Genes that modify expression of major urinary proteins in mice.

A survey of major urinary proteins (MUPs) from eight BALB/c mouse substrains by isoelectric focusing identified a common pattern with about 10 protein bands in males. One substrain, BALB/cJPt, differed in that it expressed two variant MUP patterns, designated 4.1lo and null. To find the chromosomal location of the gene which determines the 4.1lo phenotype, BALB/cJPt-MUP-4.1lo was crossed with a wild-derived Mus musculus domesticus inbred strain (CLA) that expresses the common BALB/c MUP pattern. The F1 phenotype revealed that the gene(s) controlling the MUP-4.1lo trait was recessive. A restriction fragment polymorphism between these strains found with a MUP cDNA probe allowed us to establish that a gene determining the MUP-4.1lo trait was not linked to the MUP structural genes on chromosome 4. Assays for other chromosomal marker loci revealed that a gene determining the MUP-4.1lo trait, designated Mupm-1, was closely linked to Myc-1 on chromosome 15. To determine the genetic basis of the null trait, BALB/cJPt-MUP-null mice were crossed with BALB/cJPt-MUP-4.1lo mice. A MUP restriction fragment polymorphism between these two lines was tightly linked to a gene or genes involved in determining the MUP-null phenotype. The two variant MUP phenotypes in BALB/cJ mice are determined by separate genes, one of which is located on chromosome 4 and the other on chromosome 15. The chromosomal location of Mupm-1 suggests that it produces a trans-acting factor which regulates MUP expression.     

Preference for social contact versus environmental enrichment in male laboratory mice

Due to their aggressive nature, male mice are less frequently used than female mice in biomedical research. When aggressive males are being used, individual housing is common practice. The question arises whether this is an acceptable housing for a social species. The present study was designed to gain more insight into the nature of inter-male social contact and into the potential of a form of environmental enrichment (nesting material) to compensate for the lack of social contact. In a series of tests, we analysed whether male mice of different ages preferred to spend time (1) near a familiar cage mate versus an empty cage, or (2) near to a familiar cage mate versus direct contact with nesting material (tissues). Dwelling time in each of the test cages and sleeping sites was recorded, as was the behaviour of the test mice. Results indicated that when other conditions were similar, male mice preferred to sleep in close proximity to their familiar cage mate. Furthermore, the need to engage in active social behaviour increased with age. Tissues were used to a large extent for sleeping and sleep-related behaviour. It is concluded that single housing in order to avoid aggression between male mice is a solution with evident negative consequences for the animals. When individual housing is inevitable due to excessive aggressive behaviour, the presence of nesting material could partly compensate for the deprivation of social contact.     

Analysis of epididymal proteins during sexual maturation in male albino mice.

Androgen dependent epididymal proteins act as antigen to produce autoantibodies and affect normal fertility. In the present study, epididymal proteins were analyzed during the time of sexual maturation and their androgen dependency was studied in male albino mice. Epididymis of 21 days (Pre-pubertal), 45 days (Pubertal), 60 days (Post-pubertal), orchidectomized (15 days after surgery) and orchidectomized with testosterone-treated (15 days after treatment) mice were dissected out and analyzed. Caput, corpus and cauda epididymidis were separated and the protein extract was prepared with 0.1 M PBS for 10% SDS-PAGE analysis. Testosterone assay was performed in the experimental groups except the testosterone treated group. The electrophoretic analysis of proteins in caput, corpus and cauda epididymidis of orchidectomized animals showed the disappearance of several proteins as compared to the adult. However, the disappeared proteins started to reappear in testosterone treated animals. The results suggest that removal of testis depletes the testosterone level and causes significant alteration in epididymal proteins. These proteins need further investigation for the purpose of immunocontraception by using them as antigens.     

