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.     

Variation in the Major Urinary Protein Multigene Family in Wild-Derived Mice

The levels of expression and genomic organization of genes coding for the major urinary proteins (MUPs) were examined in several stocks of wild-derived mice. Levels of MUP mRNA in the liver varied considerably with M. musculus Brno and M. castaneus males having several-fold more MUP RNA than inbred C57BL/6 males, whereas M. hortulanus, M. caroli and M. cervicolor displayed levels much lower than C57BL/6. Analysis of RNA with MUP cDNAs specific to two different subfamilies of MUP genes revealed that M. caroli and M. cervicolor primarily expressed a MUP mRNA that was less abundant in C57BL/6, suggesting differential expression of subfamilies of genes within the MUP multigene complex. Although inbred males usually have five-fold more MUP mRNA than inbred females, male to female ratios for wild-derived stocks ranged from one to several hundred. Southern blots of genomic DNA hybridized to MUP subfamily probes revealed differences in restriction fragment sizes as well as possible variation in the number of MUP genes in some species. Analysis of urinary proteins from hybrids between C57BL/6 and M. spretus suggested that low MUP expression in M. spretus females was due to cis-acting genetic elements.     

Structural genes of the mouse major urinary protein are on chromosome 4

The major urinary proteins (MUPs) of mouse are a family of at least three major proteins which are synthesized in the liver of all strains of mice. The relative levels of synthesis of these proteins with respect to each other in the presence of testosterone is regulated by the Mup-a locus located on chromosome 4. In an effort to determine the mechanism of this regulation in molecular terms, a cDNA clone containing most of the coding region of a MUP protein has been isolated and identified by partial DNA sequence analysis. Using a combination of hybridization analysis and somatic cell genetics, the structural gene family has been unambiguously mapped to mouse chromosome 4. These data suggest that Mup-a regulation operates in a cis fashion and that models proposing trans regulation of MUP protein synthesis are unlikely.     

Variation in mouse major urinary protein (MUP) genes and the MUP gene products within and between inbred lines

The mouse major urinary proteins (MUPs) and the unprocessed in vitro translation products of MUP mRNA were each resolved by isoelectric focusing (IEF). The urinary MUPs showed about 15 distinct components, and the unprocessed MUPs about 20. In each case wide variation was observed in the relative intensities of individual bands. A comparison of three inbred lines (C57BL, BALB/c and JU) showed inter-line variation in the patterns both of the urinary MUPs and of the unprocessed MUPs. A series of experiments was carried out with a cloned MUP cDNA probe. All three inbred lines contain the same number (about 20) of MUP genes per haploid genome. In Southern blot analysis of genomic DNA the MUP genes displayed complex patterns which we interpret as showing variation on a common basic MUP gene sequence. For each combination of restriction enzymes tested, one size of fragment carried more than half of the total label, and this fragment was always the same in the three inbred lines. Inter-line differences were observed in the patterns of some of the less reactive fragments. MUP mRNA consists of at least two distinct species with sizes of 1 and 1.2 kb, which reacted with the probe in a label ratio of about 0.5 to 1. In the three inbred lines this ratio was essentially the same.     

Differential, multihormonal regulation of the mouse major urinary protein gene family in the liver.

The hormonal requirements for the regulation of the major urinary protein (MUP) mRNA levels in mouse liver have been examined. Previous experiments have shown that administration of testosterone to female or castrated male mice increases MUP mRNA levels approximately fivefold to normal male levels. We have found that thyroxine and the peptide hormone, growth hormone, each had a pronounced effect on MUP mRNA levels. MUP mRNA was reduced 150-fold in growth-hormone-deficient mutant mice (little). The administration of growth hormone and thyroxine induced MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. testosterone administration. When administered separately to these mice, growth hormone and thyroxine induced with MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. Testicular feminized mice, which lack a functional major testosterone receptor protein, can also be induced to male levels by treatment with both growth hormone and thyroxine. In addition, we present evidence which indicates that growth hormone, thyroxine, and testosterone differentially regulate the levels of distinct MUP mRNA species.     

Nucleotide sequences of liver, lachrymal, and submaxillary gland mouse major urinary protein mRNAs: mosaic structure and construction of panels of gene-specific synthetic oligonucleotide probes.

