Biological Roles of Lipocalins in Chemical Communication, Reproduction, and Regulation of Microbiota.

Major evolutionary transitions were always accompanied by genetic remodelling of phenotypic traits. For example, the vertebrate transition from water to land was accompanied by rapid evolution of olfactory receptors and by the expansion of genes encoding lipocalins, which - due to their transporting functions - represent an important interface between the external and internal organic world of an individual and also within an individual. Similarly, some lipocalin genes were lost along other genes when this transition went in the opposite direction leading, for example, to cetaceans. In terrestrial vertebrates, lipocalins are involved in the transport of lipophilic substances, chemical signalling, odour reception, antimicrobial defence and background odour clearance during ventilation. Many ancestral lipocalins have clear physiological functions across the vertebrate taxa while many other have - due to pleiotropic effects of their genes - multiple or complementary functions within the body homeostasis and development. The aim of this review is to deconstruct the physiological functions of lipocalins in light of current OMICs techniques. We concentrated on major findings in the house mouse in comparison to other model taxa (e.g., voles, humans, and birds) in which all or most coding genes within their genomes were repeatedly sequenced and their annotations are sufficiently informative.

Transcriptomic and Proteomic Profiling Revealed High Proportions of Odorant Binding and Antimicrobial Defense Proteins in Olfactory Tissues of the House Mouse.

Mammalian olfaction depends on chemosensory neurons of the main olfactory epithelia (MOE), and/or of the accessory olfactory epithelia in the vomeronasal organ (VNO). Thus, we have generated the VNO and MOE transcriptomes and the nasal cavity proteome of the house mouse, Mus musculus musculus. Both transcriptomes had low levels of sexual dimorphisms, while the soluble proteome of the nasal cavity revealed high levels of sexual dimorphism similar to that previously reported in tears and saliva. Due to low levels of sexual dimorphism in the olfactory receptors in MOE and VNO, the sex-specific sensing seems less likely to be dependent on receptor repertoires. However, olfaction may also depend on a continuous removal of background compounds from the sites of detection. Odorant binding proteins (OBPs) are thought to be involved in this process and in our study Obp transcripts were most expressed along other lipocalins (e.g., Lcn13, Lcn14) and antimicrobial proteins. At the level of proteome, OBPs were highly abundant with only few being sexually dimorphic. We have, however, detected the major urinary proteins MUP4 and MUP5 in males and females and the male-biased central/group-B MUPs that were thought to be abundant mainly in the urine. The exocrine gland-secreted peptides ESP1 and ESP22 were male-biased but not male-specific in the nose. For the first time, we demonstrate that the expression of nasal lipocalins correlates with antimicrobial proteins thus suggesting that their individual variation may be linked to evolvable mechanisms that regulate natural microbiota and pathogens that regularly enter the body along the 'eyes-nose-oral cavity' axis.

Differential regulation of vaginal lipocalins (OBP, MUP) during the estrous cycle of the house mouse.

Female house mice produce pheromone-carrying major urinary proteins (MUPs) in a cycling manner, thus reaching the maximum urinary production just before ovulation. This is thought to occur to advertise the time of ovulation via deposited urine marks. This study aimed to characterize the protein content from the house mouse vaginal flushes to detect putative vaginal-advertising molecules for a direct identification of reproductive states. Here we show that the mouse vaginal discharge contains lipocalins including those from the odorant binding (OBP) and major urinary (MUP) protein families. OBPs were highly expressed but only slightly varied throughout the cycle, whilst several MUPs were differentially abundant. MUP20 or 'darcin', was thought to be expressed only by males. However, in females it was significantly up-regulated during estrus similarly as the recently duplicated central/group-B MUPs (sMUP17 and highly expressed sMUP9), which in the mouse urine are male biased. MUPs rise between proestrus and estrus, remain steady throughout metestrus, and are co-expressed with antimicrobial proteins. Thus, we suggest that MUPs and potentially also OBPs are important components of female vaginal advertising of the house mouse.

On the tear proteome of the house mouse (Mus musculus musculus) in relation to chemical signalling.

Mammalian tears are produced by lacrimal glands to protect eyes and may function in chemical communication and immunity. Recent studies on the house mouse chemical signalling revealed that major urinary proteins (MUPs) are not individually unique in Mus musculus musculus. This fact stimulated us to look for other sexually dimorphic proteins that may-in combination with MUPs-contribute to a pool of chemical signals in tears. MUPs and other lipocalins including odorant binding proteins (OBPs) have the capacity to selectively transport volatile organic compounds (VOCs) in their eight-stranded beta barrel, thus we have generated the tear proteome of the house mouse to detect a wider pool of proteins that may be involved in chemical signalling. We have detected significant male-biased (7.8%) and female-biased (7%) proteins in tears. Those proteins that showed the most elevated sexual dimorphisms were highly expressed and belong to MUP, OBP, ESP (i.e., exocrine gland-secreted peptides), and SCGB/ABP (i.e., secretoglobin) families. Thus, tears may have the potential to elicit sex-specific signals in combination by different proteins. Some tear lipocalins are not sexually dimorphic-with MUP20/darcin and OBP6 being good examples-and because all proteins may flow with tears through nasolacrimal ducts to nasal and oral cavities we suggest that their roles are wider than originally thought. Also, we have also detected several sexually dimorphic bactericidal proteins, thus further supporting an idea that males and females may have adopted alternative strategies in controlling microbiota thus yielding different VOC profiles.

On the saliva proteome of the Eastern European house mouse (Mus musculus musculus) focusing on sexual signalling and immunity.

Chemical communication is mediated by sex-biased signals abundantly present in the urine, saliva and tears. Because most studies concentrated on the urinary signals, we aimed to determine the saliva proteome in wild Mus musculus musculus, to extend the knowledge on potential roles of saliva in chemical communication. We performed the gel-free quantitative LC-MS/MS analyses of saliva and identified 633 proteins with 134 (21%) of them being sexually dimorphic. They include proteins that protect and transport volatile organic compounds in their beta barrel including LCN lipocalins, major urinary proteins (MUPs), and odorant binding proteins (OBPs). To our surprise, the saliva proteome contains one MUP that is female biased (MUP8) and the two protein pheromones MUP20 (or 'Darcin') and ESP1 in individuals of both sex. Thus, contrary to previous assumptions, our findings reveal that these proteins cannot function as male-unique signals. Our study also demonstrates that many olfactory proteins (e.g. LCNs, and OBPs) are not expressed by submandibular glands but are produced elsewhere-in nasal and lacrimal tissues, and potentially also in other oro-facial glands. We have also detected abundant proteins that are involved in wound healing, immune and non-immune responses to pathogens, thus corroborating that saliva has important protective roles.