The pattern of major urinary proteins (MUPS) expression during postnatal ontogenesis of the laboratory mouse depends on genotype and sex

We investigated the specific pattern of major urinary proteins (MUPs) expression in 3-, 4-, and 12-week old mice of CBA/LacY and C57BL/6JY inbred strains using polyacrylamide gel electrophoresis. Quantitative evaluation of 8 protein fractions A-H with regard to sex, age, and genotype of the animals is presented for the first time. Actual problems of genetic control and neuroendocrine regulation of MUPs expression during ontogenesis are discussed. In the light of current views on MUPs as a key component in intrapopulation information exchange via pheromones, we put forward the idea that the genetically determined structure of the olfactory code of the definitive type is formed at an early ontogenetic stage on the basis of the MUPs combinatorial pattern.     

Multiple genes coding for the androgen-regulated major urinary proteins of the mouse.

We have purified a cDNA fragment complementary to the mRNA coding for one of the major urinary proteins (MUPs) synthesized in the mouse liver. Using this cDNA as a hybridization probe, we have shown that the level of MUP mRNA is lower in the livers of females and castrated males than in those of males. The addition of testosterone to females and castrated males results in an increase in the concentration of the mRNA to levels found in males. There are approximately 15 gene per haploid genome coding for the MUPs; this allows a possible new interpretation of some of the genetic data concerning the regulation of levels of the different MUPs in the urine (Szoka and Paigen, 1978). Finally, we have shown that mouse MUP and rat alpha 2u-globulin mRNA share common sequences, but that there are surprising differences in gene number and regulation of the genes in these two closely related animals.     

The reproductive correlates of social hierarchy in laboratory male mice

In laboratory male mice the effects of social hierarchy on hormonal and spermatogenic testicular function, accessory organs and testicular weights, sexual behaviour have been investigated using an experimental model of social hierarchy, which is characterised by a minimal size (two male mice) and 5 days period of social interactions. The social rank of the partners was detected by asymmetry in aggressive behaviour. Using the experimental condition, when the both partners have no preferences for exclusive use of area we demonstrated that there were no rank differences in the number of mounts and testicular testosterone content. Nevertheless a rank asymmetry in the male sniffing behaviour towards a receptive female, weights of the testes, seminal vesicles, epididymes and the number of epididymal sperm was kept up in a stable social group. Social dominance was found to affect negatively on testicular testosterone increase in response to introduction of a receptive female and sexual attractiveness of male to a receptive female in both dominant and subordinate males. The results obtained demonstrate the impact of social hierarchy on reproduction in laboratory male mice, particular in respect of spermatogenesis and the testicular testosterone in response to a receptive female.     

Comparative investigations on cytogenetic effects of X-irradiation on the germinal epithelium of male mice and Chinese hamsters

Summary In one short-term-experiment and one long-term-experiment spermatogonia of mice and Chinese hamsters were compared for their sensitivity of X-ray induced chromosome aberrations.Short-term-experiment: Six hours after varying doses of X-rays the spermatogonia of both species were analysed and the number of induced chromatid breaks determined. At the dose range from 25–125 R the number of induced chromatid breaks per cell per roentgen is 0.01 in mice. In Chinese hamsters this value is 0.0072.The frequencies of chromatid breaks were studied in both species after a single dose of 100 R until 48 h p.i. The frequency in mice decreased more slowly than in hamster spermatogonia. After 12 h p.i. the ratio breaks in mice cells: breaks in hamster cells was 3.5:1, after 24 h this ratio was 5.2:1 after 48 h both frequencies were on the same level.Long-term-experiment: Analysis of spermatogonia and primary spermatocytes has been done 5 weeks after irradiation of the mice and 2, and 4 months after irradiation of the Chinese hamsters. The number of observed reciprocal translocations turned out to be higher in spermatogonial mitoses than in diakinesis-metaphases I in each animal.The conclusion is drawn for mice that a selection against abnormal cells is taking place already during pre-meiosis. In hamster pre-meiosis, the results are only indicative for a similar effect.These investigations were sponsored by the Deutsche Forschungsgemeinschaft within the SFB 35 (Klinishe Genetik).     