The mouse major urinary proteins (MUPs) are encoded by a gene family of about 35 to 40 members. MUPs are synthesized in at least six secretory tissues under a variety of developmental and endocrine controls, but the identities of the individual genes expressed in each tissue have not previously been established. In this article, we present the nucleotide sequences of five MUP mRNAs which we designate MUP I through V. MUPs I, II, and III are the most abundant MUP mRNA species in the liver, and MUPs IV and V are the most abundant MUP mRNA species in the lachrymal gland and the submaxillary gland, respectively. The sequence data show that each of the five mRNAs is encoded by a distinct member of the gene family. The structures of the MUP mRNA consist of interspersed segments of variable and conserved sequences. On the basis of the sequences of the variable segments, gene-specific panels of synthetic oligonucleotide probes were prepared. The gene-specific panels were used to identify cloned genes and, as described in the accompanying paper (K. Shahan, M. Denaro, M. Gilmartin, Y. Shi, and E. Derman, Mol. Cell. Biol. 7:1947-1954, 1987), to characterize the expression of MUP genes I through V.     

An abundant androgen-regulated mRNA in the mouse kidney.

We have identified an abundant 20,000 dalton protein (KAP) by in vitro translation of male mouse kidney mRNA. This protein is synthesized in reduced amounts from female kidney mRNA. A KAP cDNA fragment was purified and used for nucleic acid hybridization studies. Females and castrated males have 10 and 200 fold lower levels, respectively, of KAP mRNA relative to males. The administration of testosterone to females or castrated males results in the induction of KAP mRNA to normal male levels. Testicular feminized (Tfm) mice have 3 fold lower levels of KAP mRNA relative to normal males and are not induced by testosterone. KAP mRNA is not found in significant amounts in tissues other than the kidney, and the KAP gene renatures with kinetics similar to single-copy DNA. With the rapidly expanding knowledge of mouse genetics, KAP should prove useful in determining genetic factors which regulate the inducibility and tissue specificity of a hormonally regulated gene.     

Biosynthesis of the major urinary proteins in mouse liver: A biochemical genetic study

By labeling liver protein in vivo with [³H]leucine, the relative biosynthetic rate has been measured for the major urinary proteins (MUPs), three closely related, androgen-regulated proteins that are synthesized in mouse liver, secreted into the bloodstream, and excreted into the urine. In livers from females of strain C57BL/6J, total MUP synthesis represents about 0.6–0.9% of the total protein synthesis; in males and testosterone-treated females of the same strain, synthesis increases to about 3.5–4.0% of the total. This 4-to 6-fold induction of total MUP synthesis is similar to the androgen-mediated increase in MUP-specific messenger RNA reported by others, and indicates that the previously observed 20- to 25-fold induction of total MUP excretion into urine is generated partly at the posttranslational level. By measuring the ratio of synthesis of the individual MUPs, it was determined that the testosterone-mediated change in the relative levels of the MUPs in urine reflects a similar change in the pattern of MUP synthesis, indicating that the posttranslational processes operate on the quantity, and not the nature, of MUPs excreted. A survey of seven inbred mouse strains revealed polymorphism for the rate of total MUP synthesis in untreated females. Two classes could be distinguished on the basis of a 3- to 5-fold difference in the rate. This variation does not correlate with variation at Mup-a, a locus that controls the ratio of the three MUPs in urine from androgen-induced mice. These findings are consistent with the notion that MUP expression is controlled by a variety of independently assorting genes.     

From sexual attraction to maternal aggression: When pheromones change their behavioural significance

This article is part of a Special Issue “Chemosignals and Reproduction”.This paper reviews the role of chemosignals in the socio-sexual interactions of female mice, and reports two experiments testing the role of pup-derived chemosignals and the male sexual pheromone darcin in inducing and promoting maternal aggression. Female mice are attracted to urine-borne male pheromones. Volatile and non-volatile urine fractions have been proposed to contain olfactory and vomeronasal pheromones. In particular, the male-specific major urinary protein (MUP) MUP20, darcin, has been shown to be rewarding and attractive to females. Non-urinary male chemosignals, such as the lacrimal protein ESP1, promote lordosis in female mice, but its attractive properties are still to be tested. There is evidence indicating that ESP1 and MUPs are detected by vomeronasal type 2 receptors (V2R).When a female mouse becomes pregnant, she undergoes dramatic changes in her physiology and behaviour. She builds a nest for her pups and takes care of them. Dams also defend the nest against conspecific intruders, attacking especially gonadally intact males. Maternal behaviour is dependent on a functional olfactory system, thus suggesting a role of chemosignals in the development of maternal behaviour. Our first experiment demonstrates, however, that pup chemosignals are not sufficient to induce maternal aggression in virgin females. In addition, it is known that vomeronasal stimuli are needed for maternal aggression. Since MUPs (and other molecules) are able to promote intermale aggression, in our second experiment we test if the attractive MUP darcin also promotes attacks on castrated male intruders by lactating dams. Our findings demonstrate that the same chemosignal, darcin, promotes attraction or aggression according to female reproductive state.     

