique with alpha-32*P-labelled r-prepro-ANP-cDNA probe. 2. The ANP-mRNA contents of 14-18 month rats were remarkably less than that of 2-3 month rats. The levels of male and female rats' atria at 14-18 months were about 15 and 60%, the content of male and female rats at 2-3 months respectively. 3. G-PSL and G-PR increased the ANP-mRNA content of male rats at 14-18 months 1- and 2-fold, respectively, whereas G-PSL and G-PR decreased the ANP-mRNA content in male rats of age 2-3 months. 4. These results revealed that the ANP gene expression declined during ontogenic ageing development and ginsenosides possessed anti-ageing effects in the heart endocrineous function aspect.