Dietary fats influence 'open-field' behaviour of male and female laboratory mice

Swiss mice of differing ages (juvenile and adult) and sexes were fed four specially formulated, pelleted diets containing respectively 8% saturated vegetable fat, 8% soya oil, 8% olive oil and 2% soya oil (with identities hidden from the experimenter) or a local commercial chow (3% crude fat) for 3 or 6 weeks. Subjects were individually housed and were assessed under red lighting for behaviour in a modified 'open field' (a 30 x 20 cm box with a black floor). Videotaped records were analysed using 'The Observer' system, quantifying transitions between inner and outer zones, rearing, freezing, grooming and defaecation as well as location in the two equal-sized zones. Clearly, these non-isocaloric diets differed in palatability, producing complex effects on growth as well as physiological and behavioural measures. Many indices were influenced by age, sex, and the duration of dietary exposure. Interactions between factors were common. Defaecation does not seem to provide a useful index of 'emotionality' in this type of study and investigations lacking a wide range of indices seem unlikely to provide unequivocal support for postulated links between dietary lipids and behaviour. The study broadly supports the contention that dietary fats subtly influence mood in mice.     

Effects of a major androgen-dependent urinary protein, alpha 2u-globulin on the pituitary-gonadal axis and hypothalamic monoamines in adult male mice.

The purpose of the present study was to evaluate the effects of alpha-2u-globulin, a sex-dependent male rat urinary protein on pituitary-gonadal functions and hypothalamic monoamine contents in male mice. Adult male mice, maintained under standardized laboratory conditions (L:D, 14:10) were injected subcutaneously with alpha-2u-globulin at a dose of 1 mg/animal/day or with vehicle daily for 14 days and killed 16 h after the last injection. Plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T) and testicular levels of T were measured by radioimmunoassays. The concentrations of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in medial basal hypothalamus (MBH) and anterior hypothalamus (AH) were measured by high performance liquid chromatography. Administration of alpha-2u-globulin led to a significant increase in plasma FSH and LH levels (P less than 0.05) as well as in plasma and testicular T levels (P less than 0.025). In the MBH of alpha-2u-globulin treated mice, there were significant elevations of NE (P less than 0.025), DA (P less than 0.01) and 5-HT (P less than 0.025) contents. In the AH, both DA (P less than 0.025) and 5-HT (P less than 0.01) contents were decreased while NE content remained unaltered. These results indicate that administration of alpha-2u-globulin can lead to a significant stimulation of pituitary-testicular axis and that this effect may be mediated through alteration of hypothalamic monoamines.     

Attempts to determine the status of electroshock-induced attack in male laboratory mice

An attempt was made using a combination of simple experimental manipulations and videotape recorder (VTR) analysis of bite targets employed to determine whether electroshock-induced attack on anosmic opponents in laboratory mice was an offensive or a defensive behaviour. VTR analysis suggested that ventral surface biting was more evident in this form of attack than in social conflict. Individually- and group-housed males showed similar levels of fighting on exposure to electroshock, but dominant males from pairs showed greater attack than their subordinate partners. Zinc sulphate-induced anosmia, 36 h of food deprivation, castration and lithium chloride treatment reduced electroshock-induced attack. Although significant changes were not obtained, there was some evidence that acute treatment with dexamethasone or ACTH augmented this behaviour. The direction of these changes is similar to that seen with social conflict, and it is suggested that electroshock-induced attack in the mouse (unlike the rat) is largely an aggressive offensive behaviour. The high incidence of ventral surface biting may be a consequence of the upright postures assumed on subjecting the animals to electroshock.     

The gene family for major urinary proteins: Expression in several secretory tissues of the mouse

The major urinary proteins (MUPs) of the mouse are encoded by a multigene family located at the Mup a locus on chromosome 4. Previous investigations have shown that the MUPs are synthesized in the liver, secreted and then excreted in the urine. We have found significant levels of MUP mRNA in several secretory tissues: the liver and the submaxillary, lachrymal and mammary glands. There are striking differences in hormonal and developmental regulation of MUP gene expression in these tissues. Furthermore, each tissue appears to express a characteristic pattern of MUP mRNAs. In particular, the lachrymal glands appear to express an entirely different set of MUP mRNAs. These results are discussed in relation to the organization of the MUP gene cluster and a possible function of the MUPs.     