Protein pheromone expression levels predict and respond to the formation of social dominance networks

Communication signals are key regulators of social networks and are thought to be under selective pressure to honestly reflect social status, including dominance status. The odours of dominants and nondominants differentially influence behaviour, and identification of the specific pheromones associated with, and predictive of, dominance status is essential for understanding the mechanisms of network formation and maintenance. In mice, major urinary proteins (MUPs) are excreted in extraordinary large quantities and expression level has been hypothesized to provide an honest signal of dominance status. Here, we evaluate whether MUPs are associated with dominance in wild‐derived mice by analysing expression levels before, during and after competition for reproductive resources over 3 days. During competition, dominant males have 24% greater urinary MUP expression than nondominants. The MUP darcin, a pheromone that stimulates female attraction, is predictive of dominance status: dominant males have higher darcin expression before competition. Dominants also have a higher ratio of darcin to other MUPs before and during competition. These differences appear transient, because there are no differences in MUPs or darcin after competition. We also find MUP expression is affected by sire dominance status: socially naive sons of dominant males have lower MUP expression, but this apparent repression is released during competition. A requisite condition for the evolution of communication signals is honesty, and we provide novel insight into pheromones and social networks by showing that MUP and darcin expression is a reliable signal of dominance status, a primary determinant of male fitness in many species.     

Identification and characterization of functional genes encoding the mouse major urinary proteins.

Mouse Ltk- cells were stably transfected with cloned genes encoding the mouse major urinary proteins (MUPs). C57BL/6J MUP genomic clones encoding MUP 2 (BL6-25 and BL6-51), MUP 3 (BL6-11 and BL6-3), and MUP 4 (BL6-42) have been identified. In C57BL/6J mice, MUP 2 and MUP 4 are known to be synthesized in male, but not female, liver, and MUP 3 is known to be synthesized in both male and female liver and mammary gland. A BALB/c genomic clone (BJ-31) was shown to encode a MUP that is slightly more basic than MUP 2 and was previously shown to be synthesized in both male and female liver of BALB/c but not C57BL/6 mice. Comigration on two-dimensional polyacrylamide gels of the MUPs encoded by the transfecting gene provides a basis for tentative identification of the tissue specificity and mode of regulation of each gene. DNA sequence analysis of the 5' flanking region indicates that the different MUP genes are highly homologous (0.20 to 2.40% divergence) within the 879 base pairs analyzed. The most prominent differences in sequence occur within an A-rich region just 5' of the TATA box. This region (from -47 to -93) contains primarily A or C(A)N nucleotides and varies from 15 to 46 nucleotides in length in the different clones.     

Tissue-specific transcription initiation and effects of growth hormone (GH) deficiency on the regulation of mouse and rat GH-releasing hormone gene in hypothalamus and placenta

Hypothalamic GRH gene expression has been shown to be negatively regulated by GH in both rat and mouse. The recent reports of different 5' untranslated sequences in mouse GRH cDNA from hypothalamus and placenta have raised the possibility of tissue-specific regulation of the GRH gene. To provide support for this possibility, we have studied rodent models with GH deficiency due to genetic defects in the pituitary. Complementary DNA probes for the hypothalamic and placental 5' regions were used to determine the tissue specificity of each mRNA. Although the hypothalamic form of GRH mRNA was detected in placenta, it constituted less than 0.7% of total placental GRH mRNA. A placental 5' probe (based on the previously reported sequence) hybridized only with a larger mRNA species and was not tissue specific, indicating that it was not related to GRH and was derived possibly from a cloning artifact. The correct 5' sequence of mouse placental GRH cDNA was determined and shown to be distinct from both that previously reported and the hypothalamic sequence. Although the placental form of GRH mRNA was detected in hypothalamus using the polymerase chain reaction, its levels were undetectable by Northern blotting. The 5' end of rat placental GRH cDNA was similarly sequenced and shown to exhibit no homology with the rat 5' hypothalamic sequence, but a high degree of homology with the corresponding mouse placental sequence. In GH-deficient dwarf (dw/dw) rats, hypothalamic GRH mRNA levels were significantly increased above control levels in both females and males, and pregnancy did not alter the levels in either (dw) or control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue-specific expression of major urinary protein (MUP) genes in mice: characterization of MUP mRNAs by restriction mapping of cDNA and by in vitro translation.

The major urinary proteins (MUPs) in mice are coded for by a gene family which consists of ca. 30 members. The number of MUP genes that are expressed is not known. Previous studies have shown that MUP mRNAs are present in several secretory tissues in addition to the liver, in which they were originally identified. In this paper we show, through restriction analysis of MUP cDNAs, that distinct sets of MUP mRNAs are synthesized in each of the tissues studied and that these mRNAs are most likely coded for by different genes. As is shown, MUP mRNAs of different tissues are related to an extent that precludes the use of gene-specific probes in differentiating among them. The regions of homology also include the 3' untranslated regions of MUP mRNAs. The question of differential expression was thus investigated by searching for restriction polymorphisms in MUP mRNAs. We demonstrate that subtle differences in the sequences of even scarce mRNAs can be recognized by this particular approach. In addition, it is shown that MUP mRNAs of different tissues code for different, nonoverlapping sets of polypeptides, as determined by gel electrophoresis of in vitro-translated precursors to MUPs. The relevance of these results to models of evolution of tissue-specific regulation in a multigene family is discussed.     

Male pheromones and their reception by females are co-adapted to affect mating success in two subspecies of brown rats

Pheromonal communication plays a key role in the sociosexual behavior of rodents. The coadaptation between pheromones and chemosensory systems has been well illustrated in insects but poorly investigated in rodents and other mammals. We aimed to investigate whether coadaptation between male pheromones and female reception might have occurred in brown rats Rattus norvegicus. We recently reported that major urinary protein (MUP) pheromones are associated with male mating success in a brown rat subspecies, R. n. humiliatus (Rnh). Here, we discovered that MUPs were less polymorphic and occurred at much lower concentrations in males of a parapatric subspecies, R. n. caraco (Rnc), than in Rnh males, and found no association between pheromones and paternity success. Moreover, the observation of Rnc males that experienced chronic dyadic encounters and established dominance–submission relationships revealed that the dominant males achieved greater mating success than the subordinate males, but their MUP levels did not differ by social status. These findings suggest that male mating success in Rnc rats is related to social rank rather than to pheromone levels and that low concentration of MUPs might not be a reliable signal for mate choice in Rnc rats, which is different from the findings obtained in Rnh rats. In addition, compared with Rnh females, Rnc females exhibited reduced expression of pheromone receptor genes, and a lower number of vomeronasal receptor neurons were activated by MUP pheromones, which imply that the female chemosensory reception of pheromones might be structurally and functionally coadapted with male pheromone signals in brown rats.     

Major urinary proteins, alpha(2U)-globulins and aphrodisin

The major urinary proteins (MUPs) are proteins secreted by the liver and filtered by the kidneys into the urine of adult male mice and rats, the MUPs of rats being also referred to as alpha(2U)-globulins. The MUP family also comprises closely related proteins excreted by exocrine glands of rodents, independently of their sex. The MUP family is an expression of a multi-gene family. There is complex hormonal and tissue-specific regulation of MUP gene expression. The multi-gene family and its outflow are characterized by a polymorphism which extends over species, strains, sexes, and individuals. There is evidence of evolutionary conservation of the genes and their outflow within the species and evidence of change between species. MUPs share the eight-stranded beta-barrel structure lining a hydrophobic pocket, common to lipocalins. There is also a high degree of structural conservation between mouse and rat MUPs. MUPs bind small natural odorant molecules in the hydrophobic pocket with medium affinity in the 10(4)-10(5) M(-1) range, and are excreted in the field, with bound odorants. The odorants are then released slowly in air giving a long lasting olfactory trace to the spot. MUPs seem to play complex roles in chemosensory signalling among rodents, functioning as odorant carriers as well as proteins that prime endocrine reactions in female conspecifics. Aphrodisin is a lipocalin, found in hamster vaginal discharge, which stimulates male copulatory behaviour. Aphrodisin does not seem to bind odorants and no polymorphism has been shown. Both MUPs and aphrodisin stimulate the vomeronasal organ of conspecifics.     

Structure of mouse major urinary protein genes: different splicing configurations in the 3'-non-coding region.

The multigene family which codes for the mouse major urinary proteins (MUPs) consists of approximately 35 genes. Most of these are members of two different groups, Group 1 and Group 2, which can be distinguished by nucleic acid hybridisation. Here we describe the structure of a Group 1 gene and show that two size classes of MUP mRNA which are found in mouse liver result from different splicing events in the 3''-non-coding region and contain different polyadenylation sites. Short mRNA is approximately 750 nucleotides long, contains six exons, and is the main product of the Group 2 genes. Long mRNA is approximately 880 nucleotides long, contains seven exons and is the main product of the Group 1 genes. Five exons and part of the sixth are common to long and short mRNA and contain the coding region. This codes for an acidic protein of 180 amino acids containing an 18 residue signal peptide. A comparison of the mouse sequence with a homologous rat alpha 2u-globulin sequence shows that the rate of evolutionary divergence of the two proteins has been high. Silent sites have diverged four times more rapidly than replacement sites, showing that there has been selection against change in the protein sequence.     

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.