Maturation of sexual behavior in laboratory male mice: a role of genotype

Sexual behaviour and testosterone output in response to a receptive female were investigated in male mice of three inbred strains BALB/cLac, CBA/Lac and PT at puberty (45 days of age) and in adulthood (90 days of age). The animals were exposed for 10 min to a receptive female separated by a plastic grill, which would not allow contact between male and female. Male and female behaviour was recorded by measuring the time the male or female spent at the grill and the number of approaches to it (sexual motivation). The grill was then removed and the number of mounts and chemoinvestigatory behavior towards a female (nasal and anogenital sniffing) was recorded for each male. An increase in serum concentration and testicular content of testosterone was used as an endocrine index of the sensitivity to female pheromones. It has been shown the significant genotype and developmental effects on sexual behaviour and the hormonal response to sexual stimuli. The pubertal BALB/cLac males were characterised by the adult pattern of sexual motivation, chemoinvestigatory behaviour and the evident testosterone respond to a female. Males of the strain PT showed the lowest sexual motivation, chemoinvestigatory behavior towards a receptive female and no testosterone responses at both ages. This is a very different situation with the CBA/Lac's who showed the developmental increase in the sexual motivation, sniffing behaviour and the endocrine reflex, and the highest level of sexual behaviour but the moderate testosterone respond to a female at adulthood. The data obtained suggest genotype related asynchrony in maturation of the olfactory system, pituitary-gonadal axis and neural circuits of sexual behavior, and their independent genetic control. So, the set of mice strains investigated represents a useful tool for genetic and endocrine study of sexual behavior and the chemosensory control of testicular steroidogenesis.     

Inhibition of sexual maturation by a urinary pheromone in male prairie deer mice

Male prairie deer mice (Peromyscus maniculatus bairdii) were treated from weaning until 8 weeks of age with chemical stimuli from conspecifics or with control substances. Growth of the testes and seminal vesicles was retarded in males that were reared in contact with soiled bedding material transferred from cages of adult males. No additional inhibition resulted from the physical presence of an adult male either continuously or for 1 hr per day. Application of urine collected from adult males to the noses of young males retarded seminal vesicle growth. Removal of the olfactory bulbs of males at 3 weeks of age blocked the inhibitory influence of urine on sexual maturation. Exposure to urine from adult females did not alter the growth of the reproductive organs in young males. The ability of a male deer mouse to retard the sexual maturation of young male conspecifics (Bediz, G. M., and Whitsett, J. M., 1979, J. Comp. Physiol. Psychol.93, 493–500) appears to be a consequence of chemical stimuli excreted in its urine.     

Butylated hydroxytoluene is a ligand of urinary proteins derived from female mice

Mice secrete substantial amounts of protein, particularly proteins called the major urinary proteins (MUPs), in urine. One function of MUPs is to sequester volatile pheromone ligands, thereby delaying their release and providing a stable long-lasting signal. Previously, only MUPs isolated from male mice have been used to identify ligands. Here, we tested the hypothesis that MUPs derived from females may also sequester volatile organic compounds. We identified butylated hydroxytoluene (BHT), a synthetic antioxidant present in the laboratory rodent diet, as a major ligand bound to urinary proteins derived from C57BL/6J female urine. BHT was also bound to the male-derived proteins, but the binding was less prominent than that in female urine, even though males express approximately 4 times more proteins than females. We confirmed that the majority of BHT in female urine was associated with the high molecular weight fraction (>10 kDa) and the majority of the proteins that sequestered BHT were MUPs as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequestration of BHT by MUPs was further confirmed by employing the recombinant MUP8 whose natural analogue has been reported in both sexes. Therefore, our data indicate that MUPs expressed in both sexes can bind, transport, and excrete xenobiotics into urine and raise the possibility that in addition to the known role in chemical communication, MUPs function as a defense mechanism against exogenous toxins.